Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Ames Assay: Negative in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium both with and without metabolic activation.
In Vitro Transformation Assay: Inactive in mouse BALB/3T3 cells.
In Vitro Mouse Lymphoma Forward Mutation Assay: Weakly active with metabolic activation in L5178Y TK+/- cells.

In Vitro Chromosome Aberration Test: Negative with and without metabolic activation in cultured peripheral human lymphocytes.

Link to relevant study records

Reference

Reference 1

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

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

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 )

Target gene:

structural chromosome aberrations

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 20, 1981 to July 11, 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed to recognised test method in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: Clive, D. and Spector, J.F.S: Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y louse lymphoma cells. Mutation Res., 31:17-29. 1975.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The mouse lymphoma cell line, L5178Y TK+/-, used in this assay is derived from the Fischer L5178Y line of Dr. Donald Clive. Stocks are maintained in liquid nitrogen and laboratory cultures are periodically checked for the absence of mycoplasma contamination by culturing methods. To reduce the negative control frequency (spontaneous frequency) of TK-/- mutants to as low level as possible, cell cultures are exposed to conditions which select against the TK-/- phenotype (exposure to methotrexate) and are then returned to normal growth medium for three or more days before use. The cells are maintained in Fischer’s mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10% by volume). Cloning medium consists of the preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium is cloning medium contained 100 μg/ml of BrdU or 3 μg/ml of TFT.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

100, 200, 400, 600, 800 and 1000 μg/ml

Vehicle / solvent:

The test material was insoluble in water, dimethylsulfoxide (DMSO) and acetone at 100 mg/ml but dissolved in acetone at 50 mg/ml. Acetone was used as the vehicle as specified by the client.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylnitrosamine (DMN)

Details on test system and experimental conditions:

Negative Controls: A negative control consisting of assay procedures performed on untreated cells is performed in all cases. If the test compound is not soluble in growth medium, an organic solvent (normally DMSO) is used; the final concentration of solvent in the growth medium will be 1% or less. Cells exposed to solvent in the medium are also assayed as the solvent negative control to determine any effects on survival or mutation caused by the solvent alone. For test substances assayed with activation, the untreated and solvent negative control will include the activation mixture. Positive Controls: Ethylmethane sulfonate (EMS) is highly mutagenic via alkylation of cellular DNA and will be used at 0.5 μl/ml as a positive control for non-activation studies.Dimethylnitrosamine (DMN) requires metabolic activation by microsomal enzymes to become mutagenic and will be used at 0.3 μl/ml as a positive control for assays performed with activation. Sample Forms: Solid materials are dissolved in growth medium. If possible. Or in DMSO, unless another solvent is requested. Dosage Selection (Cytotoxicity Testing): The solubility of the test chemical in growth medium and/or DMSO is first determined. Then a wide range of chemical concentrations is tested for cytotoxicity, starting with a maximum applied dose of 10 mg/ml for test chemicals soluble in media of 1 mg/ml for solutions in organic solvents. After an exposure time of four hours, the cells are washed and a viable cell count is obtained the next day. Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells are used to select seven to ten doses that cover the range from 0 to 50-90% reduction in 24-hour growth. These selected doses are subsequently applied to cell cultures prepared for mutagenicity testing, but only four or five of the doses will be carried through the mutant selection process. This procedure compensates for daily variations in cellular cytotoxicity and ensures the choice if four or five doses spaced from 0 to 50-90% reduction in cell growth. Mutagenicity TestingNon-activation Assay: The procedure used is based on that reported by Clive and Spector (1975) and is summarised as follows. Cultures exposed to the test chemical for four hours at the preselected doses are washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates. At the end of the expression period, 3 x 106 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (non-selective). The ratio of resistant colonies to total viable cell number is the mutant frequency. Activation Assay: The activation assay can be run concurrently with non-activation assay. The only difference is the addition of the S9 fraction of rat liver homogenate and necessary cofactors (CORE) during the four-hour treatment period. CORE consists of NADP (sodium salt) and isocitric acid. The final concentrations of the activation system components in the cell suspension are: 2.4 mg NADP/ml; 4.5 mg isocitric acid/ml; and 50 μl S9/ml. S9 Homogenate: A 9,000 x g supernatant prepared from Fischer 344 adult male rat liver induced by Aroclor 1254 (described by Ames et al., 1975) is purchased from Bionetics Laboratory Products, Litton Bionetics, Inc. and used in this assay. The screened doses, cell counts, and mutant and viable colony counts will be entered into a computer program. The results are analysed and printed.The suspension growth of each culture is calculated as (Day 1 Cell Count/3) x (Day 2 Cell Count/3) x (Day 3 Cell Count/3) when the cultures are split back to 3 x e+5 cells/ml after the daily count. If the cell count is less than 4 x e+5 cells/ml, the culture is not split back and the cell count is substituted for 3 in the denominator of the next daily count. In most assays, 3-day expressions are not used, so only the first two factors in the preceding calculations are used. The suspension growth is calculated for each solvent control and then averaged. Relative suspension growth values are derived by dividing the suspension growth values by the average solvent control and multiplying by 100%.The average cloning efficiency for the negative controls in an assay is the average number of viable colonies for the solvent and untreated controls, divided by 300 and multiplied by 100%. In the computer tables, the cloning efficiency of each culture is expressed relative to the average solvent control cloning efficiency. Whenever the number of cells seeded for viable colony counts differs from 300, the computer calculation of the relative cloning efficiency is adjusted by the factor (300/cells seeded).A percent relative growth value is calculated as (relative suspension growth) x (relative cloning efficiency/100). Corrected values for the relative cloning efficiency are used in the cases where the number of cells seeded for viable colonies differs from 300. The mutant frequency is calculated as the ratio of mutant colonies to viable colonies times e-4. This calculation is unaffected by changes in the number of cells seeded for viable count because the number of cells seeded for mutant selection is changes by the same factor. Thus, as an example, if 250 cells are seeded for viable count, 2.5 x e+6 cells are seeded for mutant selection; the e-4 factor remains constant.

Evaluation criteria:

The following test results must be obtained to reach this conclusion for either activation or non-activation conditions:-A dose-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three doses, but this depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears. -An increase in mutant frequency may be followed by only small or no further increases at higher concentrations of toxicities. However, a decrease in mutant frequency to values below the minimum criterion is not acceptable in a single assay for classifying the test material as a mutagen. If the mutagenic activity at lower concentrations or toxicities was large, a repeat assay will be performed to confirm the mutagenic activity. -If an increase of about two time the minimum criterion or greater is observed for a single dose near the highest testable toxicity, as defined in the Assay Acceptance Criteria, the test material will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by repeat assay. -For some test materials, the correlation between toxicity and applied concentration is poor. The proportion of the applied material that effectively interacts with the cells to cause genetic alterations is not always repeatable or under control. Conversely, measurable changes in the frequency of induced mutants may occur with concentration changes that cause only small changes in observable toxicity. Therefore, either parameter, applied concentration or toxicity *percent relative growth), can be used to establish whether the mutagenic activity is related to an increase in effective treatment. A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.

Statistics:

statistical analysis for the confidence limits is not yet available.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Three trials of the mutation assay were initiated and the third trial was performed only in the presence of S9 microsomal activation. The first trial is not represented because the positive control cloning efficiencies in the non-activation and activation assays were below 10%, therefore the validity of the assay was questionable. Under non-activation conditions the treatments were initiated over a concentration range of 31.3 μg/ml to 1000 μg/ml. Since moderate inhibition of suspension growth was observed at the highest concentration if test material, four treatments from 700 μg/ml to 1000 μg/ml were chosen for mutant analysis on order to cover a wide range of toxic action. In this trial, the minimum criterion for mutagenesis was a mutant frequency exceeding 48.1 x e-6 and none of the observed mutant frequencies approached this value. The percent relative growths ranged from 23.3% (1000 μg/ml) to 41.3% (800 μg/ml). Results from treatments that demonstrate high toxicity (percent relative growths 10% to 15%) are desirable for evaluation of the assay. However since 1000 μg/ml represents the highest concentration of test material routinely assayed in an organic solvent and the limit of solubility had been reached, further trials at higher concentrations of test material were not indicated. Therefore, under non-activation conditions, the test material was considered non-mutagenic in this assay in the presence of high concentrations of test material. In the presence of S-9 microsomal activation, the test material was more toxic than without activation, indicating an interaction of the test material with the activation mix. The second trial in the presence of S-9 activation mix was initiated with treatments in the moderate to highly toxic range. Four treatments were analysed for mutant induction and three demonstrated weak mutagenesis. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 90.3 x e-6. At very high toxic treatments (percent relative growths, 5.9% to 16%) mutant frequencies were observed that were 1.8-fold to 3.4-fold above background (average of the negative control mutant frequencies). Even though a positive response was demonstrated according to our criteria for mutagenesis, this is a borderline response with weak mutagenesis occurring only at highly toxic treatments. Therefore a third trial in the presence of S9 microsomal activation was considered necessary for evaluation of the test material. In the third trial with S9 microsomal activation, six treatments ranging from 100 μg/ml to 1000 μg/ml were analysed for mutant induction. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 27.3 x e-6. Four of the six assayed treatments induced mutant frequencies slightly above the minimum criterion and ranged from 3.6-fold to 4.2-fold above the background (average of the solvent and negative control mutant frequencies). The two least-toxic treatments (percent relative growths 47% and 56.2%) were not mutagenic and the four treatments (400 μg/ml to 1000 μg/ml) with percent relative growths ranging from 22.4% to 26.4% induced significant, although small, increases over the backgrounds. Therefore, the test material is considered weakly active in the presence of S9 microsomal activation in this assay. The average cloning efficiencies for the solvent and untreated negative controls varied from 119.3% without activation to 88.9% and 88.9% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. (However, the relative cloning efficiencies of both positive controls were exceptionally low in the first trial which necessitated initiation of a second and third trial for evaluation of the test article.)

Remarks on result:

other: all strains/cell types tested

Summary of Mouse Lymphoma (L5178Y) Results

Test	S-9		Daily Counts (cells/ml x 10E+5)			Relative Suspension Growth (% of Control)	Total Mutant Clones	Total Viable Clones	Relative Cloning Efficiency (% of Control)	Percent Relative Growth*	Mutant Frequency**
	Source	Tissue	1	2	3
NON-ACTIVATION
Solvent Control	---	---	10.3	14.7		100.0	98.0	335.0	100.0	100.0	29.3
Solvent Control	---	---	14.9	15.5		100.0	107.0	403.0	100.0	100.0	26.6
Untreated Control	---	---	15.4	14.6		117.6	68.0 +	336.0	91.1	107.1	20.2
EMS .5 μl/ml	---	---	11.4	8.7		51.9	499.0	69.0	18.7	9.7	723.2
EMS .5 μl/ml	---	---	8.7	8.6		39.1	462.0	78.0	21.1	8.3	592.3
Test Compound            700.000 μg/ml             800.000 μg/ml             900.000 μg/ml           1000.000 μg/ml	--- --- --- ---	--- --- --- ---	9.7 11.1 10.5 7.6	10.5 10.8 7.0 4.9		53.3 62.7 38.4 19.5	***90.0 ***90.0 ***86.0+ ***124.0	225.0 243.0 237.0+ 442.0	61.8 65.9 64.2 119.8	32.5 41.3 24.7 23.3	40.0 37.0 36.3 28.1
ACTIVATION
Solvent Control	Rat	Liver	10.2	10.2		100.0	128.0	266.0	100.0	100.0	48.1
Solvent Control	Rat	Liver	12.0	10.8		100.0	165.0+	309.0	100.0	100.0	53.4
Untreated Control	Rat	Liver	10.0	10.0		85.6	133.0	225.0	89.3	67.0	59.1
DMN .3 μL/ml	Rat	Liver	7.4	6.1		38.6	65.0+	22.0	7.7	3.0	295.5
DMN .3 μL/ml	Rat	Liver	6.8	5.7		33.2	95.0	27.0	9.4	3.1	351.9
Test Compound           700.000 μg/ml           800.000 μg/ml           900.000 μg/ml         1000.000 μg/ml	Rat Rat Rat Rat	Liver Liver Liver Liver	5.6 4.6 4.4 2.7	10.1 5.1 6.4 4.5		48.4 20.1 24.1 11.6	***139.0 ***245.0+ ***185.0 ***205.0	175.0 135.0 191.0 147.0	60.9 47.0 66.4 51.1	29.5 9.4 16.0 5.9	79.4 181.5 96.9 139.5

 * (relative suspension growth x relative cloning efficiency) / 100

** The ratio of cells seeded for mutant selection to cells seeded for cloning efficiency is 10E+4.

Therefore the mutant frequency is: (total mutant clones/total viable clones) *10E-4.

The mutant frequency is given in units of 10E-6.

*** Precipitate was present. Colonies were counted visually as well as mechanically.

+ One plate contaminated; value based on remaining two plates.

 

Summary of Mouse Lymphoma (L5178Y) Results

Test	S-9		Daily Counts (cells/ml x 10E+5)			Relative Suspension Growth (% of Control)	Total Mutant Clones	Total Viable Clones	Relative Cloning Efficiency (% of Control)	Percent Relative Growth*	Mutant Frequency**
	Source	Tissue	1	2	3
ACTIVATION
Solvent Control	Rat	Liver	15.1	10.6		100.0	33.0	279.0	100.0	100.0	11.8
Solvent Control	Rat	Liver	18.4	9.1		100.0	31.0	252.0	100.0	100.0	12.3
Untreated Control	Rat	Liver	17.5	10.5		11.2	28.0	269.0	101.3	113.7	10.4
DMN .3 μL/ml	Rat	Liver	9.4	6.6		37.9	154.0	40.0	15.1	5.7	285.0
Test Compound           100.000 μg/ml           200.000 μg/ml           400.000 μg/ml           600.000 μg/ml           800.000 μg/ml         1000.000 μg/ml	rat rat rat rat rat rat	Liver Liver Liver Liver Liver Liver	13.1 12.4 8.3 9.1 9.2 7.5	8.4 8.0 6.6 7.7 6.4 8.2		67.2 70.6 33.5 42.8 36.0 37.6	58.0 56.0 80.0 74.0 93.0 67.0	222.0 206.0 189.0 164.0 193.0 158.0	83.6 77.6 71.2 61.8 72.7 59.5	56.2 47.0 23.8 26.4 26.1 22.4	26.1 27.2 42.3 45.1 48.2 42.4

 * (relative suspension growth x relative cloning efficiency) / 100

** The ratio of cells seeded for mutant selection to cells seeded for cloning efficiency is 10E+4.

Therefore the mutant frequency is: (total mutant clones/total viable clones) *10E-4.

The mutant frequency is given in units of 10E-6.

Conclusions:

The test material is considered to be weekly active in the Mouse Lymphoma Forward Mutation Assay with metabolic activation.

Executive summary:

The objective of the study is to evaluate the test material for its ability to induce forward mutation in the L5178Y TK+/- mouse lymphoma cell line, as assessed by colony growth in the presence of 5-bromo-2’-deoxyuridine (BrdU) or 5-trifluorothymidine (TFT).

 

The test material, BXA, was insoluble in water, dimethylsulfoxide (DMSO) and acetone at 100 mg/ml but dissolved in acetone at 50 mg/ml. Acetone was used as the vehicle for this study as specified by the client. Just prior to each testing purpose, stock solutions were prepared and serial solutions were performed in acetone. The stocks were then diluted 1:100 into tubes of culture media containing the cells to initiate the treatments. A fine precipitate was noted in the culture medium at concentrations as low as 31.3 μg/ml and the precipitate increased with increasing concentrations of test material. The treatments ranged from 0.061 μg/ml to 1000 μg/ml in the first trial.

 

Three trials of the mutation assay were initiated and the third trial was performed only in the presence of S9 microsomal activation. The first trial is not represented because the positive control cloning efficiencies in the non-activation and activation assays were below 10%, therefore the validity of the assay was questionable.

 

Under non-activation conditions the treatments were initiated over a concentration range of 31.3 μg/ml to 1000 μg/ml. Since moderate inhibition of suspension growth was observed at the highest concentration if test material, four treatments from 700 μg/ml to 1000 μg/ml were chosen for mutant analysis on order to cover a wide range of toxic action. In this trial, the minimum criterion for mutagenesis was a mutant frequency exceeding 48.1 x 10^-6and none of the observed mutant frequencies approached this value. The percent relative growths ranged from 23.3% (1000 μg/ml) to 41.3% (800 μg/ml). Results from treatments that demonstrate high toxicity (percent relative growths 10% to 15%) are desirable for evaluation of the assay. However since 1000 μg/ml represents the highest concentration of test material routinely assayed in an organic solvent and the limit of solubility had been reached, further trials at higher concentrations of test material were not indicated. Therefore, under non-activation conditions, the test material was considered non-mutagenic in this assay in the presence of high concentrations of test material.

 

In the presence of S-9 microsomal activation, the test material was more toxic than without activation, indicating an interaction of the test material with the activation mix. The second trial in the presence of S-9 activation mix was initiated with treatments in the moderate to highly toxic range. Four treatments were analysed for mutant induction and three demonstrated weak mutagenesis. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 90.3 x 10^-6. At very high toxic treatments (percent relative growths, 5.9% to 16%) mutant frequencies were observed that were 1.8-fold to 3.4-fold above background (average of the negative control mutant frequencies). Even though a positive response was demonstrated according to our criteria for mutagenesis, this is a borderline response with weak mutagenesis occurring only at highly toxic treatments. Therefore a third trial in the presence of S9 microsomal activation was considered necessary for evaluation of the test material.

 

In the third trial with S9 microsomal activation, six treatments ranging from 100 μg/ml to 1000 μg/ml were analysed for mutant induction. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 27.3 x 10^-6. Four of the six assayed treatments induced mutant frequencies slightly above the minimum criterion and ranged from 3.6-fold to 4.2-fold above the background (average of the solvent and negative control mutant frequencies). The two least-toxic treatments (percent relative growths 47% and 56.2%) were not mutagenic and the four treatments (400 μg/ml to 1000 μg/ml) with percent relative growths ranging from 22.4% to 26.4% induced significant, although small, increases over the backgrounds. Therefore, the test material is considered weakly active in the presence of S9 microsomal activation in this assay.

 

The average cloning efficiencies for the solvent and untreated negative controls varied from 119.3% without activation to 88.9% and 88.9% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. (However, the relative cloning efficiencies of both positive controls were exceptionally low in the first trial which necessitated initiation of a second and third trial for evaluation of the test article.)

 

The test material, BXA, induced small increases in the mutant frequency of the TK locus in L5178Y mouse lymphoma cells only in the presence of rat liver S-9 microsomal activation. Without activation, concentrations up to 1000 μg/ml were moderately toxic and were not detectably mutagenic. With activation, only highly toxic treatments at concentrations at or above 800 μg/ml were weakly mutagenic in the second trial. In the third trial, concentrations of test material from 400 μg/ml to 1000 μg/ml were moderately toxic and induced weak but significant increases in the mutant frequency. The results from the first trial were not discussed because of technical problems with the assay. Therefore, the test material is considered to be weakly active in the Mouse Lymphoma Forward Mutation Assay with metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 25, 1981 to April 16, 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Ames, Bruce N., Joyce McCann, and Edith Yamasaki - Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research 31: (1975) 347-364.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine auxotrophs

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1000, 333, 100, 33 and 10 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data;

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: N-Methyl-NPR-Nitro-N-Nitrosoquanidine; 2-aminofluorene

Details on test system and experimental conditions:

Working cultures: Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50ml of oxoid broth and grown for 16 hours at 37°C in a New Brunsiwck Scientific Model G24 Environmental Incubator Shaker.Master Plates: In order to avoid the effects of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing. Master plates were prepared by spreading 0.1ml of 0.1M histidine and 0.5nM biotin on the surface of minimal glucose agar plates using a sterile glass spreader.For the R-factor strains, 0.1ml of ampicillin (8 mg/ml) was spread in addition to the histidine and biotin as pressure for retaining the plasmid. The plates were prepared 24 to 48 hours before use. Overnight cultures prepared from frozen stock cultures were then streaked onto the surface of the plates. The plates were incubated over night at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures.Tester strains were routinely checked for the presence of the appropriate genetic markers.Negative (Solvent) Controls: Tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were plated with the appropriate solvent, both with and without metabolic activation, to obtain background lawn and relevant colony formation to serve as negative solvent controls. Positive Controls: Positive controls were strain specific and were as follows:TA 1535 – without activation – N-Methyl-NPR-Nitro-N-Nitrosoquanidine (20 μg/ml). TA 1537 – without activation – 9-aminoacridine (1500 μg/ml).TA 1538 – with activation – 2-aminofluorene (100 μg/ml).TA 98 – with activation – 2-aminofluorene (100 μg/ml).TA 100 – without activation – N-Methyl-NPR-Nitro-N-Nitrosoquanidine (20 μg/ml). Replication: Tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with five levels of the test compound. All negative and positive controls were plated in triplicate. All treated groups were plated in duplicate. Preliminary Toxicity Screen: The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA 1538 and TA 100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium teste strains. The test compound was prepared to a concentration of 50 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 16.6, 5.0, 1.66 and 0.5 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5nM Histidine-0.5nM Biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile tubes (13 x 100 mm) with kaputs were labelled and placed into a Fisher Isotemp ® Dry Bath (No. 145) at 45°C. All control and treated tubes and plated were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and place in a dark 37°C incubator (GCA/Precision Scientific Thelco ® 32M). The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria.Dose Range: The concentration giving moderate inhibition of bacterial lawn growth served as the high dose in the plate incorporation assay. If no toxicity was produced, the high dose chosen for the plate incorporation assay will approach or be a saturated solution of the test article. Lower doses will be at log dilutions. Plate Incorporation Assay: The mutagenesis plate incorporation assay used the five histidine auxotrophs of Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The genetic description of these strains can be found in table form (see Any other information). Top Agar: Top agay, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 nM histidine-0.5nM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. Metabolic Activation System: Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:0.4 M MgCl2; 1.65M KCL20 μl1M Glucose-6-Phosphate5 μl0.1M NADP40 μl0.2M Phosphate buffer pH 7.4500 μlSterile distilled H2O335 – 395 μlS-9 Fraction40 – 100 μl. The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which are then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco ® 32M). The plates were incubated for 48 – 72 hours, checked for uniform background lawn, and scored by counting revertant colonies.

Evaluation criteria:

Data Reporting: In scoring the assay, the positive and negative control were first evaluated. If the control values did not fall within the acceptable historical values, the remaining plates were not scored and the assay was repeated.Evaluation Criteria: In most tests with the Salmonella/microsome assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean for control values and the test chemical produces the highest increase equal to or greater than three time the solvent control value, the test chemical will be considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

None specified

Key result

Species / strain:

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

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 1538

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

Additional information on results:

There was no apparent change in the physical state of the test article and control articles during administration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

CONTROLS

			Spontaneous Revertants (x)
		S-9	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Negative Controls		(-)^1	13	11	16	27	134
		(+)^2	10	12	24	32	120
Positive Controls
N-Methyl-N-Nitro-N-Nitrosoguanidine	2 μg/plate	(-)	1194				1170
9-Aminoacridine	150 μg/plate	(-)		873
2-Aminofluorene	10 μg/plate	(+)			942	1003

 

TEST COMPOUND

		Revertant Colonies/Plate (x)
Dose Levels (μg/plate)	S-9	TA 1535	TA 1537	TA 1538	TA 98	TA 100
1000	-/+	7/14	9/11	18/23	24/40	150/153
333	-/+	11/10	10/10	17/25	27/34	114/108
100	-/+	4/9	11/12	11/21	31/31	144/109
33	-/+	7/10	8/10	14/20	28/33	148/104
10	-/+	10/11	13/11	17/24	35/31	116/110

¹(-) Without S-9 Aroclor-induced rat liver metabolic activation

²(+) With S-9 Aroclor-induced rat liver metabolic activation

Conclusions:

The results for test article BXA, were negative in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium both with and without metabolic activation.

Executive summary:

Study purpose was to evaluate BXA for genetic activity in the Ames Salmonella typhimurium Assay with and without metabolic activation preparation.

 

Test article BXA, Lot #34, was soluble in DMSO. Dose level for the Preliminary Toxicity Screen were 5000, 1666, 500, 166 and 50 μg/plate. Strain TA 1538 showed extreme inhibition at 5000 and 1666 μg/plate, moderate inhibition at 500 μg/plate and slight inhibition at 166 and 50 μg/plate. Strain TA 100 showed moderate inhibition at 5000 μg/plate and slight inhibition at 1666 μg/plate. Dose levels in the plate assay were 1000, 333, 100, 33 and 10 μg/plate. There were 0.06 ml of S-9 supernatant (37 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

 

There was no apparent change in the physical state of the test article and control articles during administration.

 

The results for test article BXA, were negative in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium both with and without metabolic activation preparation at 1000, 333, 100, 33 and 10 μg/plate.

All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical mean data.

Reference 4

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 24, 1981 to May 4, 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in recognised test method in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: Kakunaga, T.: A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. Int. J. Cancer, 12:463-473, 1973.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

foci of transformed cells, recognized by dense, piled-up colonies on a monolayer of normal cells.

Species / strain / cell type:

mammalian cell line, other: BALB/3T3 mouse cells

Details on mammalian cell type (if applicable):

Clone I of BALB/3T3 mouse cells was obtained from Dr. Takeo Kakunaga. Further sub-clones, selected for low spontaneous frequencies of foci formation, are used for assays. Stocks are maintained in liquid nitrogen and laboratory cultures are check periodically to ensure the absence of mycoplasma contamination. Cultures are grown and passaged weekly in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Concentration range of 10.0 μg/ml to 1000.0 μg/ml

Vehicle / solvent:

Solid materials are dissolved in growth medium, if possible, or in DMSO, unless another solvent is requested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Control CompoundsNegative Controls: A negative control consisting of assay performed in untreated cells is performed. If the test compound is not soluble in growth medium, an organic solvent (normally DMSO) is used; the final concentration of solvent in the growth medium will be 1% or less. Cells exposed to solvent on the medium are assayed as the solvent negative control to determine any effects on survival or transformation caused by the solvent alone. Fifteen flasks of the appropriate type of negative control are prepared for each assay.Positive Control: 3-methylcholanthrene (MCA) is a known carcinogen and is used as a positive control for the transformation of 3T3 cells. Fifteen flasks are treated with 5 μg MCA per millilitre for each assay.Experimental DesignDosage Selection: The solubility of the test chemical in growth medium, DMSO or other solvent is first determined. Fifteen dose levels of the test compound are then chosen, starting with a maximum applied dose of 1 mg/ml for solid compounds or 1 μl/ml for liquid samples and decreasing in twofold-dilution steps.Each dose is applied to three culture dishes seeded 24 hours earlier with 200 cells per dish. After an exposure period of three days, the cells are washed and incubated in growth medium for an additional four days. The surviving colonies are stained and counted by an automatic colony counter. A relative survival for each dose is obtained by comparing the number of colonies surviving treatment to the colony counts in negative control dishes. The highest dose chosen for subsequent transformation assays should cause no more than a 50% reduction in colony forming ability and is best located near 30% reduction. Four lower doses (including one or two doses with low or no apparent toxicity) are also selected for the transformation assay. Transformation Assay: The procedure used is based on that reported by Kakunaga (1973). Twenty-four hours prior to treatment, a series of 25-cm2 flasks is seeded with 104 cells/flask and incubated. Fifteen flasks are then treated for each of the following conditions: Five preselected doses of test chemical; positive control; and solvent negative control, if applicable. The flasks are incubated for a three-day exposure period; the cells are then washed and incubation is continued for four weeks with refeeding twice a week. The assay is terminated by fixing the cell monolayers with methanol and staining with Giemsa. The stained flasks are examined by eye and by microscope to determine the number of foci of transformed cells. Scoring of Transformed Foci: At the end of the four-week incubation period, cultures of normal cells yield a uniformly stained monolayer of round, closely-packed cells. Transformed cells for a dense mass (focus or colony) that stains deeply (usually blue) and is superimposed on the surrounding monolayer of normal cells. The foci are variable in size.Scored foci have several variations in morphological features. Most foci consist of a dense piling up of cells and exhibit a random, criss-cross orientation of fibroblastic cells at the periphery of the focus. Other scored foci are composed of more rounded cells with little criss-crossing at the periphery but with necrosis at the centre cause by the dense piling up of a large number of cells. A third variation is a focus without the necrotic centre and large number of cells but which exhibit the criss-cross pattern of overlapping cells throughout most of the colony. Some foci are not scored. These include small foci of transformed morphology that are found in close proximity to larger foci; these foci are regarded as being formed from cells which migrated from the larger colony. Other unscored foci are small areas where some piling up of rounded cells has occurred but the random orientation of fibroplastic cells is not observed. Microscopic examination is employed for scored foci and in the final judgement of transformed character for any marginal foci.

Evaluation criteria:

In many cases, no transformed foci will be observed in the set of flasks comprising the negative control. This does not necessarily mean that any foci found in the treated flasks constitutes a positive response in this assay. In order to determine what minimum number of foci will allow a conclusion that the frequency of transformed foci has been elevated over the negative control, a historical negative control data base in used. This data base consists of the ten most recent assays in which 100 to 150 negative control flasks have been scored. The total number of flasks and transformed foci in this set will be provided in each report. If the negative control is found by the same test to be significantly different from the historical control (p≤0.05), the assay will be evaluated independently. Comparisons between the current negative control and tested dose levels will be analysed by the Kastenbaum-Bowman tables.The number of induced foci usually does not increase proportionately with the applied dose in this assay. In fact, above a minimum dose level where the number of foci is elevated, further increases in dose may result in little or no increase in the number of foci. The number of foci can be reduced at the highest dose assayed if the toxicity is too high. A response at only one dose level (other than the highest dose tested) that just meets the 95% confidence level will normally not be considered sufficient evidence for activity in this assay. All other degrees of response will usually provide evidence for classifying a test substance as active.

Statistics:

The statistical tables provided by Kastenbaum and Bowman are used to determine whether the results at each dose level are significantly different from the historical control at the 95% or 99% confidence levels. This test compares variables distributed according to Poissonian expectations by summing the probabilities in the tails of two binomial distributions. The 95% confidence level must be met to consider the test substance active at a particular dose level.

Key result

Species / strain:

mammalian cell line, other: BALB/3T3 mouse cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The relative survivals observed for test material concentrations from 1.0 mg/ml to 0.061 μg/ml ranged between an apparent minimum of 54.3% at 125.0 μg/ml to a maximum of 89.9% at 1.95 μg/ml. Thus, survivals below 50% were not obtained and graphic analysis of the cytotoxicity data did not reveal clear evidence of the occurrence of a dose-related response. The transformations assay is normally applied to test material concentrations that result in between 50% and 100% survival and is considered to be most sensitive near 70% survival, since the observed frequency of the appearance of transformed foci is not corrected for the number of cells surviving the treatments. In the present assay, experimental resolution of a dose-related cytotoxicity response was obviated by the extremely poor aqueous solubility of the test material and therefore the doses chosen for use in the transformation were selected so as to include concentration exhibiting a wide range if apparent solubilities. Thus, the concentration range of 1.0 mg/ml to 10.0 μg/ml, corresponding to a survival range of approximately 67% to 80% (estimated graphically) and including both aqueous dispersions (e.g. 1.0 mg/ml) and apparent solutions (e.g.: 10.0 μg/ml), was chosen for the assay. The negative (solvent) control measures the frequency of the appearance of spontaneously transformed foci. This historical negative control for the subclone of 3T3 cells used in this assay consists of 147 vessels containing a total of 21 transformed foci for an average of 0.14 focus/vessel. In the present assay, 3 foci were observed among the 20 negative control dishes for an average of 0.15 focus/dish. This result was not significantly different from the historical value showing that the frequency of spontaneous transformation was within the expected range. None of the applied concentrations of test material caused the induction of a significant number of transformed foci. In contrast, the MCA positive control treatment induced an average of 1.25 foci/dish. This result was significant at >99% confidence level using the Kastenbaum-Bowman Tables, showing that the sensitivity of the assay was adequate to resolve the positive control transforming activity. Thus, concentrations of test material from 10.0 μg/ml to 1000.0 μg/ml were clearly not transforming to 3T3 cells.

Remarks on result:

other: all strains/cell types tested

Toxicity Test in BALB 3T3 Cells

Test Compound Doses Tested		Average Number of Colonies/Plate	% Survival Relative to Control
1.	1000.0 μg/ml	61.7	67.0%
2.	500.0 μg/ml	55.7	60.5%
3.	250.0 μg/ml	59.7	64.9%
4.	125.0 μg/ml	50.0	54.3%
5.	62.5 μg/ml	70.3	76.4%
6.	31.3 μg/ml	75.7	82.3%
7.	15.6 μg/ml	67.3	73.2%
8.	7.81 μg/ml	76.3	83.0%
9.	3.91 μg/ml	73.0	79.3%
10.	1.95 μg/ml	82.7	89.9%
11.	0.98 μg/ml	71.0	77.2%
12.	0.49 μg/ml	74.3	80.8%
13.	0.24 μg/ml	70.3	76.4%
14.	0.12 μg/ml	74.7	81.2%
15.	0.06 μg/ml	64.7	70.3%
16.	0 (control)	92.0	100.0%

 

Summary of Data from Transformation Assay

Test Doses Tested	Number of Foci per Flask Scored																				Total No. of Foci	No. of Foci/ Flask
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
Negative Control (Acetone) 0.5% v/v	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	0	1	3	0.15
Positive Control (MCA) 5.0 μg/ml	0	0	2	3	1	1	0	0	3	0	1	2	1	3	3	1	0	1	3	0	25**	1.25
Test Material
1000.0 μg/ml	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	C	C	C	1	0.06
300.0 μg/ml	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	0	0	C	3	0.16
100.0 μg/ml	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	1	1	3	0.15
30.0 μg/ml	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	1	3	0.15
10.0 μg/ml	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

MCA = 3-Methylcholanthrene

C = Flask was contaminated and not scored

** = Data significant from the negative control at p ≤ 0.01.

Conclusions:

Interpretation of results (migrated information):negativeThe test material, BXA, Batch #34, did not induce the appearance of a significant number of transformed foci over the applied concentration range of 10.0 μg/ml to 1000.0 μg/ml. This concentration range encompassed the range of apparent solubilities of the test material and corresponded to approximately 80% to 67% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in the BALB/3T3 In Vitro Transformation Assay.

Executive summary:

The assay evaluates the carcinogenic potential of test materials using muose BALB/3T3 cells in culture. The objective of this semi-quantitative assay is to evaluate the test material for its ability to induce foci of transformed cells, recognised by dense, piled-up colonies on a monolayer of normal cells.

 

BALB/3T3 mouse cells will multiply in culture until a monolayer is achieved and will then cease further division. These cells, if injected into immunosuppressed, syngeneic host animals, will not produce neoplastic tumors. However, cells treated in vitro with chemical carcinogens will give rise to foci of cellular growth super-imposed on the cell monolayer. If these foci are picked from the cultures, grown to larger numbers and injected into animals, a malignant tumor will in most cases be obtained. Thus, the appearance if piled-up colonies in treated cell cultures at a higher frequency than in control cultures is highly correlated with malignant transformation.

 

The test material, BXA, Batch #34, was dissolved in acetone to a maximum concentration of 200 mg/ml. Serial 1:2 dilutions were performed in acetone to obtain a series of 15 stock concentrations ranging between 200 mg/ml and 0.012 mg/ml. Additional 1:200 dilutions were performed in culture medium to obtain working solutions ranging in concentration from 1.0 mg/ml to 0.061 μg/ml for use in the preliminary cytotoxicity test. The test material exhibited very poor solubility in culture medium at the concentration of 1.0 mg/ml and marked turbidity was observed in dilutions containing 500.0 μg/ml through 31.3 μg/ml. Evidence of precipitated material was not observed at test material concentrations below 15.6 μg/ml. The preliminary cytotoxicity test measures the ability of 3T3 cells to form colonies in the presence of various concentrations of the test material and is used to select concentrations for use in the transformation assay.

 

The relative survivals observed for test material concentrations from 1.0 mg/ml to 0.061 μg/ml ranged between an apparent minimum of 54.3% at 125.0 μg/ml to a maximum of 89.9% at 1.95 μg/ml. Thus, survivals below 50% were not obtained and graphic analysis of the cytotoxicity data did not reveal clear evidence of the occurrence of a dose-related response. The transformation assay is normally applied to test material concentrations that result in between 50% and 100% survival and is considered most sensitive near 70% survival, since the observed frequency of the appearance of transformed foci is not corrected for the number of cells surviving the treatments. In the present assay, experimental resolution of a dose-related cytotoxicity response was obviated by the extremely poor aqueous solubility of the test material and therefore the doses chosen for use in the transformation were selected so as to include concentrations exhibiting a wide range of apparent solubilities. Thus, the concentration range of 1.0 mg/ml to 10.0 μg/ml, corresponding to a survival range of approximately 67% to 80% (estimated graphically) and including both aqueous dispersions (e.g. 1.0 mg/ml) and apparent solutions (e.g.: 10.0 μg/ml), was chosen for the assay.

 

The negative (solvent) control measures the frequency of the appearance of spontaneously transformed foci. The historical negative control for the subclone of 3T3 cells used in this assay consists of 147 vessels containing a total of 21 transformed foci for an average of 0.14 focus/vessel. In the present assay, 3 foci were observed among 20 negative control dishes for an average of 0.15 focus/dish. This result was not significantly different from the historical value showing that the frequency of spontaneous transformation was within the expected range.

 

None of the applied concentrations of test material caused the induction of a significant number of transformed foci. In contrast, the MCA positive control treatment induced an average 1.25 foci/dish. This result was significant at >99% confidence level using the Kastenbaum-Bowman Tables, showing that the sensitivity of the assay was adequate to resolve the positive control transforming activity. Thus, concentrations of test material from 10.0 μg/ml to 1000.0 μg/ml were clearly not transforming to 3T3 cells.

 

The test material, BXA, Batch #34, did not induce the appearance of a significant number of transformed foci over the applied concentration range of 10.0 μg/ml to 1000.0 μg/ml. This concentration range encompassed the range of apparent solubilities of the test material and corresponded to approximately 80% to 67% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in the BALB/3T3 In Vitro Transformation Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro Ames Assay

Dose levels for the preliminary toxicity screen were 5000, 1666, 500, 166 and 50 μg/plate. Strain TA 1538 showed extreme inhibition at 5000 and 1666 μg/plate, moderate inhibition at 500 μg/plate and slight inhibition at 166 and 50 μg/plate. Strain TA 100 showed moderate inhibition at 5000 μg/plate and slight inhibition at 1666 μg/plate. Dose levels in the plate assay were 1000, 333, 100, 33 and 10 μg/plate. There were 0.06 ml of S-9 supernatant (37 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

There was no apparent change in the physical state of the test article and control articles during administration. The results for test article BXA, were negative in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium both with and without metabolic activation preparation at 1000, 333, 100, 33 and 10 μg/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical mean data.

In Vitro Transformation Assay

The relative survivals observed for test material concentrations from 1.0 mg/ml to 0.061 μg/ml ranged between an apparent minimum of 54.3% at 125.0 μg/ml to a maximum of 89.9% at 1.95 μg/ml. Thus, survivals below 50% were not obtained and graphic analysis of the cytotoxicity data did not reveal clear evidence of the occurrence of a dose-related response. The transformation assay is normally applied to test material concentrations that result in between 50% and 100% survival and is considered most sensitive near 70% survival, since the observed frequency of the appearance of transformed foci is not corrected for the number of cells surviving the treatments. In the present assay, experimental resolution of a dose-related cytotoxicity response was obviated by the extremely poor aqueous solubility of the test material and therefore the doses chosen for use in the transformation were selected so as to include concentrations exhibiting a wide range of apparent solubilities. Thus, the concentration range of 1.0 mg/ml to 10.0 μg/ml, corresponding to a survival range of approximately 67% to 80% (estimated graphically) and including both aqueous dispersions (e.g. 1.0 mg/ml) and apparent solutions (e.g.: 10.0 μg/ml), was chosen for the assay.

The negative (solvent) control measures the frequency of the appearance of spontaneously transformed foci. The historical negative control for the subclone of 3T3 cells used in this assay consists of 147 vessels containing a total of 21 transformed foci for an average of 0.14 focus/vessel. In the present assay, 3 foci were observed among 20 negative control dishes for an average of 0.15 focus/dish. This result was not significantly different from the historical value showing that the frequency of spontaneous transformation was within the expected range.

None of the applied concentrations of test material caused the induction of a significant number of transformed foci. In contrast, the MCA positive control treatment induced an average 1.25 foci/dish. This result was significant at >99% confidence level using the Kastenbaum-Bowman Tables, showing that the sensitivity of the assay was adequate to resolve the positive control transforming activity. Thus, concentrations of test material from 10.0 μg/ml to 1000.0 μg/ml were clearly not transforming to 3T3 cells.

The test material, BXA, Batch #34, did not induce the appearance of a significant number of transformed foci over the applied concentration range of 10.0 μg/ml to 1000.0 μg/ml. This concentration range encompassed the range of apparent solubilities of the test material and corresponded to approximately 80% to 67% survival in the cytotoxicity test. Therefore, the test material is considered to be inactive in the BALB/3T3 In Vitro Transformation Assay.

In Vitro Mouse Lymphoma Forward Mutation Assay

Three trials of the mutation assay were initiated and the third trial was performed only in the presence of S9 microsomal activation. The first trial is not represented because the positive control cloning efficiencies in the non-activation and activation assays were below 10%, therefore the validity of the assay was questionable.

Under non-activation conditions the treatments were initiated over a concentration range of 31.3 μg/ml to 1000 μg/ml. Since moderate inhibition of suspension growth was observed at the highest concentration if test material, four treatments from 700 μg/ml to 1000 μg/ml were chosen for mutant analysis on order to cover a wide range of toxic action. In this trial, the minimum criterion for mutagenesis was a mutant frequency exceeding 48.1 x 10^-6and none of the observed mutant frequencies approached this value. The percent relative growths ranged from 23.3% (1000 μg/ml) to 41.3% (800 μg/ml). Results from treatments that demonstrate high toxicity (percent relative growths 10% to 15%) are desirable for evaluation of the assay. However since 1000 μg/ml represents the highest concentration of test material routinely assayed in an organic solvent and the limit of solubility had been reached, further trials at higher concentrations of test material were not indicated. Therefore, under non-activation conditions, the test material was considered non-mutagenic in this assay in the presence of high concentrations of test material.

In the presence of S-9 microsomal activation, the test material was more toxic than without activation, indicating an interaction of the test material with the activation mix. The second trial in the presence of S-9 activation mix was initiated with treatments in the moderate to highly toxic range. Four treatments were analysed for mutant induction and three demonstrated weak mutagenesis. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 90.3 x 10^-6. At very high toxic treatments (percent relative growths, 5.9% to 16%) mutant frequencies were observed that were 1.8-fold to 3.4-fold above background (average of the negative control mutant frequencies). Even though a positive response was demonstrated according to our criteria for mutagenesis, this is a borderline response with weak mutagenesis occurring only at highly toxic treatments. Therefore a third trial in the presence of S9 microsomal activation was considered necessary for evaluation of the test material.

In the third trial with S9 microsomal activation, six treatments ranging from 100 μg/ml to 1000 μg/ml were analysed for mutant induction. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 27.3 x 10^-6. Four of the six assayed treatments induced mutant frequencies slightly above the minimum criterion and ranged from 3.6-fold to 4.2-fold above the background (average of the solvent and negative control mutant frequencies). The two least-toxic treatments (percent relative growths 47% and 56.2%) were not mutagenic and the four treatments (400 μg/ml to 1000 μg/ml) with percent relative growths ranging from 22.4% to 26.4% induced significant, although small, increases over the backgrounds. Therefore, the test material is considered weakly active in the presence of S9 microsomal activation in this assay.

The average cloning efficiencies for the solvent and untreated negative controls varied from 119.3% without activation to 88.9% and 88.9% with activation which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. (However, the relative cloning efficiencies of both positive controls were exceptionally low in the first trial which necessitated initiation of a second and third trial for evaluation of the test article.)

The test material, BXA, induced small increases in the mutant frequency of the TK locus in L5178Y mouse lymphoma cells only in the presence of rat liver S-9 microsomal activation. Without activation, concentrations up to 1000 μg/ml were moderately toxic and were not detectably mutagenic. With activation, only highly toxic treatments at concentrations at or above 800 μg/ml were weakly mutagenic in the second trial. In the third trial, concentrations of test material from 400 μg/ml to 1000 μg/ml were moderately toxic and induced weak but significant increases in the mutant frequency. The results from the first trial were not discussed because of technical problems with the assay. Therefore, the test material is considered to be weakly active in the Mouse Lymphoma Forward Mutation Assay with metabolic activation.

In Vitro Chromosome Aberration Test

An evaluation of the ability of formaldehyde, oligomeric reaction products with acetone and diphenylamine to induce chromosome aberrations in cultured peripheral human lymphocytes was conducted. This study investigated 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.

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.

Justification for classification or non-classification

Based on the results of the available in vitro studies, the substance is not classified as mutagenic.