Bis(2-ethylhexyl) phosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2017 - 09 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayer, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phosphonate

EC Number:

222-904-6

EC Name:

Bis(2-ethylhexyl) phosphonate

Cas Number:

3658-48-8

Molecular formula:

C16H35O3P

IUPAC Name:

bis(2-ethylhexyl) phosphonate

Test material form:

liquid

Method

Target gene:

His operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced male rats

Test concentrations with justification for top dose:

Experiment I (the incorporation test)
0.01, 0.0316, 0.1, 0.316, 1.0, 2.5 and 5.0 μL/plate

Experiment II (the pre-incubation test)
0.00316, 0.01, 0.0316, 0.1, 0.316, 1.0, 2.5 and 5.0 μL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 40 µg/plate for TA 1537, without metabolic activation; 2-aminoanthracene, 2.5 µg/plate; 10 µg/plate for TA 102, with metabolic activation

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 – 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the diminution of the background lawn or a significant reduction in the number of revertants in solvent control.

Evaluation criteria:

Negative control data within historical range. Positive controls show a distinct enhancement of revertant rates over the control plate. At least five different concentrations of each tester strain are analyzable.
Positive response: a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as:
- if in tester strains TA 98 and TA 100 the number of reversions is at least and twice as high
- if in tester strains TA 102 the number of reversions is at least 1.5-fold higher
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Negative response: neither a dose related increase in the number of revertants nor a reproducible biologically relevant increase.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A pre-experiment for toxicity was performed at 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate with tester strains TA 98 and TA 100. The test substance were observed toxic effects at the concentration of 0.316 µL/plate or higher.

HISTORICAL CONTROL DATA
- Positive historical control data: Positive controls were met the criteria of validity
- Negative (solvent/vehicle) historical control data: Negative controls were within historical norms

Any other information on results incl. tables

Table 1: Results Experiment I

 

TesterStrain:TA98                                                                                    Experiment:1

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts			Mean	SD		Counts			Mean	SD		-S9		+S9
				27						24
A. dest.				29	26			3.1		17	25			8.0		1.1	1.1
				23						33
				23						21
DMSO				31	24			7.0		26	23			2.5		1.0	1.0
				17						23
				24						28
Test Item	0.0100 µL			22	24			1.5		27	38			17.6		1.0	1.6
				25						58
				23						23
Test Item	0.0316 µL			20	18			6.8		23	24			1.7		0.7	1.0
				10						26
				16						19
Test Item	0.100 µL			34	23			9.9		28	26			5.9		1.0	1.1
				18						30
				46						18
Test Item	0.316 µL			26	33			11.0		22	20			2.1		1.4	0.9
				28						21
				17						24
Test Item	1.0 µL			13	17			4.5		27	25			2.1		0.7	1.1
				22						23
				9						27
Test Item	2.5 µL			15	12			3.0		24	23			5.1		0.5	1.0
				12						17
				6						21
Test Item	5.0 µL			11	8			2.6		20	22			2.1		0.3	0.9
				7						24
				367						/
4-NOPD	10 µg			489	451			73.2		/	/			/		19.1	/
				498						/
				/						3261
2-AA	2.5 µg			/	/			/		2784	2986			246.9		/	128.0
				/						2912

TesterStrain:TA100                                                                                      Experiment:1

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE											MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean	SD		-S9			+S9
				103						95
A. dest.				95		94		9.5		87	93		5.3		1.1	1.0
				84						97
				75						93
DMSO				103		89		14.0		81	92		10.5		1.0	1.0
				90						102
				87						63
Test Item	0.0100 µL			69		82		11.0		80	71		8.5		0.9	0.8
				89						71
				74						92
Test Item	0.0316 µL			60		66		7.1		104	91		13.5		0.7	1.0
				65						77
				52						92
Test Item	0.100 µL			112		73		34.1		71	75		15.0		0.8	0.8
				54						63
				35						64
Test Item	0.316 µL			44		39		4.5		61	67		7.4		0.4	0.7
				39						75
				44						31
Test Item	1.0 µL			53		44		9.5		66	51		18.2		0.5	0.6
				34						57
				46 B						39
Test Item	2.5 µL			48 B		46		2.5		51	49		9.6		0.5	0.5
				43 B						58
				39 B						54 B
Test Item	5.0 µL			64 B		49		13.4		57 B	59		5.7		0.5	0.6
				43 B						65 B
				726						/
NaN3	10 µg			732		726		6.0		/	/		/		8.1	/
				720						/
				/						1900
2-AA	2.5 µg			/		/		/		2754	2233		457.0		/	24.3
				/						2045

TesterStrain:TA1535                                                                                    Experiment:1

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATIONFACTOR
			Withoutactivation(-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean		SD		-S9		+S9
				24						32
A. dest.				26		25		1.2		19		23		7.5		1.0		1.0
				24						19
				28						20
DMSO				24		25		2.3		32		23		7.6		1.0		1.0
				24						18
				21						25
Test Item	0.0100 µL			25		23		2.1		12		23		10.1		0.9		1.0
				24						32
				33						12
Test Item	0.0316 µL			21		27		6.0		25		23		10.1		1.1		1.0
				26						32
				32						17
Test Item	0.100 µL			20		26		6.0		22		19		2.9		1.0		0.8
				26						17
				16						21
Test Item	0.316 µL			25		22		4.9		17		19		2.1		0.9		0.8
				24						18
				23						17
Test Item	1.0 µL			18		23		5.0		21		19		2.1		0.9		0.8
				28						18
				27						16
Test Item	2.5 µL			25		27		2.0		19		18		1.7		1.1		0.8
				29						19
				10 B						2 B
Test Item	5.0 µL			8 B		7		3.1		8 B		5		3.1		0.3		0.2
				4 B						4 B
				928						/
NaN3	10 µg			933		942		20.1		/		/		/		37.2		/
				965						/
				/						199
2-AA	2.5 µg			/		/		/		202		201		2.1		/		8.6
				/						203

TesterStrain:TA1537                                                                                  Experiment:1

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean		SD		-S9			+S9
				15						11
A. dest.				17		15		1.5		15		13		2.1		1.1	0.9
				14						12
				18						12
DMSO				12		14		3.2		17		14		2.5		1.0	1.0
				13						14
				13						12
Test Item	0.0100 µL			15		14		1.0		15		14		2.1		1.0	1.0
				14						16
				13						14
Test Item	0.0316 µL			12		14		2.1		13		14		1.5		1.0	1.0
				16						16
				18						17
Test Item	0.100 µL			11		14		3.6		12		13		3.2		1.0	0.9
				13						11
				15						15
Test Item	0.316 µL			14		14		0.6		14		14		1.5		1.0	1.0
				14						12
				13						16
Test Item	1.0 µL			19		14		4.6		14		15		1.0		1.0	1.0
				10						15
				15						12
Test Item	2.5 µL			18		15		3.0		14		14		1.5		1.0	1.0
				12						15
				5 B						2 B
Test Item	5.0 µL			3 B		4		1.0		4 B		4		1.5		0.3	0.3
				4 B						5 B
				111						/
4-NOPD	40 µg			88		95		14.2		/		/		/		6.6	/
				85						/
				/						312
2-AA	2.5 µg			/		/		/		407		361		47.6		/	25.2
				/						364

TesterStrain:TA102                                                                                      Experiment:1

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATIONFACTOR
			Withoutactivation(-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean		SD		-S9		+S9
				246						336
A. dest.				321		283		37.5		321		337		17.0		1.2		1.1
				282						355
				230						285
DMSO				206		230		24.5		302		298		11.9		1.0		1.0
				255						308
				232						260
Test Item	0.0100 µL			297		261		33.0		253		252		8.0		1.1		0.8
				255						244
				362						309
Test Item	0.0316 µL			324		335		23.5		282		293		14.0		1.5		1.0
				319						289
				287						295
Test Item	0.100 µL			282		288		6.0		289		278		25.0		1.2		0.9
				294						249
				334						230
Test Item	0.316 µL			270		304		32.1		264		253		20.2		1.3		0.8
				307						266
				335						288
Test Item	1.0 µL			317		331		12.5		336		339		52.1		1.4		1.1
				341						392
				313						333
Test Item	2.5 µL			289		298		13.3		337		338		5.0		1.3		1.1
				291						343
				77 B						75 B
Test Item	5.0 µL			81 B		77		3.5		64 B		71		6.1		0.3		0.2
				74 B						74 B
				980						/
MMS	1 µL			902		918		55.7		/		/		/		4.0		/
				872						/
				/						700
2-AA	10 µg			/		/		/		729		799		147.1		/		2.7
				/						968

Table 2: Results Experiment II

 

TesterStrain:TA98                                                                                               Experiment:2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE											MUTATIONFACTOR
		Without activation(-S9)						With activation (+S9)
		Counts	Mean		SD			Counts	Mean		SD		-S9		+S9
20							29
A.dest.                                        16				20		4.0	28			30		2.1		0.9		1.4
24							32
22							22
DMSO                                         31				23		7.5	24			22		2.5		1.0		1.0
16							19
26							30
Test Item              0.00316µL           29				29		2.5	29			27		4.9		1.2		1.2
31							21
32							25
TestItem                0.0100µL           25				27		4.4	25			23		3.5		1.2		1.1
24							19
20							19
TestItem                0.0316µL           25				21		4.0	22			25		8.5		0.9		1.2
17							35
21							26
TestItem                  0.100µL           22				23		2.6	19			24		4.7		1.0		1.1
26							28
26							32
TestItem                  0.316µL           18				21		4.6	31			31		1.5		0.9		1.4
18							29
21							20
TestItem                       1.0µL           32				25		6.4	29			25		4.5		1.1		1.1
21							25
15 B							11 B
TestItem                       2.5µL           10B				13		2.5	9 B			11		1.5		0.6		0.5
13 B							12 B
12 B							5 B
TestItem                       5.0µL           10B				10		2.0	4 B			4		0.6		0.4		0.2
8 B							4 B
419							/
4-NOPD                 10µg         356				388		31.5	/			/		/		16.9		/
389							/
/							1169
2-AA                       2.5µg              /				/		/	943			1022		127.4		/		47.2
/							954

TesterStrain:TA100                                                                             Experiment:2

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean		SD		-S9			+S9
				96						89
A. dest.				63		87		21.0		92		87		5.7		1.2	1.2
				102						81
				66						68
DMSO				78		73		6.1		75		70		4.4		1.0	1.0
				74						67
				87						71
Test Item	0.00316 µL			87		89		2.9		70		72		3.2		1.2	1.0
				92						76
				87						72
Test Item	0.0100 µL			82		84		2.5		75		74		1.5		1.2	1.1
				84						74
				92						71
Test Item	0.0316 µL			109		92		16.5		71		73		2.9		1.3	1.0
				76						76
				67						68
Test Item	0.100 µL			89		82		13.0		93		83		13.2		1.1	1.2
				90						88
				75						91
Test Item	0.316 µL			93		83		9.1		64		76		13.7		1.1	1.1
				82						73
				46						27
Test Item	1.0 µL			36		36		9.5		60		49		19.1		0.5	0.7
				27						60
				10 B						20 B
Test Item	2.5 µL			8 B		9		1.0		18 B		17		4.2		0.1	0.2
				9 B						12 B
				2 B						5 B
Test Item	5.0 µL			2 B		2		0.6		1 B		2		2.3		0.0	0.0
				1 B						1 B
				635						/
NaN3	10 µg			770		678		80.0		/		/		/		9.3	/
				628						/
				/						979
2-AA	2.5 µg			/		/		/		740		936		178.9		/	13.4
				/						1090

TesterStrain:TA1535                                                                             Experiment:2

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts		Mean		SD		Counts		Mean		SD		-S9		+S9
				14						19
A. dest.				18		16		2.1		13		16		3.1		1.1		1.0
				17						15
				20						15
DMSO				11		15		4.6		21		15		5.5		1.0		1.0
				14						10
				14						12
Test Item	0.00316 µL			17		16		1.5		12		14		2.9		1.0		0.9
				16						17
				15						14
Test Item	0.0100 µL			14		15		0.6		17		16		1.5		1.0		1.0
				15						16
				21						14
Test Item	0.0316 µL			14		16		4.0		21		18		3.5		1.1		1.2
				14						18
				21						17
Test Item	0.100 µL			12		17		4.5		19		17		2.5		1.1		1.1
				17						14
				12						14
Test Item	0.316 µL			16		15		2.6		14		16		3.5		1.0		1.0
				17						20
				14						13
Test Item	1.0 µL			14		15		1.7		18		17		3.2		1.0		1.1
				17						19
				7 B						6 B
Test Item	2.5 µL			8 B		7		0.6		9 B		7		1.5		0.5		0.5
				7 B						7 B
				3 B						5 B
Test Item	5.0 µL			3 B		2		1.2		1 B		3		2.1		0.2		0.2
				1 B						2 B
				210						/
NaN3	10 µg			213		214		5.1		/		/		/		14.3		/
				220						/
				/						85
2-AA	2.5 µg			/		/		/		116		105		17.6		/		6.9
				/						115

TesterStrain:TA1537                                                                              Experiment:2

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATION FACTOR
			Without activation (-S9)						With activation (+S9)
			Counts			Mean	SD		Counts		Mean		SD		-S9		+S9
				22						15
A. dest.				20	19			3.6		17		17		1.5		0.8		1.0
				15						18
				29						15
DMSO				22	23			6.0		16		16		1.0		1.0		1.0
				17						17
				18						21
Test Item	0.00316 µL			17	18			1.0		18		19		1.7		0.8		1.2
				19						18
				20						17
Test Item	0.0100 µL			15	17			2.5		17		18		1.2		0.8		1.1
				17						19
				18						21
Test Item	0.0316 µL			19	19			1.0		17		18		2.6		0.8		1.1
				20						16
				17						18
Test Item	0.100 µL			16	17			1.0		17		18		0.6		0.8		1.1
				18						18
				20						15
Test Item	0.316 µL			21	19			2.1		18		16		2.1		0.9		1.0
				17						14
				16						15
Test Item	1.0 µL			18	18			1.5		16		15		0.6		0.8		1.0
				19						15
				20						15
Test Item	2.5 µL			20	19			1.7		16		16		1.0		0.8		1.0
				17						17
				2 B						5 B
Test Item	5.0 µL			3 B	2			0.6		4 B		4		0.6		0.1		0.3
				2 B						4 B
				178						/
4-NOPD	40 µg			191	185			6.6		/		/		/		8.2		/
				186						/
				/						157
2-AA	2.5 µg			/	/			/		129		153		21.8		/		9.5
				/						172

TesterStrain:TA102                                                                                  Experiment:2

Treatment		Dose/plate	REVERTANT COLONIES PER PLATE												MUTATIONFACTOR
			Withoutactivation(-S9)						With activation (+S9)
			Counts			Mean	SD		Counts		Mean		SD		-S9		+S9
				245						228
A. dest.				272	260			13.7		293		264		33.1		1.2		1.1
				263						271
				234						245
DMSO				175	216			35.9		242		250		11.9		1.0		1.0
				240						264
				198						274
Test Item	0.00316 µL			222	214			14.2		267		273		5.1		1.0		1.1
				223						277
				258						280
Test Item	0.0100 µL			237	245			11.6		309		310		30.5		1.1		1.2
				239						341
				218						321
Test Item	0.0316 µL			200	202			14.6		322		312		15.9		0.9		1.2
				189						294
				198						319
Test Item	0.100 µL			194	195			2.6		290		314		22.4		0.9		1.3
				193						334
				204						301
Test Item	0.316 µL			202	203			1.2		295		285		22.1		0.9		1.1
				204						260
				226						279
Test Item	1.0 µL			288	264			33.3		304		284		17.6		1.2		1.1
				278						270
				273						331
Test Item	2.5 µL			246	255			15.9		337		328		10.8		1.2		1.3
				245						316
				25 B						15 B
Test Item	5.0 µL			24 B	23			2.6		10 B		12		2.5		0.1		0.0
				20 B						12 B
				1656						/
MMS	1 µL			2276	2100			387.6		/		/		/		9.7		/
				2369						/
				/						817
2-AA	10 µg			/	/			/		914		865		48.5		/		3.5
				/						865

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative. The substance is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A mutagenicity study with two experiments (used incorporation and pre-incubation method respectively) was performed on the test substance using S. typhimurium in the presence and absence of metabolic activation. Concentrations of 0.0100, 0.0316, 0.100,0.316, 1.0, 2.5 and 5.0 µL/plate in incorporation experiment and the same as pre-incubation experiment but added the 0.00316 µL/plate, each was tested in triplicate. Cytotoxicity was observed from concentrations of 0.316µL/plate (without metabolic activation) and from 2.5 µL/plate (with metabolic activation). All criteria of validity were met. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed at any concentration level, neither in the presence nor absence of metabolic activation in two experiments. The substance is considered to be non-mutagenic in this bacterial reverse mutation assay.