Tributyl citrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2000-05-12 - 2000-11-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum (adopted 1997-07-21)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A, Annex V, B14, dated December 29, 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA7J2-C-96-219, June 1996 (Public Draft)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate

EC Number:

205-617-0

EC Name:

Tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate

Cas Number:

144-15-0

Molecular formula:

C32H58O8

IUPAC Name:

tris(2-ethylhexyl) 2-acetoxypropane-1,2,3-tricarboxylate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): ATEHC
- Physical state: liquid
- Expiration date of the lot/batch: November 01, 2000
- Stability under test conditions: pure at RT: min. 6 month
- Storage condition of test material: room temperature, dry, closed containers
- Other: Chemical Name: Acetyl-tri-(2-ethylhexyl)-citrat
- Other: Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

TA 1537: his C 3076; rfa; uvrB: frame shift mutations
TA 98: his D 3052; rfa"; uvrB"; R-factor: frame shift mutations
TA 1535: his G 46; rfa"; uvrB": base-pair substitutions
TA 100: his G46; rfa"; uvrB"; R-factor: base-pair substitutions
TA102: his G 428 (pAQl); rfa"; R-factor: base-pair substitutions

Additional strain / cell type characteristics:

other: The strains are constructed to differentiate between base pair (TA 1535, TA 100, TA 102) and frameshift (TA 1537, TA 98) mutations.

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsome enzyme activation mixture

Test concentrations with justification for top dose:

100, 316.2, 1000, 2000, 3000 and 4000 µg/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:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: NaN3 (sodium azide), 4-NOPD (4-nitro-o-phenylene-diamine), MMS (methyl methane sulfonate) and 2-AA (2-aminoanthracene)

Details on test system and experimental conditions:

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, preculture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item solution was preincubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (range of spontaneous reversion frequencies, mean of three replicates):
TA 1535: 5-30
TA 1537: 4-32
TA98: 18-63
TA 100: 79-197
TA 102: 173-396
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement over the control plate.

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the test points occurs in at least one strain with or without metabolic activation.
According to the new OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
A biologically relevant increase is described as follows:
- if in strain TA 100 and TA 102 the number of reversions is at least twice as high
- if in strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the spontaneous reversion rate.

Statistics:

According to the new OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Slight toxic effects of the test item (indicated by a reduction of the spontaneous rate) were observed at the highest investigated dose without metabolic activation in the tester strains TA 100 (2nd experiment) and TA 1537 (1st experiment).
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with ATEHC at any concentration level, either in the presence or absence of metabolic activation (S9-mix) in experiment I and II. There was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in both experiments independently performed.
The reference mutagens showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I (Plate-incorporation Test)

Test Strain : TA 1535

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		15	15	4	9	9	2	1.8	0.8
		12			7
		19			10
DMSO		12	9	3	10	11	2	1.0	1.0
		6			13
		8			10
Test item	100.0 µg/plate	9	11	2	13	10	4	1.2	0.9
		12			5
		11			11
Test item	316.2 µg/plate	14	13	2	11	8	4	1.5	0.8
		11			10
		13			4
Test item	1000 µg/plate	9 P	9	4	7	9	2	1.0	0.9
		5 P			11
		13 P			10
Test item	2000 µg/plate	10 P	9	3	14	13	2	1.1	1.2
		12 P			11
		6 P			14
Test item	3000 µg/plate	8 P	9	1	6	9	4	1.0	0.9
		9 P			14
		10 P			8
Test item	4000 µg/plate	12 P	11	2	5	9	4	1.3	0.8
		12 P			10
		9 P			12
Na-azide	10 µg/plate	1161	1118	94	*	/	/	128.9	/
		1182
		1010
2 -AA	2.5 µg/plate	* /		/	195	227	28	/	20.6
					238
					248

M: Mean

+/-SD: Standard-deviation

B : Background lawn reduced

N : No background lawn

C: Contamination

P: Precipitation

+S9: With S9 mix

-S9: Without S9 mix

*: Not tested

Mutation factor = mean revertants (test item) / mean revertants (solvent control)

Test Strain : TA 1537

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		11	7	4	14	13	2	1.0	1.4
		4			14
		7			11
DMSO		8	8	2	7	9	3	1.0	1.0
		6			12
		9			9
Test item	100.0 µg/plate	13	9	4	15	10	4	1.1	1.1
		8			8
		5			7
Test item	316.2 µg/plate	10	8	3	9	11	3	1.0	1.2
		4			14
		9			11
Test item	1000 µg/plate	13 P	9	4	7	6	1	1.1	0.7
		5 P			5
		8 P			7
Test item	2000 µg/plate	12 P	11	2	8	7	1	1.4	0.8
		9 P			7
		12 P			6
Test item	3000 µg/plate	8 P	10	3	11	7	4	1.4	0.8
		14 P			7
		9 P			4
Test item	4000 µg/plate	6 P	5	1	11	15	4	0.6	1.6
		4			17
		4			18
4 -NOPD	40 µg/plate	368	334	30	*	/	/	43.6	/
		313
		321
2 -AA	2.5 µg/plate	*	/	/	308	328	18	/	35.2
					339
					338

Test Strain : TA 102

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		283	295	17	306	315	9	1.1	1.0
		315			323
		287			316
DMSO		273	263	10	316	330	15	1.0	1.0
		253			329
		264			346
Test item	100.0 µg/plate	263	256	7	287	301	27	1.0	0.9
		250			332
		254			285
Test item	316.2 µg/plate	284	281	6	312	322	17	1.1	1.0
		274			342
		285			312
Test item	1000 µg/plate	282 P	282	39	335	334	4	1.1	1.0
		321 P			337
		243 P			330
Test item	2000 µg/plate	238 P	241	31	348	328	39	0.9	1.0
		273 P			352
		212 P			283
Test item	3000 µ/plate	237 P	252	30	371 P	350	26	1.0	1.1
		233 P			358 P
		287 P			321 P
Test item	4000 µg/plate	235 P	246	11	294 P	333	36	0.9	1.0
		246 P			339 P
		256 P			365 P
MMS	1 µg/plate	2009	2031	149	*	/	/	7.7	/
		1895
		2190
2 -AA	10 µg/plate	*	/	/	507	531	36	/	1.6
					513
					573

Test Strain : TA 98

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		20	23	3	36	39	3	1.1	1.0
		25			41
		24			40
DMSO		18	21	3	34	38	4	1.0	1.0
		23			41
		21			40
Test item	100.0 µg/plate	20	24	6	43	41	3	1.2	1.1
		22			38
		31			41
Test item	316.2 µg/plate	21	26	4	44	39	8	1.2	1.0
		28			29
		28			43
Test item	1000 µg/plate	22	27	6	40	38	2	1.3	1.0
		34			36
		25			37
Test item	2000 µg/plate	26	24	2	35	40	7	1.2	1.0
		23			37
		24			48
Test item	3000 µ/plate	22	27	5	27	35	7	1.3	0.9
		28			41
		31			38
Test item	4000 µg/plate	30	28	4	37	35	5	1.4	0.9
		24			30
		31			39
4 -NOPD	10 µ/plate	1558	1695	125	*	/	/	82.0	/
		1804
		1724
2 -AA	2.5 µg/plate	*	/	/	827	918	222	/	24.0
					1171
					757

Test Strain : TA 100

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		117	107	8	93	104	10	1.2	1.0
		102			107
		103			112
DMSO		92	89	6	105	103	10	1.0	1.0
		83			92
		93			112
Test item	100.0 µg/plate	100	94	9	105	92	13	1.1	0.9
		84			92
		97			79
Test item	316.2 µg/plate	75	86	10	67	83	16	1.0	0.8
		92			99
		92			83
Test item	1000 µg/plate	112	104	17	84	88	10	1.2	0.9
		84			99
		115			80
Test item	2000 µg/plate	107	105	5	85	96	10	1.2	0.9
		108			105
		99			98
Test item	3000 µg/plate	100	97	12	91	96	5	1.1	0.9
		84			101
		107			95
Test item	4000 µg/plate	77	91	12	89	86	3	1.0	0.8
		97			85
		99			83
Na-azide	10 µg/plate	968	1012	48	*	/	/	11.3	/
		1063
		1006
2 -AA	2.5 µg/plate	*	/	/	2100	2030	98	/	19.7
					2071
					1918

Experiment II (Pre-incubation Test)

Test Strain : TA 1535

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		12	11	3	10	14	3	0.8	1.0
		13			15
		8			16
DMSO		12	14	3	9	14	6	1.0	1.0
		18			20
		13			13
Test item	100.0 µg/plate	13	12	2	10	11	4	0.8	0.8
		10			8
		12			15
Test item	316.2 µg/plate	9	11	3	14	13	4	0.7	1.0
		9			17
		14			9
Test item	1000 µg/plate	13	13	1	16	17	2	0.9	1.2
		14			16
		12			19
Test item	2000 µg/plate	15	14	2	20	16	4	1.0	1.1
		15			15
		11			13
Test item	3000 µ/plate	4	10	6	9	13	4	0.7	0.8
		16			13
		10			16
Test item	4000 µg/plate	12 P	10	3	9 P	11	2	0.7	0.8
		7 P			11 P
		12 P			13 P
Na-azide	10 µ/plate	1391	1307	77	*	/	/	91.2	/
		1240
		1289
2 -AA	2.5 µg/plate	*	/	/	193	167	31	/	12.0
					133
					176

M: Mean

+/-SD: Standard-deviation

B : Background lawn reduced

N : No background lawn

C: Contamination

P: Precipitation

+S9: With S9 mix

-S9: Without S9 mix

*: Not tested

Mutation factor = mean revertants (test item) / mean revertants (solvent control)

Test Strain : TA 1537

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		8	7	1	16	16	2	0.6	1.5
		6			14
		7			18
DMSO		8	11	4	13	11	3	1.0	1.0
		15			12
		10			8
Test item	100.0 µg/plate	9	10	1	13	10	3	0.9	0.9
		10			8
		11			10
Test item	316.2 µg/plate	14	12	3	6	10	5	1.1	0.9
		14			9
		8			15
Test item	1000 µg/plate	11	9	2	6	10	4	0.9	0.9
		10			10
		7			14
Test item	2000 µg/plate	13	14	5	13	10	3	1.3	0.9
		10			7
		19			10
Test item	3000 µg/plate	9	12	4	10	9	2	1.1	0.9
		11			7
		16			11
Test item	4000 µg/plate	11 P	11	3	14 P	10	4	1.0	0.9
		8 P			6 P
		14 P			9 P
4 -NOPD	40 µg/plate	179	181	5	*	/	/	16.5	/
		178
		187
2 -AA	2.5 µg/plate	*	/	/	445	417	26	/	37.9
					411
					394

Test Strain : TA 102

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		285	299	12	478	461	15	1.0	1.2
		306			453
		305			451
DMSO		304	307	32	377	389	40	1.0	1.0
		277			434
		340			355
Test item	100.0 µg/plate	316	307	11	270	291	25	1.0	0.8
		294			285
		310			319
Test item	316.2 µg/plate	343	322	18	333	344	13	1.1	0.9
		309			358
		315			340
Test item	1000 µg/plate	321	289	29	340	361	19	0.9	0.9
		264			376
		283			366
Test item	2000 µg/plate	301	293	8	353	339	29	1.0	0.9
		292			358
		286			306
Test item	3000 µg/plate	316	278	33	355	351	12	0.9	0.9
		261			337
		256			361
Test item	4000 µg/plate	283 P	289	9	325 P	351	23	0.9	0.9
		286 P			358 P
		299 P			370 P
MMS	1 µg/plate	1459	1378	78	*	/	/	4.5	/
		1371
		1303
2 -AA	10 µg/plate	*	/	/	937	960	20	/	2.5
					974
					969

Test Strain : TA 98

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		30	35	5	49	47	4	1.6	1.2
		39			42
		36			50
DMSO		19	21	2	42	41	5	1.0	1.0
		23			36
		22			45
Test item	100.0 µg/plate	12	16	11	51	50	1	0.8	1.2
		28			49
		8			50
Test item	316.2 µg/plate	19	19	4	50	49	4	0.9	1.2
		23			53
		16			45
Test item	1000 µg/plate	17	20	4	44	42	4	1.0	1.0
		24			45
		20			37
Test item	2000 µg/plate	17	22	5	34	41	10	1.0	1.0
		27			52
		21			37
Test item	3000 µ/plate	17	24	8	45	47	2	1.1	1.1
		24			47
		32			48
Test item	4000 µg/plate	19 P	19	2	40 P	44	8	0.9	1.1
		21 P			38 P
		17 P			53 P
4 -NOPD	10 µg/plate	2639	2743	92	*	/	/	128.6	/
		2812
		2778
2 -AA	2.5 µg/plate	*	/	/	2338	2555	207	/	62.3
					2576
					2751

Test Strain : TA 100

Dose Group		Revertants per plate						Mutation Factor
	dose/plate	-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
Water		108	113	15	116	125	11	1.1	1.0
		102			121
		130			137
DMSO		103	101	2	121	122	12	1.0	1.0
		101			111
		100			134
Test item	100.0 µg/plate	101	95	9	102	109	6	0.9	0.9
		99			113
		85			113
Test item	316.2 µg/plate	135	124	11	103	109	7	1.2	0.9
		124			116
		114			109
Test item	1000 µg/plate	132	112	17	117	117	7	1.1	1.0
		100			124
		105			110
Test item	2000 µg/plate	115	103	12	103	112	8	1.0	0.9
		92			118
		102			115
Test item	3000 µ/plate	84	89	14	107	125	16	0.9	1.0
		79			131
		105			136
Test item	4000 µg/plate	93	52	42	127 P	112	15	0.5	0.9
		53 P			97 P
		9 P			111 P
Na-azide	10 µg/plate	772	741	28	*	/	/	7.3	/
		734
		718
2 -AA	2.5 µg/plate	*	/	/	2340	2224	324	/	18.2
					2474
					1858

Applicant's summary and conclusion

Conclusions:

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

The test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used during the described mutagenicity test and under the experimental conditions reported.

Executive summary:

In order to investigate the potential of the test substance for its ability to induce gene mutations the plate incorporation test (experiment I) and the preincubation test (experiment II) were performed with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (2000, GLP study according to OECD 471 and EU method B.14). The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 100.0, 316.2, 1000, 2000, 3000 and 4000 µg/plate. Slight toxic effects of the test item (indicated by a reduction of the spontaneous rate) were observed at the highest investigated dose without metabolic activation in the tester strains TA 100 (2nd experiment) and TA 1537 (1st experiment). No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay. As positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies.

It can be assumed that the same applies to tributyl citrate (CAS 77-94-1) as it is a near analogue to the test substance acetyltri-2 -ethylhexylcitrat (ATEHC).