Tris(2-ethylhexyl) phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Negative; Herbold (1982)

Chromosome Aberration: Negative; CIPC Japan (1995)

Mouse Lymphome Assay: Negative; Myhr (1991)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD or GLP study defined.

Principles of method if other than guideline:

bacterial reverse mutation assay (e.g. Ames test)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

ames assay: detection of base pair substitutions and frameshift mutations

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Negative control: 0 µg/plate
TOF: 12500 µg/plate
TOF: 2500 µg/plate
TOF: 500 µg/plate
TOF: 100 µg/plate
TOF: 20 µg/plate
Positive control Endoxan: 435 µg/plate (only TA 1535 and TA 100)
Positive control Trypaflavin: 200 µg/plate (only TA 1537 and TA 98)
Positive control 2-Aminoantrazen: 10 µg/plate

Vehicle / solvent:

DMSO for TOF, Trypaflavin and 2-Aminoantrazen.
Mineral free water for Endoxan.

Untreated negative controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

IUCLID 4 Type: Ames test

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Remarks on result:

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

Remarks:

Migrated from field 'Test system'.

In doses up to 12500 µg/plate no bacteria-toxic effects could be observed.

Executive summary:

The test substance was tested in the Ames test in doses up to 12500 µg/plate in 4 Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98). The positive controls Endoxan, Trypaflavin and 2 -Aminoanthrazen were tested positive, but the test substance Disflamoll TOF showed no mutagen effects.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline defined.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosome Aberration Test in CHL Cells

Species / strain / cell type:

other: Chinese hamster CHL/IU cells

Metabolic activation:

with and without

Metabolic activation system:

-S9 (continuous treatment): 0, 0.003, 0.006, 0,011 mg/ml; -S9 (shortterm treatment): 0, 1.1, 2.2, 4.4 mg/ml; +S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml

Test concentrations with justification for top dose:

-S9 (continuous treatment): 0, 0.003, 0.006, 0,011 mg/ml; -S9 (short-term treatment): 0, 1.1, 2.2, 4.4 mg/ml; +S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

-S9: Mitomycin C; +S9: Cyclophosphamide

Positive control substance:

not specified

Species / strain:

other: Chinese hamster CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: strain/cell type: Chinese hamster CHL/IU cells

Remarks:

Migrated from field 'Test system'.

Test results:

Lowest concentration producing cytogenetic effects in vitro:

without metabolic activation (continuous treatment): >0.01 mg/ml

without metabolic activation (short-term treatment): >4.4 mg/ml

with metabolic acivtion (short-term treatment): >4.4 mg/ml

Genotoxic effects:

	Clastogenicity			Polyploidy
	+	?	-	+	?	-
without metabolic activation	/	/	X	/	/	X
with metabolic activation	/	/	X	/	/	X

Executive summary:

In a chromosomal aberrations test in CHL/IU cells following concentrations were tested: -S9 (continuous treatment): 0, 0.003, 0.006, 0.011 mg/ml; -S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml; +S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml. The highest dose was selected based on cytotoxic effects. Neither structural nor numerical chromosomal aberrations were induced, in the absence or presence of an exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD Guideline or GLP defined.

Principles of method if other than guideline:

other: L5178Y mouse lymphoma cell mutation assay; similar to OECD TG 476

GLP compliance:

not specified

Type of assay:

other: L5178Y mouse lymphoma cell mutation assay

Target gene:

L5178Y mouse lymphoma cell mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

up to and exceeding the apparent solubility limit of 62.5 nl/ml

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Remarks:

see: Any other information on materials and methods

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Summary of Mutagenic Activities

Chemical	Test	LEC or [HTC] µg/ml	RTG (%) at LEC or [HTC]	Max. foldchange in MF	Evaluation
Tris(2-ethylhexyl)phosphate	-S9	[56]	[53 -70]	1.0	-
	+S)	[56]	[33 -97]	0.8 -1.3	-

Executive summary:

In an in vitro mammalian cell gene mutation test similar to OECD TG 476 the test substance did not induce any increases in mutation frequencies (MF) at the thymidine kinase (TK) locus in L5178Y cells, with or without rat liver S9 mix, for concentrations up to and exceeding the apparent solubility limit of 62.5 nl/ml in culture medium. The toxicity at 60 -80 nl/ml was highly variable and random, ranging from 27% to 121% relative total growth (RTG) for unknown reasons. However, the toxicity caused no increases in MF. Two different batches of S9 mix were used, but the chemical remained ineffective in causing detectable mutations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Bone Marrow Micronucleus Test: Negative; Shelby (1993, 1995)

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: other: Mikronuclei

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline or GLP defined.

Principles of method if other than guideline:

other; Micronucleus assay

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Duration of treatment / exposure:

5 d

Frequency of treatment:

injection once daily on 3 consecutive days

Remarks:

Doses / Concentrations:

Basis:
other: intraperitoneal injection of 0, 500, 1000, 2000 mg/kg test substance

No. of animals per sex per dose:

5-6

Control animals:

yes

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Tris(2 -Ethylhexyl)Phosphate

Overall result: Negative

The initial test gave a positive trend to 2,000 mg/kg with 1.1 MN-PCE/1,000 PCE in the control and 3.9 MN-PCE/1,000 PCE in the high dose group. Two repeat studies, one

to 2,000 mg/kg and one to 3,000 nng/kg were not positive by trend analysis and no dose groups were significantly elevated. Based on the lack of reproducibility of the effect seen in the initial test, this chemical is considered negative.

Initial Test:

Chemical^a)	Tissue^b)	Trend^c)p value	Dose^d) (mg/kg)	MN-PCE/1000^e) (No.animals)	Pair-wise^f)	Survival^g)	%PCE^h)
Tris(2 -ethylhexyl)phosphate (C)	BM	<0.001	0	1.10 +/-0.40(5)		5/5	49.9
Negative -/-			500	1.90 +/-0.10(5)	0.0719	5/5	46.4
			1000	3.20 +/- 0.82 (5)	<0.001	5/5	54.7
			2000	3.90 +/- 0.62(5)	<0.001	6/6	46.7

^a) Chemical name

^b) Tissue used (BM=bone marrow)

^c) Value of P for trend analysis alpha= .05

^d) chemical concentration administered i.p. daily to each animal

^e) Micronucleated PCEs per 1000 PCE scored (+/- Standard Error of the Means) (Number of the Animals Scored).

^f) The value of P fpr pair-wise comparisons between each treatment group and the concurrent solvent group alpha= .05.

^g) No. of animals surviving treatment over number of animals treated

^h) Percentage of erythrocytes that were polychromatic

Executive summary:

In an mouse bone marrow micronucleus assay the test substance was tested with following doses administered intraperitoneally to mice: 0, 500, 1000, 2000 mg/kg bw. The result was negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not relevant, no adverse effects observed.

Additional information

In vitro:

Several in vitro studies were performed:

The test substance was tested in the Ames test in doses up to 12500 µg/plate in 4 Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98). The positive controls Endoxan, Trypaflavin and 2 -Aminoanthrazen were tested positive, but the test substance Disflamoll TOF showed no mutagen effects (Herbold, Bayer AG, 1982).

The test substance was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

No toxicity was observed up to a concentration of 5000 µg/plate with or without metabolic activation (CICP, 1995).

In a chromosomal aberrations test in CHL/IU cells following concentrations were tested:

-S9 (continuous treatment): 0, 0.003, 0.006, 0.011 mg/ml;

-S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml;

+S9 (short term treatment): 0, 1.1, 2.2, 4.4 mg/ml.

The highest dose was selected based on cytotoxic effects. Neither structural nor numerical chromosomal aberrations were induced, in the absence or presence of an exogenous metabolic activation system (CICP, 1995).

In an in vitro mammalian cell gene mutation test similar to OECD TG 476 the test substance did not induce any increases in mutation frequencies (MF) at the thymidine kinase (TK) locus in L5178Y cells, with or without rat liver S9 mix, for concentrations up to and exceeding the apparent solubility limit of 62.5 nl/ml in culture medium. The toxicity at 60 -80 nl/ml was highly variable and random, ranging from 27% to 121% relative total growth (RTG) for unknown reasons. However, the toxicity caused no increases in MF. Two different batches of S9 mix were used, but the chemical remained ineffective in causing detectable mutations (Myhr, 1991).

In an in vitro sister chromatid exchange (SCE) assay the test substance was tested up to 5 mg/ml with and without metabolic activation. The SCE was negative for the test substance. In the SCE trial without activation severe cell cycle delay was observed at 16.7 µg/ml and no M2 cells were available for analyses. In all other trials there was reduction in cell confluence at the highest doses scored. The chemical precipitated at doses of 251 µg/ml and above (Benigni, 1989; Ivett, 1989; Tennant, 1987).

In vivo:

In a mouse bone marrow micronucleus assay the test substance was tested with following doses administered intraperitoneally to mice: 0, 500, 1000, 2000 mg/kg bw. The result was negative (Shelby, 1993, 1995).

Justification for classification or non-classification

The substance did not meet the classification criteria in accordance with Regulation (EC) No 1272/2008.