Phosphoric acid, mixed esters with Bu alc. and ethylene glycol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Please refer to additional information

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

Study period:

2012 -02-08 till 2012-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimurium
Tryptophan for E. Coli

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Better solubility than other

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
PRECIPITATION:
No precipitation of the test item occurred up to the highest investigated dose.
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

   Summary of Results Pre-Experiment and Experiment I

Study Name: 1465902	Study Code: Harlan CCR 1465902
Experiment: 1465902 VV Plate	Date Plated: 08/02/2012
Assay Conditions:	Date Counted: 14/02/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			21 ± 1	14 ± 2	28 ± 6	96 ± 4	55 ± 6
	Untreated			22 ± 3	14 ± 1	31 ± 2	91 ± 4	49 ± 8
	test item	3 µg		25 ± 5	14 ± 1	33 ± 6	97 ± 10	47 ± 13
		10 µg		21 ± 5	16 ± 4	31 ± 5	95 ± 3	50 ± 5
		33 µg		22 ± 4	14 ± 3	28 ± 2	93 ± 13	39 ± 5
		100 µg		28 ± 11	14 ± 7	29 ± 7	109 ± 10	53 ± 2
		333 µg		26 ± 9	15 ± 5	30 ± 4	83 ± 8	49 ± 7
		1000 µg		24 ± 9	15 ± 2	31 ± 5	100 ± 4	45 ± 15
		2500 µg		21 ± 12	14 ± 4	30 ± 4	110 ± 2	45 ± 10
		5000 µg		27 ± 12	16 ± 3	37 ± 3	120 ± 14	54 ± 14
	NaN3	10 µg		1887 ± 165			2477 ± 173
	4-NOPD	10 µg				395 ± 25
	4-NOPD	50 µg			75 ± 5
	MMS	3.0 µL						931 ± 72
With Activation	DMSO			33 ± 6	16 ± 4	37 ± 4	110 ± 12	42 ± 5
	Untreated			31 ± 2	19 ± 2	34 ± 6	114 ± 3	57 ± 3
	test item	3 µg		36 ± 3	23 ± 4	40 ± 4	102 ± 8	50 ± 9
		10 µg		34 ± 6	19 ± 3	34 ± 6	111 ± 5	53 ± 12
		33 µg		34 ± 8	17 ± 2	33 ± 6	108 ± 8	46 ± 5
		100 µg		33 ± 2	17 ± 6	33 ± 9	116 ± 10	42 ± 7
		333 µg		38 ± 0	24 ± 6	42 ± 10	105 ± 23	51 ± 2
		1000 µg		32 ± 1	19 ± 4	35 ± 6	124 ± 13	56 ± 7
		2500 µg		38 ± 4	17 ± 5	35 ± 8	104 ± 15	55 ± 4
		5000 µg		32 ± 3	17 ± 3	44 ± 7	112 ± 4	44 ± 7
	2-AA	2.5 µg		390 ± 45	313 ± 27	1778 ± 488	2031 ± 252
	2-AA	10.0 µg						267 ± 44

 

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

  Summary of Results Experiment II

Study Name: 1465902	Study Code: Harlan CCR 1465902
Experiment: 1465902 HV2 Pre	Date Plated: 22/02/2012
Assay Conditions:	Date Counted: 27/02/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			15 ± 2	15 ± 1	26 ± 6	93 ± 1	46 ± 5
	Untreated			14 ± 2	14 ± 3	28 ± 4	112 ± 12	40 ± 3
	test item	33 µg		18 ± 1	13 ± 1	23 ± 3	93 ± 9	41 ± 6
		100 µg		14 ± 4	16 ± 4	31 ± 3	86 ± 2	43 ± 1
		333 µg		17 ± 3	13 ± 2	25 ± 6	92 ± 6	39 ± 9
		1000 µg		15 ± 2	13 ± 3	29 ± 2	89 ± 17	44 ± 6
		2500 µg		18 ± 4	11 ± 5	28 ± 5	92 ± 2	45 ± 2
		5000 µg		15 ± 5	12 ± 5	31 ± 3	116 ± 19	43 ± 8
	NaN3	10 µg		1846 ± 76			2129 ± 116
	4-NOPD	10 µg				365 ± 10
	4-NOPD	50 µg			98 ± 8
	MMS	3.0 µL						330 ± 12
With Activation	DMSO			17 ± 4	22 ± 6	45 ± 4	128 ± 8	57 ± 10
	Untreated			22 ± 3	24 ± 5	46 ± 10	132 ± 9	64 ± 1
	test item	33 µg		20 ± 4	21 ± 2	50 ± 7	122 ± 10	51 ± 3
		100 µg		23 ± 3	18 ± 2	43 ± 7	111 ± 8	52 ± 6
		333 µg		21 ± 3	22 ± 6	43 ± 5	129 ± 5	56 ± 2
		1000 µg		18 ± 2	21 ± 8	39 ± 6	114 ± 6	54 ± 7
		2500 µg		21 ± 5	24 ± 4	41 ± 5	119 ± 7	55 ± 3
		5000 µg		16 ± 2	22 ± 7	50 ± 2	117 ± 10	65 ± 7
	2-AA	2.5 µg		298 ± 12	218 ± 22	1550 ± 46	1890 ± 37
	2-AA	10.0 µg						330 ± 23

 

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Conclusions:

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

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) usingSalmo­nella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                    33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

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

Study period:

2012-01-20 to 2012-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP conform guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 35.8, 62.7, 109.7, 191.9, 335.9, 587.8, 1028.7, 1800.2, 3150.3, 5513.0 µg/mL
Experiment II: 587.8, 1028.7, 1800.2, 3150.3, 5513.0 µg/mL

Without metabolic activation:
Experiment I: 35.8, 62.7, 109.7, 191.9, 335.9, 587.8, 1028.7, 1800.2, 3150.3, 5513.0 µg/mL
Experiment II: 35.8, 62.7, 109.7, 191.9, 335.9, 587.8, 1028.7, 1800.2, 3150.3, 5513.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5513.0 µg/mL was chosen with regard to the purity (94.5 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
No visible precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed.
In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In Experiment II after continuous treatment with 5513.0 µg/mL the mitotic index was reduced to 65.7 % of control.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, excluding gaps) were within the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770 or 660 µg/mL) or CPA (15.0 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study with the test item

Exp.		Preparation			Test item	Mitotic indices	Aberrant cells
		interval			concentration	in %	in %
					in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
I		22 hrs			Solvent control^1	100.0	3.0	2.5	0.0
					Positive control^2	71.5	14.5	14.0^S	3.5
					1800.2	92.7	0.0	0.0	0.0
					3150.3	106.2	1.0	1.0	0.0
					5513.0	81.1	2.0	2.0	0.0
			Exposure period 22 hrs without S9 mix
II		22 hrs			Solvent control^1	100.0	0.5	0.5	0.0
					Positive control^3#	39.1	42.0	40.0^S	13.0
					1800.2	91.0	0.5	0.5	0.0
					3150.3	86.7	1.0	0.5	0.0
					5513.0	65.7	0.5	0.5	0.0
				Exposure period 4 hrs with S9 mix
I		22 hrs			Solvent control^1	100.0	1.0	1.0	0.0
					Positive control^4	57.7	15.5	15.5^S	3.5
					1800.2	85.4	2.0	1.5	0.0
					3150.3	98.0	1.0	1.0	0.0
					5513.0	86.2	1.5	1.5	0.5
II		22 hrs			Solvent control^1	100.0	2.0	2.0	0.0
					Positive control^5	79.8	9.5	9.5^S	0.5
					1800.2	110.7	0.5	0.5	0.0
					3150.3	76.8	0.5	0.5	0.0
					5513.0	100.6	1.5	1.5	0.0

*  Including cells carrying exchanges

^#   Evaluation of 50 metaphases per culture

^S  Aberration frequency statistically significant higher than corresponding control values

¹   DMSO      1.0 % (v/v)

²     EMS    770.0 µg/mL

³     EMS    660.0 µg/mL

⁴   CPA      15.0 µg/mL

⁵   CPA        7.5 µg/mL

Conclusions:

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

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, th test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored.

The highest applied concentration in this study (5513.0 µg/mL of the test item) was chosen with regard to the purity (94.5 %) of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473.

In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

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

Study period:

11 April 2012 until 05 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 476; GLP conform

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 21.6; 43.2; 86.3; 172.5; 345.0; 517.5 µg/mL
with metabolic activation: 172.5; 345.0; 690.0; 1380.0; 2760.0; 5520.0 µg/mL
Experiment II:
without metabolic activation: 43.2; 86.3; 172.5; 345.0; 517.5; 690.0 µg/mL
with metabolic activation. 172.5; 345.0; 690.0; 1380.0; 2760.0; 5520.0 µg/mL
In experiment I and II the cultures at the lowest concentrations with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
experimental group: p-value:
experiment I, culture I without S9 mix 0.067
experiment I, culture II without S9 mix 0.647
experiment I, culture I with S9 mix 0.898
experiment I, culture II with S9 mix 0.200
experiment II, culture I without S9 mix 0.416
experiment II, culture II without S9 mix 0.108
experiment II, culture I with S9 mix 0.528
experiment II, culture II with S9 mix 0.263

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 517.5 µg/mL (without S9 mix)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
In the pre-experiment the pH at concentrations of 1380 to 5520 µg/mL without metabolic activation and 690 to 5520 µg/mL with metabolic activation was adjusted to neutral with 2 N sodium hydroxide. In experiment I adjustment of pH was performed as described at 345 µg/mL and above with and without metabolic activation. In experiment II the pH was adjusted at the concentrations of 1380 µg/mL and above with metabolic activation.

- Effects of osmolality: Not effected

- Evaporation from medium: Not examined

- Water solubility: Not indicated

- Precipitation:
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation occurred following 4 hours treatment with and without of metabolic activation and 24 hours treatment without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four ana-lysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at sub-cultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxic-ity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.
In the range finding pre-experiment test item concentrations between 43.2 and 5520 µg/mL (≈ 5 mg pure substance) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency below 50% of the sol-vent control were noted at 345.0 µg/mL and above without metabolic activation following 4 hours treatment and at 690 µg/mL without metabolic activation following 24 hours treat-ment.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation occurred following 4 hours treatment with and without of metabolic activation and 24 hours treatment without metabolic activation.
In the pre-experiment the pH at concentrations of 1380 to 5520 µg/mL without metabolic activation and 690 to 5520 µg/mL with metabolic activation was adjusted to neutral with 2 N sodium hydroxide. In experiment I adjustment of pH was performed as described at 345 µg/mL and above with and without metabolic activation. In experiment II the pH was adjusted at the concentrations of 1380 µg/mL and above with metabolic activation.
There was no relevant shift of osmolarity of the medium even at the maximum concentra-tion of the test item in the pre-experiment. The osmolarity was generally rather high com-pared to the physiological level of approximately 300 mOsm, even in the solvent control. This effect however, is based on the solvent DMSO used at a final concentration of 1% (v/v). The osmolarity is measured by freezing point depression and organic solvents as DMSO lead to a marked depression of the freezing point of aqueous solutions.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at high concentrations without metabolic activation to cover the cytotoxic range more closely.

COMPARISON WITH HISTORICAL CONTROL DATA:
The highest solvent control in experiment II, culture I with metabolic activation slightly exceeded the historical range (47.9 versus 40.3 colonies per 106 cells). The data are acceptable however, as the mean of both parallel cultures (18.6 and 47.9, equal to a mean of 33.3) is fully acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted in the first experiment at 517.5 µg/mL without metabolic activation. The cytotoxic gradient was rather steep with virtually no cytotoxicity at 345.0 µg/mL and strong cytotoxicity at 517.5 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary Table

			relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
			%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4 h treatment			culture I					culture II
Solvent control with DMSO		-	100.0	100.0	100.0	24.0	1.0	100.0	100.0	100.0	17.0	1.0
Positive control (EMS)	150.0	-	103.2	105.8	131.7	86.1	3.6	96.7	101.0	136.0	120.9	7.1
Test item	21.6	-	98.7	culture was not continued^#				96.3	culture was not continued^#
Test item	43.2	-	95.6	122.8	124.0	20.7	0.9	96.3	101.0	119.6	32.4	1.9
Test item	86.3	-	99.0	100.6	120.8	17.1	0.7	95.7	127.8	146.1	17.6	1.0
Test item	172.5	-	96.6	107.6	132.8	15.1	0.6	95.4	108.8	75.0	44.4	2.6
Test item	345.0	-	86.3	102.6	138.5	18.8	0.8	80.5	117.2	171.6	15.9	0.9
Test item	517.5	-	25.1	95.6	136.2	11.4	0.5	25.6	126.5	112.2	35.7	2.1
Solvent control with DMSO		+	100.0	100.0	100.0	34.2	1.0	100.0	100.0	100.0	8.7	1.0
Positive control (DMBA)	1.1	+	75.1	80.9	88.4	503.7	14.7	74.2	73.9	80.7	461.2	53.0
Test item	172.5	+	102.5	culture was not continued^#				99.2	culture was not continued^#
Test item	345.0	+	100.0	94.0	112.4	8.5	0.2	99.4	89.7	103.3	9.4	1.1
Test item	690.0	+	99.6	86.9	91.7	13.8	0.4	98.8	81.1	102.0	12.0	1.4
Test item	1380.0	+	100.6	74.5	92.6	11.4	0.3	99.1	85.6	94.0	24.4	2.8
Test item	2760.0	+	100.4	92.1	96.6	26.3	0.8	94.9	80.6	103.9	16.3	1.9
Test item	5520.0	+	87.6	83.9	106.2	16.1	0.5	90.0	84.2	102.3	20.3	2.3
Experiment II / 24 h treatment			culture I					culture II
Solvent control with DMSO		-	100.0	100.0	100.0	12.8	1.0	100.0	100.0	100.0	8.5	1.0
Positive control (EMS)	150.0	-	84.4	81.3	68.0	259.9	20.4	91.8	101.1	74.9	206.3	24.2
Test item	43.2	-	100.2	culture was not continued^#				100.0	culture was not continued^#
Test item	86.3	-	101.8	92.3	83.5	11.4	0.9	97.9	100.8	93.9	10.3	1.2
Test item	172.5	-	104.0	76.6	89.1	17.0	1.3	98.3	97.9	90.3	10.1	1.2
Test item	345.0	-	101.7	84.9	92.4	11.3	0.9	98.6	108.7	96.4	14.4	1.7
Test item	517.5	-	100.7	73.3	89.1	18.2	1.4	98.4	100.4	94.4	12.6	1.5
Test item	690.0	-	101.9	85.6	79.6	14.7	1.2	96.4	94.4	84.3	12.3	1.4
Experiment II / 4 h treatment
Solvent control with DMSO		+	100.0	100.0	100.0	47.9	1.0	100.0	100.0	100.0	18.6	1.0
Positive control (DMBA)	1.1	+	65.8	123.8	124.5	920.9	19.2	70.6	132.5	80.5	907.7	48.8
Test item	172.5	+	102.2	culture was not continued^#				104.4	culture was not continued^#
Test item	345.0	+	101.3	105.5	130.3	19.7	0.4	100.6	125.8	84.0	32.4	1.7
Test item	690.0	+	97.8	71.3	133.2	14.5	0.3	97.4	114.8	90.7	28.9	1.6
Test item	1380.0	+	98.4	94.2	117.3	50.1	1.0	99.3	107.3	94.5	22.1	1.2
Test item	2760.0	+	103.6	81.4	157.4	12.6	0.3	100.7	98.7	100.8	9.3	0.5
Test item	5520.0	+	100.1	91.7	115.1	21.5	0.4	97.1	128.0	92.6	17.0	0.9

^#   culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

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

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. Experiment I was prematurely terminated due to microbial contamination. Experiment I was repeated as experiment IA. The data of experiment IA are reported as experiment I with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	43.2	86.3	172.5	345.0	517.5
4 hours	+	34.5.0	690.0	1380.0	2760.0	5520.0
		Experiment II
24 hours	-	86.3	172.5	345.0	517.5	690.0
4 hours	+	345.0	690.0	1380.0	2760.0	5520.0

 

Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted in the first experiment at 517.5 µg/mL without metabolic activation. The cytotoxic gradient was rather steep with virtually no cytotoxicity at 345.0 µg/mL and strong cytotoxicity at 517.5 µg/mL.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of 3 times the mutation frequency of the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.5 up to 47.9 mutants per 10⁶cells; the range of the groups treated with the test item was from 8.5 up to 50.1 mutant colonies per 10⁶cells.

The highest solvent control in experiment II, culture I with metabolic activation slightly exceeded the historical range (47.9 versus 40.3 colonies per 10⁶cells). The data are acceptable however, as the mean of both parallel cultures (18.6 and 47.9, equal to a mean of 33.3) is fully acceptable.

 

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Additional information

Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol also yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to 517.5 µg/ml without metabolic activation and 5520 µg/ml in the presence of metabolic activation.

 

Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol was assessed for its potential to induce chromosome aberrations in human lymphocytes in vitro. The test item did not induce chromosome aberrations. Therefore, Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol is considered to be non-mutagenic in this in vitro chromosome aberration test when tested up to 5513 µg/mL.

Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and und GLP.

Short description of key information:
Mutagenic activity of Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A), in an in vitro gene mutation study in mammalian cells (Chinese Hamster V79 cells) and in one in vitro chromosome aberration study in human lymphocytes. Negative results were obtained in all tests with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In conclusion, Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in Chinese hamster V79 cells and the in vitro chromosome aberration in the presence and absence of metabolic activation up to the tested concentrations. 

Phosphoric acid, mixed esters with Bu.Alc. and Ethylene glycol does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 5520 µg/mL) or in the in vitro chromosome aberration study in concentrations up to 5513 µg/mL.