Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mutagenicity: Negative (non-mutagenic); OECD 471; Dreher, D, 2017

In vitro gene mutation: Negative (non-mutagenic); OECD 473; Lacey F E, Durward R, 2011 (read-across to Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate)

In vitro mamalian cell Chromosome Aberration Test: Negative (non-mutagenic); OECD 476; Morris A, 2012 (read-across to Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 - 18 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate.
Experiment 2: 40, 80, 160, 250, 500, 1600 and 5000 µg/plate.

5000 µg/plate is the guideline recommended maximum concentration to be tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: test item readily soluble at testing levels in purified water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control treatments were performed using the same addition volumes per plate as the test article treatments.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Mutation Experiments

The test article was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45 ± 1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution

This was followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 ± 1°C protected from light for 2 to 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step in order to increase the sensitivity of the test system. Quantities of test article, vehicle control solution or positive control, bacteria and S-9 mix, were mixed together and incubated for 20 minutes at 37 ± 1°C, with shaking, before the addition of 2 mL molten agar at 45 ± 1 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

Toxicity Assessment

The background lawns of the plates were examined for thinning as sign of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration

Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.

2. Any observed response was reproducible under the same treatment conditions.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met.

Statistics:

Statistical analysis using Dunnett’s test was used to aid evaluation of a concentration response, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight lawn thinnning observed at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight and very thinned lawns present at 1600 and 5000 µg/plate, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight thinning of lawn at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at 5000 µg/plate - slight and very thinned lawns present at 500 and 1600 µg/plate with S9 mix, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at 5000 µg/plate - slight and very thinned lawns present at 500 and 1600 µg/plate with S9 mix, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: test item readily soluble at highest tested concentration
- Precipitation: not obsered
- Definition of acceptable cells for analysis: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).
- Other confounding effects: n/a

RANGE-FINDING/SCREENING STUDIES: no

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: visual inspection of bacterial lawn
- Other observations when applicable: n/a

Table 2:       Mean number of revertants per plate (experiment 1 – pre-incubation not conducted)

Conc. (µg/plate)	TA98			TA100			TA1535			TA1537			TA102
	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)	S9 -	S9 +	T (without / with S9 mix)
Neg. control	36.7	46.7	- / -	115.0	122.7	- / -	24.3	19.0	- / -	10.0	27.3	- / -	338.7	264.0	- / -
5	42.0	50.3	- / -	93.0	137.7	- / -	23.0	25.0	- / -	10.3	28.7	- / -	365.0	293.3	- / -
16	43.3	43.0	- / -	102.7	155.7	- / -	20.7	25.3	- / -	14.7	34.3	- / -	410.7^*	251.7	- / -
50	43.0	49.3	- / -	98.0	146.3	- / -	23.3	20.7	- / -	14.0	28.3	- / -	375.7	272.3	- / -
160	49.3	53.0	- / -	98.0	141.0	- / -	21.7	17.0	- / -	12.3	25.3	- / -	507.3^**	236.7	- / -
500	39.0	40.7	- / -	171.7^*	157.3	- / -	14.7	24.0	- / -	11.7	27.3	- / -	386.3	275.7	- / -
1600	38.7	40.7	- / -	99.7	162.0	S / -	19.3	19.3	- / -	9.7	20.0	- / -	349.7	246.3	S / -
5000	38.3	36.3	- / -	92.7	111.3^*	S / S	17.7	14.3	S / -	9.0	13.7	S / -	257.7	217.0	S / S
Pos. control	1074.3	476.0	- / -	887.3	1915.7	- / -	642.0	343.3	- / -	321.0	224.3	- / -	992.7	2352.7	- / -

T= cytotoxic, no sign of revertant colonies

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

^*= p<0.05

^**= p<0.01

 

Table 3: Mean number of revertants per plate (experiment 2 – pre-incubation conducted)

Conc. (µg/plate)	TA98			TA100			TA1535			TA1537			TA102
	S9 -	S9 +	Toxicity (without / with S9 mix)	S9 -	S9 +	Toxicity (without / with S9 mix)	S9 -	S9 +	Toxicity (without / with S9 mix)	S9 -	S9 +	Toxicity (without / with S9 mix)	S9 -	S9 +	Toxicity (without / with S9 mix)
Neg. control	13.7	27.0	- / -	87.7	145.0	- / -	12.3	12.7	- / -	7.3	11.3	- / -	286.7	252.7	- / -
40	15.0	27.7	- / -	124.0^*	148.0	- / -	14.0	16.0	- / -	6.3	8.0	- / -	315.0	251.3	- / -
80	17.3	27.0	- / -	111.3	147.7	- / -	10.7	22.0^*	- / -	7.0	10.3	- / -	278.0	265.0	- / -
160	15.7	21.0	- / -	101.3	159.0	- / -	10.3	16.0	- / -	5.3	5.0	- / -	286.7	201.0	- / -
250	14.0	19.3	- / -	104.7	155.0	- / -	10.7	18.0	- / -	9.0	9.7	- / -	272.7	199.3	- / -
500	18.3	20.3	- / -	87.0	129.3	- / -	13.0	14.7	- / -	5.3	8.3	- / S	267.0	166.0	- / S
1600	13.0	27.7	- / -	78.3	110.7	S / S	15.7	14.0	- / -	7.3	6.7	- / V	167.3	75.7	S / V
5000	13.7	17.7	S / S	51.0	56.3	S / V	7.0	13.0	S /S	4.3	-	S / T	73.3	-	V / T
Pos. control	655.0		- / -	759.7	1706.7	- / -	595.3	271.0	- / -	137.7	350.3	- / -	723.7	1693.0	- / -

- = no sign of cytotoxicity

S= slight reduction in bacterial lawn

V= very thin background bacterial lawn

T= cytotoxic, no revertant colonies

^*= p<0.05

^**= p<0.01

Conclusions:

It was concluded that under the condition of this study, Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

Executive summary:

OECD 471 (2017) - In a reverse gene mutation assay in bacteria using strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102), Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2- ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts was assayed for mutagenic potential both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two independent experiments with the inclusion of pre-incubation step.  The test article treatments in this study were performed using formulations prepared in purified water, and all concentrations are expressed in terms of compound constituents using a correction factor of 1.3229.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test article at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 1600 μg/plate and/or 5000 μg/plate in strains TA100 and TA102, in the absence and presence of S-9, and at 5000 μg/plate in the absence of S-9 in strains TA1535 and TA1537.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 40-5000 μg/plate, in order to examine more closely those concentrations of the test article approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. All treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step.

Following these treatments, evidence of toxicity was observed at 1600 and/or 5000 μg/plate in all strains in the absence of S-9, at 1600 and/or 5000 μg/plate in strains TA98, TA100 and TA1535, in the presence of S-9 and at 500 μg/plate and above in strains TA1537 and TA102, in the presence of S-9.

No precipitation was observed on the test plates following incubation.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

In Experiment 1, there was a statistically significant increase in revertant numbers (p≤0.01 in Dunnett’s test) in strain TA102, in the absence of S-9 at a concentration of 160 μg/plate. The increase was also ≥1.5-fold that of the concurrent vehicle control. However, the response was not reproduced in Experiment 2 and did not appear to be concentration related, therefore this was considered not to be a biologically relevant effect.

No statistically significant increases in revertant numbers in strains TA98, TA100, TA1535 or TA1537 were observed following treatments.

It was concluded that under the condition of this study, the test article did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The Experimental phases of the study were performed between 19 April 2011 and 18 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same 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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions. Cell Culture:Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal, bovine serum, at 37°C with 50/0 C02 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test):The dose range of test item used was 19.53 to 5000 µg/mlExperiment 1: 4(20)-hour without S9 (µg/ml)0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*4(20)-hour with S9 (µg/ml)0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*Experiment 224-hour without S9 (µg/ml)0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*4(20)-hour with S9 (µg/ml)0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, CP 5**Dose levels selected for metaphase analysisMMC: Mitomycin CCP: Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test item was readily miscible in it.Preparation of Test Item and Control Items:The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The test item was considered to be a mixture, therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level. There was a modest decrease in pH of less than 1 pH unit when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated. The decrease in pH was within acceptable limits.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9 at 5 µg/ml.Migrated to IUCLID6: (CP)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9 at 0.4 and 0.2 µg/ml for 4(20)-hour and 24-hour culture respectively.Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 48 hrs- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.- Selection time (if incubation with a selection agent): Not applicable.- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 µg/ml)STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.NUMBER OF REPLICATIONS: Duplicate culturesNUMBER OF CELLS EVALUATED: 100/cultureDETERMINATION OF CYTOTOXICITY - Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.OTHER EXAMINATIONS: - Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(Refer to information on results and attached tables.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTSPRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST)The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). It can be seen that the test item showed clear evidence of dose-related toxicity in all three exposure groups. A greasy oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 2500 µg/ml, in all three of the exposure groups.Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 156.25 µg/ml, in all three of the exposure groups. Dose selection for Experiments 1 and 2 was based on test item induced toxicity.CHROMOSOME ABERRATION TEST - EXPERIMENT 1:The dose levels of the controls and the test item are given in the table below:GroupFinal concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (µg/ml)4(20)-hour without S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*4(20)-hour with S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 156.25 µg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 µg/ml in either exposure group.The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show that 59% growth inhibition was achieved at 156.25 µg/ml in the absence of S9 and 40% growth inhibition was achieved at 156.25 µg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either exposure group.The maximum dose level selected for metaphase analysis was based on the maximum surviving dose level and toxicity, and was 156.25 µg/ml in both of the exposure groups.The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).The polyploid cell frequency data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.CHROMOSOME ABERRATION TEST - EXPERIMENT 2:The dose levels of the controls and the test item are given in the table below:GroupFinal concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (µg/ml)24-hour without S90*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*4(20)-hour with S90*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, CP 5*The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the test item dose level of 156.25 µg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 µg/ml in either of the exposure groups.The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show that 75% growth inhibition was achieved at 156.25 µg/ml in the absence of S9 and 50% growth inhibition was achieved at 156.25 µg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either of the exposure groups.The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on toxicity at 156.25 µg/ml, for both exposure groups. In the absence of S9 the toxicity observed at 156.25 µg/ml was high at 75%, however the dose level was selected for metaphase analysis because the next dose level down demonstrated no toxicity.The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

See attached background material for:

Appendix 1: Report of Results of Chromosomal Aberration Test in Cultured Mammalian Cells

Appendix 2: Results of Chromosome Aberration Test

Appendix 3: Dose Response Curves

Conclusions:

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Test (1990), US EPA OPPTS 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration ofTest Item(µg/ml)
4(20)-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (2%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
24-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (1%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5

 

 

Results.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded the optimum 50% mitotic inhibition.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro

Executive summary:

In a one-to-one read-across approach, the substance Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (source substance) is considered appropriate for direct read-across (one-to-one) to Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts (target substance) for the endpoint in vitro genetic toxicity (in vitro gene mutation in mammalian cells). In conclusion, The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

In a one-to-one read-across approach, the substance Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (source substance) is considered appropriate for direct read-across (one-to-one) to Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts (target substance) for the endpoint in vitro genetic toxicity (in vitro gene mutation in mammalian cells). In conclusion, the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 22 June 2011 and 05 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Reason / purpose for cross-reference:

reference to same study

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)

Version / remarks:

Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for Mycoplasma contamination:yes- Periodically checked for karyotype stability: no- Periodically "cleansed" against high spontaneous background: yesCell Line :The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.Cell Culture:The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Hams F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.Cell Cleansing:Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

The test item was considered to be a mixture, therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level. The dose range of test item used in the preliminary cytotoxicity test was 19.53 to 5000 µg/ml. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-Exposure GroupFinal concentration of test item (µg/ml)4-hour without S92.5, 5, 10, 20, 30, 404-hour with S9 (2%)5, 10, 20, 40, 60, 8024-hour without S91.25, 2.5, 5, 10, 20, 30, 404-hour with S9 (1%)5, 10, 20, 30, 40, 50, 60

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)- Justification for choice of solvent/vehicle: The test material formed a solution with the solvent suitable for dosing at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

Dimethyl benzanthracene (DMBA) at 0.5 and1.0 µg/ml was used as the positive controls in cultures with S9. All positive controls were dissolved in dimethyl sulphoxide and dosed at 1%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethyl methane sulphonate (EMS) was used at 500 and 750 µg/ml as the positive control in the 4-hour cultures without S9 and at 200 and 300 µg/ml for the 24-hour cultures without S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.DURATION- Exposure duration: 4 hours (with and without S9), 24 hours (without S9)- Expression time (cells in growth medium): 7 daysSELECTION AGENT (mutation assays): 6-thioguanine (6-TG)NUMBER OF REPLICATIONS: Duplicate culturesDETERMINATION OF CYTOTOXICITY- Method: Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.ASSAY ACCEPTANCE CRITERIAAn assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test item in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Evaluation criteria:

Please see 'Assay Acceptance criteria', in details on test system and conditions section.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST:A dose range of 19.53 to 5000 µg/ml was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in the attached Table 1 (attached background material). It can be seen that there was very marked toxicity at and above 19.53 µg/ml in both the 4 hour exposure group in the absence of S9 and the 24 hour exposure group with no surviving cells above this dose level. In the 4 hour exposure group in the presence of S9 the toxicity was slightly less severe with approximately 70% survival at 39.06 µg/ml when compared to the negative control and a few cells surviving up to 78.13 µg/ml. A precipitate of the test item was noted in all three exposure groups at the end of exposure at and above 1250 µg/ml.MUTAGENICITY TEST - EXPERIMENT 1:The dose levels of the controls and the test item are given in the table below:Group Final concentration of test item (µg/ml)4-hour without S90*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 500* and 750*4-hour with S9 (2%)0*, 5*, 10*, 20*, 40*, 60*, 80, DMBA 0.5* and 1** Dose levels plated for mutant frequency.No precipitate of the test item was seen at the end of exposure in either exposure group. The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3 (attached background material). The Day 0 and Day 7 cloning efficiencies for the vehicle control groups in both the with and without S9 exposure groups did not achieve 70% cloning efficiency but all achieved at least 50% cloning efficiency and were therefore considered to be acceptable. The test item demonstrated a steep toxicity curve in both exposure groups consistent with that seen in the preliminary toxicity test. In the absence of S9 the test item achieved 58% toxicity at 30 µg/ml at Day 0 when compared to the vehicle control group. The dose level of 40 µg/ml had no surviving cells and was too toxic for plating. In the 4 hour exposure group in the presence of S9 the toxicity was too great for plating at 80 µg/ml at Day 0 with no surviving cells at this dose level. The test item achieved a 21% increase in toxicity when compared to the vehicle control group at 40 µg/ml. The dose level of 60 µg/ml was plated although it exceeded 90% toxicity as it provided an intermediate dose in a steep toxicity curve. The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3 (attached background material). In the 4 hour exposure group in the absence of S9 there was an increase in the mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 at 2.5 µg/ml, however since this increase was not dose related and the mutant frequency for this exposure group was generally high this was considered to be a random fluctuation. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 in the presence of S9 with the exception of the 60 µg/ml dose level which can be excluded on the basis of excessive toxicity.MUTAGENICITY TEST - EXPERIMENT 2:The dose levels of the controls and the test item are given in the table below:Group Final concentration of test item (µg/ml)24-hour without S90*, 1.25*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 200* and 300*4-hour with S9 (1%)0* ,5*, 10*, 20*, 30*, 40*, 50*, 60 DMBA 0.5* and 1** Dose levels plated for mutant frequency.No precipitate of the test item was seen at the end of exposure in either exposure group The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation are presented in the attached Tables 4 and 5 attached background material). The Day 0 cloning efficiencies for the vehicle control groups in both exposure groups did not achieve 70% but were considered acceptable as they did achieve the 50% minimum. It can be seen that the toxicity is similar to that seen in Experiment 1. The maximum dose plated for mutation frequency in the 4 hour exposure group in the presence of S9 was 50 µg/ml with an increase in toxicity of 66% when compared to the vehicle control group. The dose level of 60 µg/ml was not plated due to toxicity greater than 90%. The 24 hour exposure group demonstrated an increase in toxicity of 51% when compared to the vehicle control at 30 µg/ml. The dose level of 40 µg/ml was too toxic for plating with no surviving cells. The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in the attached Table 4 and 5 (attached background material). In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6.It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. In the positive control groups dosed with DMBA at 1 µg/ml and EMS at 300 µg/ml there were insufficient cells for plating 5 mutant flasks due to the toxicity of these positive control items and the mutant frequency was therefore calculated from the counts of the available flasks and adjusted according to the number of flasks. The positive response was clearly demonstrated and therefore the reduction in mutant flasks was considered to be acceptable.

See attached background material for:

Table 1: Preliminary Cytotoxicity Results

Table 2: Experiment 1 - 4 Hour Exposure Without Metabolic Activation (S9)

Table 3: Experiment 1 - 4 Hour Exposure With Metabolic Activation (S9)

Table 4: Experiment 2 - 24 Hour Exposure Without Metabolic Activation (S9)

Table 5: Experiment 2 - 4 Hour Exposure WIth Metabolic Activation (S9)

Conclusions:

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476' In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods

Chinese hamster ovary (CHO) cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration of test item (µg/ml)
4-hour without S9	2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%)	5, 10, 20, 40, 60, 80
24-hour without S9	1.25, 2.5, 5, 10, 20, 30, 40
4-hour with S9 (1%)	5, 10, 20, 30, 40, 50, 60

 

Results

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no mutagenic activity observed in available studies.

Additional information

Ames Test (OECD 471)

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were exposed to Reaction mass of Ammonium mono (2-ethylhexyl)phosphate, Ammonium bis (2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts, formulated in purified water at concentrations of 5 – 5000 µg/plate in the presence and absence of mammalian metabolic activation S9-mix. The test item was tested 5000 µg/plate, the maximum recommended concentration according to OECD 471.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence (or a concentration related positive response) of induced mutant colonies over background in the test item treated plates.  It was concluded that under the condition of this study, Reaction mass of Ammonium mono(2-ethylhexyl)phosphate, Ammonium bis(2-ethylhexyl)phosphate and 2-ethylhexyl diphosphate ammonium salts did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

In Vitro Gene Mutation Assay - Comparable to OECD 476.

In a mammalian cell gene mutation assay (HPRT ) locus Chinese hamster ovary (CHO) cells  cultured in vitro were exposed to the test item (Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate) at concentrations of  2.5, 5, 10, 20, 30, 40 µg/mL ( 4-hr -S9),  5, 10, 20, 40, 60, 80 µg/mL ( 4-hr plus 2% S9)),  5, 10, 20, 30, 40, 50, 60 µg/mL ( 4-hr plus 1% S9) and  1.25, 2.5, 5, 10, 20, 30, 40 µg/mL ( 24-hr - S9)  (Morris A, 2012).  The the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test. A full justification for the read-across approach is presented in IUCLID Section 13.2.

In Vitro Mammalian Gene mutation - Chromosome Aberration Test (OECD 473)

In a Chromosome Aberration Test  human lymphocyte cells cultured in vitro were exposed to the test item (nReaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate) at concentrations of  9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/mL ( 4(20)hr -S9), 9.77, 19.53, 39.06, 78.13, 156.25, 312.5µg/mL ( 4 (20)hr plus 2% S9),  9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/mL ( 4(20)hr plus 1% S9) and 9.77, 19.53, 39.06, 78.13, 156.25, 312.5 µg/mL ( 24-hr - S9)  (Lacey F E, Durward R, 2011).  The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Justification for classification or non-classification

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