Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium mono(2- ethylhexyl)phosphate

Molecular formula:

C8H22NO4P

IUPAC Name:

Ammonium mono(2- ethylhexyl)phosphate

Constituent 2

Reference substance name:

Ammonium bis(2-ethylhexyl) phosphate

EC Number:

225-615-3

EC Name:

Ammonium bis(2-ethylhexyl) phosphate

Cas Number:

4971-47-5

Molecular formula:

C16H38NO4P

IUPAC Name:

Ammonium bis(2- ethylhexyl)phosphate

Constituent 3

Reference substance name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Molecular formula:

C8H23NO7P2

IUPAC Name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Test material form:

liquid

Details on test material:

Storage Conditions: Room temperature.

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).