Phosphoric acid, mono- and di-C8-10-(even numbered)-alkyl esters

Genetic toxicity: in vitro

Administrative data

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:

The experimental phases of the study were performed between 13 July 2011 and 09 September 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

Phosphoric acid, mono- and di-C6-10-alkyl esters

EC Number:

269-616-7

EC Name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

Cas Number:

68307-94-8

Molecular formula:

UVCB

IUPAC Name:

Esterification Products of Phosphorus Pentoxide and Alcohols C6-C10 (even numbered)

Details on test material:

Sponsor's identification: Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered)
Description: Amber coloured slightly viscous liquid
Batch number: CI1E0447 solvent free
CAS No.: 68307-94-8
Identifier: TIS O2891
Date received: 13 June 2011
Expiry date: 29 May 2013
Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1:
Salmonella strains (with and without S9 mix): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli WP2uvrA (with and without S9 mix: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2:
0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulpoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Standard Deviation
Statistical analysis of data as determined by UKEMS

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: A test item precipitate (globular in appearance) was observed at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item exhibited toxicity at 5000 (µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (preincubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test item exhibited toxicity at 5000 µg/plate to TA100 and WP2uvrA in the presence of S9-mix only. No toxicity was noted to either strain in the absence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9‑mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	89	110	92	73	84	76	82	66	67	63	61
+	TA100	82	79	73	74	102	85	74	79	69	67	56*
-	WP2uvrA	25	28	31	25	34	20	31	31	24	27	16
+	WP2uvrA	34	19	26	20	29	29	19	32	28	17	8*

* : Partial absence of bacterial background lawn

 

Mutation Test

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4

and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4 (see attached background material for Tables and Figures).

A history profile of untreated/vehicle and positive controls (reference items) is presented in attached background material.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the 59-mix and the sensitivity of the bacterial strains.

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1 (Range-finding Test)

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
114		21		26		22		8
108	(110)	29	(25)	31	(30)	21	(21)	14	(11)
108		25		33		20		11

 

Experiment 2 (Main Test)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
104		21		25		34		14
73	(90)	29	(24)	26	(31)	37	(36)	15	(14)
93		23	41	33		36		14

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the in vitro mutagenic potential of the test substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS guidelines, in compliance with GLP. Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item,Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered), using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.In the range-finding test (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 µg/plate in both the absence and presence of S9-mix. In the main test (pre-incubation), the test item again induced a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially noted from 500 and 1500 µg/plate in the absence and presence of S9-mix respectively. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with and without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was observed at 5000 µg/plate.No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item either with or without metabolic activation or exposure method. Under the study conditions, the test substance was considered to be non-mutagenic (Bowles and Thompson, 2012).