Polyphosphoric acids, esters with triethanolamine, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four in-vitro studies, all reliability 1, have been conducted on the substance and the results are summarised below.

Ames Studies (x2):

A Bowles (2011) - key study

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item,Polyphosphoric acids, esters with triethanolamine, sodium salts, using both the Ames plate incorporation and pre-incubation methods at five 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 was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.

The test item,Polyphosphoric acids, esters with triethanolamine, sodium salts, was considered to be non-mutagenic under the conditions of this test.

Haddouk H (2002) - supporting study

The mutagenic potential of RHODAFAC BP10 was assessed in the Salmonella typhimurium microsomal assay according to the OECD Guideline Nb. 471 / EC method B13/B14, and in compliance with Good Laboratory Practice.

 

The histidine-requiring S. typhimuriummutants TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels. After 48 to 72 hours of incubation, the revertant colonies were scored.

A preliminary toxicity assay was performed to define the 5 dose levels to be used. The test substance was then tested in 2 independent experiments performed according to the direct plate incorporation method, except for the second test with S9 mix, which was performed according to the preincubation method (60 min,).

The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test article was dissolved in distilled water. Dose levels used in the main assay were 312.5, 625, 1250, 2500 and 5000 µg/plate in both assays, with and without S9-mix. All determinations were made in triplicate. Simultaneous negative (solvent) and positive controls were used in all experiments.

 

No noteworthy toxicity was observed in all strains in the absence and in the presence of S-9 mix. There was no increase in the mean number of revertant colonies, with or without S9-mix in any of the bacterial strains tested. Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

 

Under the conditions of this study, RHODAFAC BP10did not demonstrate anyin vitromutagenic activity in this bacterial test system.

Chromosome Aberration Test in Human Lymphocytes:

Bohnenberger S (2011) - key study

The test item Polyphosphoric acids, esters with triethanolamine, sodium salts, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin 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	¾ hrs	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

The highest applied concentration in this study was 5980.0 µg/mL of the test item

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

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in the absence of S9 mix in Experiment II, one statistically significant increase was observed after treatment with 5980.0 µg/mL (3.0 % aberrant cells, excluding gaps). This value was in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and is therefore considered as being biologically irrelevant.

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

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytesin vitro.

Therefore, Polyphosphoric acids, esters with triethanolamine, sodium salts is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

CHO HPRT Forward Mutation Assay:

Morris A (2011) - key study

Chinese hamster ovary (CHO) cells were treated with the test item at 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 oftest item(µg/ml)
4-hour without S9	156.25, 312.5, 625, 1250, 2500, 5000
4-hour with S9 (2%)	156.25, 312.5, 625, 1250, 2500, 5000
24-hour without S9	312.5, 625, 1250, 2500, 3750, 5000
4-hour with S9 (1%)	312.5, 625, 1250, 2500, 3750, 5000

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 in the exposure groups in the presence of metabolic activation, in either the first or second experiment. However, in the absence of S9 the test item demonstrated a statistically significant dose related increase in mutant frequency in the 24-hour exposure group of Experiment 2 and a modest increase in mutant frequency at the maximum dose tested of 5000 µg/ml in the 4-hour exposure group in the absence of S9 in Experiment 1. The response seen in the 4-hour exposure group of Experiment 1 was at the limit of acceptability for the response to be considered positive and at the upper limit for a vehicle control value but is statistically significant and is considered to be supporting evidence for the clear mutagenic response seen in the 24-hour exposure group of Experiment 2

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

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:

The experimental phase of this study was performed between 09 June 2011 and 15 August 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 no effect the quality of the relevant results.

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

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

Target gene:

Histidine for Salmonella.Tryptophan for E.Coli

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 / 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 main test: Experiment one: 50, 150, 500, 1500 and 5000 µg/plateExperiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Example:- Vehicle(s)/solvent(s) used: Sterile distilled water.- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: (BP): 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: (4NQO): 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: (9AA): 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: (ENNG): 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: (ENNG) 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: (ENNG): 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation in Experiment 2.DURATION- Preincubation period for bacterial strains: 10h- Exposure duration: Approximately 48 hours- Expression time (cells in growth medium): Not applicable- Selection time (if incubation with a selection agent): Not applicableNUMBER 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.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 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 thefollowing can be used to determine the overall result of the study:1. A dose-related increase in mutant frequency over the dose range tested 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.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.Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviationStatistical analysis of data as determined by UKEMS Dunnett's t test

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.RANGE-FINDING/SCREENING STUDIES: Preliminary Toxicity Test:The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). 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) were considered to be acceptable.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 S9-mix and the sensitivity of the bacterial strains.ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS

Preliminary ToxicityTest

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). 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	83	102	105	104	96	106	93	90	74	98	96
+	TA100	79	77	73	76	71	94	88	77	65	71	57
-	WP2uvrA	38	46	46	45	53	44	38	54	45	31	52
+	WP2uvrA	41	46	42	39	45	48	40	45	40	42	57

MutationTest

Results for the negative controls (spontaneous mutation rates) are presented in Table1 (see below) and were considered to be acceptable. These data are for concurrent untreated control plates perford on the saday as the 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 1 -5 and figures 1 -4).

A history profile of untreated/vehicle and positive controls (reference items) is presented in attached background material (Appendix 3: Historical profile 200 9/2010)

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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 S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
107		30		34		11		12
130	(110)	21	(22)	35	(30)	16	(15)	5	(8)
94		14		21		17		8

Number of revertants (mean number of colonies per plate)
						Frameshift type
						TA98
						30
						30	(27)†
						21

†         Experimental procedure performed at a later date (without S9-mix) due to contamination in the original test

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
104		18		23		44		8
95	(99)	25	(21)	20	(21)	19	(26)	8	(7)
97		20		20		15		5

 

 

 

Conclusions:

Interpretation of results (migrated information):negativeThe test item, Polyphosphoric acids, esters with triethanolamine, sodium salts, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Polyphosphoric acids, esters with triethanolamine, sodium salts, using both the Ames plate incorporation and pre-incubation methods at five 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 was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

Results.

The vehicle (sterile distilled water) 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.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.

Conclusion.

The test item, Polyphosphoric acids, esters with triethanolamine, sodium salts, was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental starting date (preliminary toxicity test treatment): 29 August 2001, Experimental completion date (last counting of revertants): 26 September 2001

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.

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

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver (S9 fraction)

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 10, 100, 500, 1000, 2500, 5000 µg/plateExperiment 1: 0, 312.5, 625, 1250, 2500, 5000 µg/plateExperiment 2: 0, 312.5, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

yes

Remarks:

(Sponteneous mutation rates)

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene and Mitomycin C

Remarks:

Positive controls used without S9 mix

Untreated negative controls:

yes

Remarks:

(Sponteneous mutation rates)

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

Positive control used with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix) and preincubation method (second experiment with S9 mix)DURATION- Preincubation period: 14 hours- Exposure duration: 48 to 72 hoursNUMBER OF REPLICATIONS: Triplicate platingDETERMINATION OF CYTOTOXICITYPreliminary toxicity test: observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.Mutagenicity experiments: In each experiment. for each strain and for each experimental point. the number of revertants per plate was scored.

Evaluation criteria:

Acceptance criteria:This study is considered valid if the following criteria are fully met:- the number of revertants in the vehicle controls is consistent with historical data- the number ofrevertants in the positive controls is higher than that of the vehicle controls and is consistent with historical data.Evaluation criteria:A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:The test substance was freely soluble in the vehicle (distilled water) at 50 mg/mL.Consequently, with a treatment volume of 100 µg/plate, the dose-levels were 10, 100, 500, 1000,2500 and 5000 µg/plate.No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.No noteworthy toxicity was induced in the three tester strains, both with and without S9 mix.MUTAGENICITY EXPERIMENTS:The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.Since the test substance was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all tester strains in both experiments with and without 59 mix.No noteworthy toxicity was induced in the tester strains in both experiments with and withoutS9 mix.The test substance did not induce any noteworthy increase in the number of revertants, in both experiments with and without S9 mix in any of the remaining tester strains.

The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence ofthe vehicle), are presented in tables 1 -5 (see attached background material).

Conclusions:

Interpretation of results (migrated information):negativeUnder the experimental conditions, the test substance RHODAFAC BPI0 does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

SUMMARY

The objective of this study was to evaluate the potential of the test substance RHODAFAC BPI0 to induce reverse mutation inSalmonella typhimurium.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of RHODAFAC BP10 to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction(S9fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

 

Five strains of bacteriaSalmonella typhimurium:TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance was dissolved in distilled water.

 

Results

Since the test substance was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all tester strains in both experiments with and without 59 mix.

 

No noteworthy toxicity was induced in the tester strains in both experiments with and without S9 mix.

 

The test substance did not induce any noteworthy increase in the number of revertants, in both experiments with and without S9 mix in any of the remaining tester strains.

 

Conclusion:

Under the experimental conditions, the test substance RHODAFAC BPI0 does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

 

Reference 3

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 July 2011 and 24 November 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 no effect the quality of therelevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according 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 Ham's F12 medium, supplemented with 5% foetal bovine 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 and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

A dose range of 19.53 to 5000 µg/ml was used in the preliminary cytotoxicity test.Mutagenicity Test - Experiment 1The dose levels of the controls and the test item are given below:Group Final concentration of test item (µg/ml)4-hour without S90*, 156.25*, 312.5*, 625*, 1250*, 2500*, 5000*, EMS 500* and 750*4-hour with S9 (2%)0*, 156.25*, 312.5*, 625*, 1250*, 2500*, 5000*, DMBA 0.5* and 1*Mutagenicity Test - Experiment 2The dose levels of the controls and the test item are given below:Group Final concentration of test item (µg/ml)24-hour without S90*, 312.5*, 625*, 1250*, 2500*, 3750*, 5000*, EMS 200* and 300*4-hour with S9 (1%)0*, 312.5*, 625*, 1250*, 2500*, 3750*, 5000*, DMBA 0.5* and 1**= Dose levels plated for mutant frequencyEMS= EthylmethanesulphonateDMBA= Dimethyl benzanthracene

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hams F12 cell culture media- Justification for choice of solvent/vehicle:The test material dissolved in Hams F12 to give a solution suitable for dosing at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: (EMS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate assay using tissue culture flasks.DURATION- Exposure duration: 4 hours (with and without S9), 24 hours (without S9 in Experiment 2)- Expression time (cells in growth medium):SELECTION 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.OTHER EXAMINATIONS:Mutant colonies were manually counted and recorded for each flask.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 article 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:

See assay acceptance criteria details (see details on test system and conditions).

Statistics:

The individual flask counts of the 4-hour exposure group in the absence of S9 in Experiement 1 and the 24 hour exposure group of Experiment 2 were adjusted for mutant frequency and were analysed using the SPSS (Version 11.5 SPSS Inc., 233 S. Wacker Drive, 11th Floor Chicago, Illinois 60606). The statistical analysis was performed using transformed data (√x+1).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

significant dose-related increase in mutant frequency per survivor in the absence of metabolic activation in the 24-hour exposure group of Experiment 2 and an increase in the 4-hour exposure group in the absence of S9 in Experiment 1 at the maximum dose.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to the attached tables

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: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 Table 1 (see attached background material). It can be seen that there was no marked reduction in the cloning efficiency (CE) at the maximum dose tested in either of the 4-hour exposure groups. The 24-hour exposure group demonstrated a marked reduction in cloning efficiency at 5000 µg/ml with 82% toxicity when compared to the vehicle control group. The maximum dose level selected for both Experiment 1 and 2 was the maximum recommended dose level of 5000 µg/ml.ADDITIONAL INFORMATION ON CYTOTOXICITY:Refer to any other information on results incl. tables section

 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 S9	0*, 156.25*, 312.5*, 625*, 1250*, 2500*, 5000*, EMS 500* and 750*
4-hour with S9 (2%)	0*, 156.25*, 312.5*, 625*, 1250*, 2500*, 5000*, DMBA 0.5* and 1*

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 Table 2 and Table 3 (see attached background material). There was no reduction in the Day 0 and Day 7 cloning efficiencies in the presence of S9. The 4-hour exposure group in the absence of S9 demonstrated an 80% reduction in cloning efficiency at 5000 µg/ml on Day 0 when compared to the vehicle control group and a modest reduction of 25% on Day 7. 

 

The Day 0 and Day 7 vehicle control cloning efficiencies for the exposure groups of Experiment 1 did not always achieve 70%, however since they all achieved at least 50% this was considered to be acceptable. 

The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3 (see attached background material). There was no indication of increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6at any dose level in the 4-hour exposure group in the presence of S9. In the 4-hour exposure group in the absence of S9 there was an increase in mutant frequency at 5000 µg/ml which although is at the lower limit of acceptability for a positive response and is only just outside the vehicle control range it is statistically significant.

The positive control in the presence of S9 (DMBA) was particularly toxic resulting in insufficient cells being available for plating 5 mutant flasks. 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 and not to affect the integrity of the study.

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 S9	0*, 312.5*, 625*, 1250*, 2500*, 3750*, 5000*, EMS 200* and 300*
4-hour with S9 (1%)	0*, 312.5*, 625*, 1250*, 2500*, 3750*, 5000*, DMBA 0.5* and 1*

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 Tables 4 and 5 (see attached background material). It can be seen that, as in Experiment 1, there was no reduction in the Day 0 and Day 7 cloning efficiency in the presence of S9 and no toxicity of the test item was observed when compared to the vehicle controls. In the 24-hour exposure group there was a reduction in cloning efficiency of approximately 50% at Day 0 when compared to the vehicle control group at 3750 and 5000 µg/ml.

The vehicle control Day 0 counts for both exposure groups did not achieve 70%, however since cloning efficiencies of at least 50% were achieved the data was considered to be acceptable. The positive control in the absence of S9 (EMS) was too toxic for plating 5 mutant flasks in some instances.  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.

The mutation frequency counts and mean mutation frequency per survivor values are presented in Tables 4 and 5 (see attached background material). There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 with the presence of S9. In the absence of S9 there was a dose related increase in mutant frequency which was statistically significant at 3750 and 5000 µg/ml. The ‘A’ culture at 5000 µg/ml demonstrated a much higher mutant frequency than the ‘B’ culture but both replicates exceeded the vehicle control value by more than 20 x 10^-6per survivor indicating a positive response. The individual flask counts of the 24‑hour exposure group were adjusted for mutant frequency andwere analysed using the SPSS (Version 11.5 SPSS Inc). The statistical analysis was performed using transformed data (√x+1). The response seen in the 24-hour exposure group in the absence of S9 in Experiment 2 is supported by the result of the 4-hour exposure group in Experiment 1 where a modest increase in mutant frequency was seen at 5000 µg/ml.

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 in both Experiment 1 and Experiment 2, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:

Interpretation of results (migrated information):positive without metabolic activation The test item was therefore considered to be mutagenic to CHO cells at the HPRT locus under the conditions of the test.The test item induced a significant dose-related increase in mutant frequency per survivor in the absence of metabolic activation in the 24-hour exposure group of Experiment 2 and demonstrated an increase in the 4-hour exposure group in the absence of S9 in Experiment 1 at the maximum dose tested. The test item was therefore considered to be 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 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	156.25, 312.5, 625, 1250, 2500, 5000
4-hour with S9 (2%)	156.25, 312.5, 625, 1250, 2500, 5000
24-hour without S9	312.5, 625, 1250, 2500, 3750, 5000
4-hour with S9 (1%)	312.5, 625, 1250, 2500, 3750, 5000

 

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 in the exposure groups in the presence of metabolic activation, in either the first or second experiment. However, in the absence of S9 the test item demonstrated a statistically significant dose related increase in mutant frequency in the 24-hour exposure group of Experiment 2 and a modest increase in mutant frequency at the maximum dose tested of 5000 µg/ml in the 4-hour exposure group in the absence of S9 in Experiment 1. The response seen in the 4-hour exposure group of Experiment 1 was at the limit of acceptability for the response to be considered positive and at the upper limit for a vehicle control value but is statistically significant and is considered to be supporting evidence for the clear mutagenic response seen in the 24-hour exposure group of Experiment 2

Conclusion. 

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

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-09 to 2011-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: -

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

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):

- 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:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:Experiment I: 38.8, 68.0, 119.0, 208.2, 364.3, 637.6, 1115.8, 1952.7*, 3417.1*, 5980.0* µg/mLExperiment II: 637.6, 1115.8, 1952.7*, 3417.1*, 5980.0* µg/mLWithout metabolic activation:Experiment I: 38.8, 68.0, 119.0, 208.2, 364.3, 637.6, 1115.8, 1952.7*, 3417.1*, 5980.0* µg/mLExperiment II: 119.0, 208.2, 364.3, 637.6, 1115.8, 1952.7*, 3417.1*, 5980.0* µg/mL*Evaluated experimental points.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used without metabolic activation Migrated to IUCLID6: (EMS)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used with metabolic activation. Migrated to IUCLID6: (CPA)

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 mediumDURATION- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)- Fixation time (start of exposure up to fixation or harvest of cells): 22 hoursSPINDLE INHIBITOR (cytogenetic assays): ColcemidSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: In each experimental concentration two parallel cultures were analysed. NUMBER OF CELLS EVALUATED: 100 per cultureDETERMINATION OF CYTOTOXICITY - Method: mitotic indexAnalysis of Metaphase CellsThe slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

Evaluation of Results:A test item is classified as non-mutagenic if:- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.- no significant increase of the number of structural chromosome aberrations is observed.A test item is classified as mutagenic if:- the number of induced structural chromosome aberrations is not in the range of the historical control data and- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:The assay can indicate an aneugenic potential of the test item if:- the number of induced numerical aberrations is not in the range of the historical control data.

Statistics:

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

Key result

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 Polyphosphoric acids, esters with triethanolamine, sodium salts, dissolved in deionised water, 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 concentration two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.The highest treatment concentration in this study, 5980.0 µg/mL was chosen with regard to the solubility properties and the water/glycol content (16.4 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.No precipitation of the test item in the culture medium was observed at any concentration. In Experiments I and II in the absence and presence of S9 mix in a range of 637.6 to 5980.0 µg/mL the pH was adjusted to physiological values using small amounts of 2 N NaOH. The osmolarity was slightly increased at the highest applied concentration. (Exp. I: solvent control: 282 mOsm, pH 7.3 versus 328 mOsm and pH 7.1 at 5980.0 µg/mL; Exp. II: solvent control: 282 mOsm, pH 7.2 versus 316 mOsm and pH 7.2 at 5980.0 µg/mL). 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 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 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. In Experiment II in the absence of S9 mix, one statistically significant increase was observed after treatment with 5980.0 µg/mL (3.0 % aberrant cells, excluding gaps). This value was in the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps) and is therefore considered as being biologically irrelevant. 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 (660 or 825 µg/mL) or CPA (2.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.In conclusion, it can be stated that under the experimental conditions reported, the test item Polyphosphoric acids, esters with triethanolamine, sodium salts did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to the highest required concentration.

Remarks on result:

other: strain/cell type: human lymphocytes

Summary of results of the chromosomal aberration study with Polyphosphoric acids, esters with triethanolamine, sodium salts

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		2.0		2.0		0.0
						Positive control^2		85.3		8.5		8.0^S		0.5
						1952.7		114.4		3.5		3.0		0.5
						3417.1		100.7		0.5		0.5		0.0
						5980.0		100.7		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		34.5		14.5		14.0^S		1.5
						1952.7		85.2		2.0		2.0		0.0
						3417.1		88.6		2.5		2.5		0.0
						5980.0		69.0		3.0		3.0^S		0.0
			Exposure period 4 hrs with S9 mix
I			22 hrs			Solvent control^1		100.0		2.0		2.0		0.0
						Positive control^4		69.4		17.0		16.5^S		2.5
						1952.7		105.9		1.0		1.0		0.0
						3417.1		90.6		0.0		0.0		0.0
						5980.0		102.8		1.5		1.0		0.0
II			22 hrs			Solvent control^1		100.0		1.0		1.0		0.0
						Positive control^5		59.8		9.5		9.5^S		1.0
						1952.7		88.7		2.0		2.0		0.0
						3417.1		77.4		0.5		0.5		0.0
						5980.0		74.4		0.5		0.5		0.0

*  Including cells carrying exchanges

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water     10.0 % (v/v)

²     EMS                    825.0 µg/mL

³     EMS                    660.0 µg/mL

⁴   CPA                      15.0 µg/mL

⁵   CPA                       2.5 µg/mL

Conclusions:

Interpretation of results (migrated information):negativeIn 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, Polyphosphoric acids, esters with triethanolamine, sodium salts is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Executive summary:

Summary

The test item Polyphosphoric acids, esters with triethanolamine, sodium salts, dissolved in deionised water, 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 concentration two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (5980.0 µg/mL of the test item) was chosen with regard to the solubility properties and the water/glycol content (16.4 %) 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.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in the absence of S9 mix in Experiment II, one statistically significant increase was observed after treatment with 5980.0 µg/mL (3.0 % aberrant cells, excluding gaps). This value was in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and is therefore considered as being biologically irrelevant.

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 (p < 0.05) in cells with structural chromosome aberrations.

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, Polyphosphoric acids, esters with triethanolamine, sodium salts is considered to benon-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo Mammalian Alkaline Comet Assay

Morris A (2014)

The Comet assay main test was conducted at the maximum recommended dose (MRD) 2000 mg/kg with 1000 mg/kg and 500 mg/kg as the lower dose levels. Further groups of rats were given an oral dose of distilled water (seven rats) or N-Nitroso-N-methylurea (MNU) (5 rats), to serve as vehicle and positive controls respectively. Animals were killed 4 hours after the second dose administration, the stomach and liver tissues were processed and the bone marrow extracted, the slides were then prepared and processed prior to scoring for the presence of Comets. 

There was no evidence of an increase in the percentage tail intensity values in the liver, glandular stomach or bone marrow in animals dosed with the test item when compared to the concurrent vehicle control group.

 

The positive control material produced a marked increase in the percentage tail intensity in the liver, glandular stomach and bone marrow, indicating that the test method was working as expected.

The test item did not induce any increases in the percentage tail intensity values in any of the tissues investigated. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 17 December 2013 and 25 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

To invetsigate the positive mutagenic result (in the absence of metabolic activation) observed in the CHO HPRT forward mutation assay a Single Cell Gel Electrophoresis (Comet) Assay in the male rat was performed to address concerns for worker safey. It also fills the REACH data requirement.

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline for the Testing of Chemicals: In vivo Mammalian Alkaline Comet Assay (2013).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the males weighed 181 to 211 g, and were approximately seven to twelve weeks old. Details of the individual animal weights, group means and standard deviations are presented in the attached Table 1 (attached background material). After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage cardThe animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Harlan Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study. The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

The vehicle control was (water for irrigation) supplied by a reputable supplier. Details as follows:Sighting Test:Identification:Water for irrigation* Supplier: Aguettant LtdSupplier’s Lot Number: 3009615 01Harlan Serial Number: V-5541Expiry Date:01 June 2014Storage conditions: Room temperatureMain Experiment:Identification:Water for irrigation*Supplier: Aguettant LtdSupplier’s Lot Number: 3010081Harlan Serial Number: V-5709Expiry Date:01 April 2015Storage Conditions: Room temperature* Referred to as distilled water throughout the report

Details on exposure:

Range-finding Toxicity Test:A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 20 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The selection of the maximum dose and the choice of vehicle were based on data from a previous study (41101794), investigating Acute Oral Toxicity in the Rat.A group of rats were dosed orally as follows:Dose Level 2000mg/kg, Concentration 200 mg/mL, Dose Volume 10 mL/kg, Number of Rats 2 All animals were dosed twice 20 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.Animals were observed approximately 1 hour after each dosing and approximately 24 hours after the initial dose. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.Comet Test:Groups each of seven males, was dosed twice with a 20 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed using a schedule 1 method approximately 4 hours following the second administration. In addition, two further groups of rats were included in the study; one group (seven male rats) was dosed twice with a 20-hour interval via the oral route with the vehicle alone (distilled water) and a second group (five male rats) was dosed twice orally with 20-hour interval with MNU. MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive controls were killed approximately 4 hours following the second administration. All animals were observed for signs of overt toxicity and death one hour after dosing and immediately prior to termination (24 hours after the initial dose).

Duration of treatment / exposure:

24-hours

Frequency of treatment:

Twice

Post exposure period:

Not applicable

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

The test item concentrations presented throughout the report are calculated based on the active ingredient (85.53%) of the test item and an allowance was made for the water/glycol content when the formulations were prepared.

No. of animals per sex per dose:

Comet Test: 7 males per dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

For the purpose of this study the positive control item N-Nitroso-N-methylurea (MNU) was freshly prepared as required as a solution at the appropriate concentration in distilled water (25 mg/Kg). Five male rats were dosed twice orally with 20-hour interval with MNU. MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.

Tissues and cell types examined:

Tissue Sample RequirementsSamples of liver, bone marrow and glandular stomach were obtained from each animal. Sub-samples of the primary tissues taken from the vehicle control animals and the dose group animals were processed and preserved for possible histopathology investigations. The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay as follows:Liver - A small piece of liver was excised (approximately 1 cm3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension. Glandular Stomach – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides. Bone Marrow – A femur was dissected from each animal and aspirated with PBS to provide a single cell suspension. The cell suspension was filtered through gauze prior to use for the comet slides. Whole Blood - Samples of whole blood (5 µL per slide) were also processed, fixed and stored but not scored. The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed at approximately 4 °C. Blood samples (approximately 1 mL) were taken from the vehicle control animals and the dose group animals into heparinized tubes, centrifuged and the supernatant removed and stored frozen at approximately -20 ºC prior to analysis for presence of the test item.

Details of tissue and slide preparation:

Slide Preparation:Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. The slides were labelled for animal number, project number and tissue type prior to use for the comet assay.Once the cell suspensions had been obtained, approximately 30 µL was added to 270 µL of 0.5% low melting point (LMP) agarose and mixed thoroughly and 50 µL of this cell suspension was placed onto one half of a pre-coated slide, 2 slides per animal (4 gels), for each tissue. The agarose was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing. Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated at approximately 4 °C in the dark overnight.After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralization buffer (0.4M Tris, pH 7.5) and placed onto the platform of a electrophoresis bath, which was filled with chilled electrophoresis buffer (pH >13), until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The aim was to induce sufficient migration of the DNA so that minimal sized comets are produced in the nuclei of vehicle control cells.At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer (0.4M Tris pH 7.5) and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer performed twice. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.Scoring:The slides were coded using a computer generated code prior to scoring. To each dry slide, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV.Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data for each tissue was processed using the Excel program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups would be made. The primary end-point was percentage DNA in the tail (percentage Tail intensity), although other endpoints such as tail moment and length may also be utilized. A positive response is demonstrated when there is clear dose-related change in the defined measurement between the vehicle control and the test item at single sampling time, or a change in the defined measurement in a single dose group at least at a single sampling time. If no marked change in the defined measurement is observed then the test item will be judged as not causing DNA damage in vivo in the tissues evaluated. Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgment. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues see if cell toxicity may be the causative agent rather than any genotoxic mechanism.Histopathology:Samples of the above primary tissues taken from the vehicle control animals and the dose group animals were preserved in buffered 10% formalin and stored for possible histopathology processing. Plasma Analysis:Frozen blood serum/plasma samples were analyzed by the Department of Analytical Services for triethanolamine concentration by High Performance Liquid Chromatography using Mass Spectrometry (HPLC MS). Validation of the analytical method was performed by spiking in known amounts of the suspected hydrolysis product, triethanolamine, into control plasma and determining accuracy and precision data. Full details of the analytical method used is documented in the original data, approved prior to use and detailed in the Plasma Analysis Report (Attached Appendix 1 - attached background material).

Evaluation criteria:

Refer to 'scoring' section above.

Statistics:

No statistical analysis was performed

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-Finding Toxicity TestThe mortality data are summarized as follows: In animals dosed with test item there were no premature deaths, and no clinical signs observed. Based on the above data the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels. Comet Assay:Mortality Data and Clinical Observations:There were no premature deaths seen in any of the test item dose groups. No clinical signs were observed in animals dosed with the test item at any of the dose levels or time points. Evaluation of Comet Assay Slides:A table of the combined group data for each tissue scored for the Comet Assay; glandular stomach, liver and bone marrow, is given in the attached Table 2. Individual animal data for each tissue are presented in the attached Tables 3 to 5 with the means, medians and standard deviations calculated from the individual animal data and group data calculated from the combined individual animal data.The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control group induced marked increases in percentage tail intensity in all tissues analyzed which were consistent with the laboratory historical positive control range. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.There was no marked increase in percentage tail intensity for any of the test item dose levels in any of the tissues analyzed when compared to the vehicle control. Confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated. The hedgehog frequency data for each tissue is presented in the attached Tables 6 to 8. Tables 2-8 in attached background material.

Discussion

The test item did not demonstrate any increases in percentage tail intensity over the vehicle controls in any of the tissues investigated. There was also no dose related increase in the group median values for any of the tissues investigated indicating no shift in the percentage tail intensity between test item groups. 

 

The percentage tail intensity for the vehicle control in the glandular stomach was higher than that observed in the dose groups but this considered to be as a result of high values in three animals (animals 3, 4 and 7) and confirmed by the high standard deviations in these animals. The gastro-epithelial lining has a high rate of proliferation and therefore cell destruction and it is considered that in these animals a higher proportion of dying or damaged cells were sampled resulting in the higher overall percentage tail intensity values. Although the increase was not so marked in the 500 mg/Kg dose level there were also two animals with elevated percentage tail intensities (animals 32 and 33) which increased the overall percentage tail intensity for the group. However, the overall group values for the vehicle control and the 500 mg/Kg dose level were well below the response seen in the positive control group, were within the laboratory historical range for a vehicle control and were not considered to be part of a positive response.

Low percentage tail intensity values were achieved in the vehicle control and the test item dose levels in the liver and bone marrow indicating that there was consistently little damage in these tissues at the test item dose levels tested. Although some of the group and individual median values reported were zero these were rounded values and reflected very low levels of migration rather than no migration or negative migration.

The hedgehog frequency was seen to be higher in all the positive control groups compared to the vehicle control and the dose groups, possibly as a result of cytotoxicity, but did not show any dose related increases in the test item dose groups for any of the tissues investigated. The glandular stomach demonstrated higher levels of hedgehog frequency generally but this is considered to be due in part to the high turnover of cells in the glandular stomach with high numbers of dying cells being constantly present. 

Analysis of the blood serum from the animals of the 2000 mg/Kg dose group demonstrated the presence of triethanolamine indicating that the test item had been absorbed into the blood stream and therefore the liver and bone marrow will have been exposed. The glandular stomach will have been exposed to the test item as it was dosed orally. It is assumed that with evidence of test item absorption in the highest dose group, that the lower dose groups will have also been absorbed.

Conclusions:

The test item did not induce any increases in the percentage tail intensity values of the liver, glandular stomach or bone marrow. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.

Executive summary:

Introduction

This test method has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC in 1999, as described by Ticeet al.(2000). At the time a method for this assay had not yet been adopted by the OECD, active discussions were in progress to develop an approved OECD Method. The test method was designed to be compatible with the procedures indicated in the Draft OECD Guideline for the Testing of Chemicals: In vivo Mammalian Alkaline Comet Assay (2013). 

The method detects evidence of primary DNA damage, as opposed to any of the potential resultant biological effects (e.g. micronuclei, mutations, structural chromosomal aberrations). The assessment of damage to the DNA in single cells is made using an alkaline gel electrophoresis method. The method is capable of detecting DNA single strand breaks (SSB), and double strand breaks (DSB) and alkali labile sites (ALS). Under modified conditions DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites can be detected. Advantages of the method over other genotoxicity tests include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample and its flexibility including adaptation to multiple target tissues.

 

The primary target tissues of this assay were liver, glandular stomach and bone marrow.

Methods

The maximum dose selected for the range-finding test was the maximum recommended dose of 2000 mg/kg. The selection of the maximum dose and the vehicle was based on data from a previous study (41101794), investigating Acute Oral Toxicity in the Rat. The Comet assay main test was conducted at the maximum recommended dose (MRD) 2000 mg/kg with 1000 mg/kg and 500 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the stomach and liver tissues were processed and the bone marrow extracted, the slides were then prepared and processed prior to scoring for the presence of Comets. Comet slides were also prepared from blood sampled but not scored. At the request of the Sponsor blood samples were taken from the vehicle control and test item dose groups and stored for possible future analysis. The samples from the vehicle control group and the high dose group (2000 mg/kg) were analysed for the presence of the test item hydrolysis product, triethanolamine.

Further groups of rats were given an oral dose of distilled water (seven rats) or N-Nitroso-N-methylurea (MNU) (5 rats), to serve as vehicle and positive controls respectively.

Results

There was no evidence of an increase in the percentage tail intensity values in the liver, glandular stomach or bone marrow in animals dosed with the test item when compared to the concurrent vehicle control group.

 

The positive control material produced a marked increase in the percentage tail intensity in the liver, glandular stomach and bone marrow, indicating that the test method was working as expected.

 

Conclusion

The test item did not induce any increases in the percentage tail intensity values in any of the tissues investigated. The test item was considered to be non-genotoxic to the rat tissue investigatedin vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Four in-vitro studies have been conducted on the substance:
- Two Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli
- Chromosome aberration test in human lymphocytes
- CHO HPRT forward mutation assay.
The two Ames studies and chromosome aberration test gave negative result. The CHO HPRT forward mutation assay gave a positive (in the absence of metabolic activation).

One in-vivo study has been conducted on the substance

- A comet study examined the liver, glandular stomach and bone marrow of treated rats
The Comet study gave a negative result.

Justification for classification or non-classification

The test item, Polyphosphoric acids, esters with triethanolamine, sodium salts, was considered to be non-mutagenic under the conditions of two separate in-vitro Ames studies.

The test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Polyphosphoric acids, esters with triethanolamine, sodium salts is considered to be non-clastogenic in the chromosome aberration test, when tested up to the highest required concentration.

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

The test item did not induce any increases in the percentage tail intensity values in the liver, glandular stomach or bone marrow of treated rats and therefore was considered to be non-genotoxic to the rat tissue investigated in vivo.

An in vivo Comet study gave a negative result. Although a positive result was observed from the in-vitro CHO HPRT forward mutation assay study this was not confirmed in vivo and all other in vitro results were negative.It is therefore considered that the substance does not meet the criteria for mutagenicity in accordance with Regulation (EC) No 1272/2008.