[No public or meaningful name is available]

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:

From 2015-08-05 to 2015-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study, OECD 471 compliant

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-05-06

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium sulphate (2:1) and its oligomerisation products with urea

Molecular formula:

Not applicable

IUPAC Name:

Tetrakis(hydroxymethyl)phosphonium sulphate (2:1) and its oligomerisation products with urea

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding 1: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate without and with metabolic activation (TA 100 and WP2uvrA; direct plate assay)
Experiment 1: 5.4, 17, 52, 164, 512 and 1600 μg/plate without and with metabolic activation (TA1535, TA1537 and TA98; direct plate assay)
Dose range finding 2: 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/plate without and with metabolic activation (TA 100 and WP2uvrA; pre-incubation assay)
Experiment 2: 5.4, 17, 52, 164, and 512 μg/plate without and with metabolic activation (TA1535, TA1537 and TA98; pre-incubation assay)

Vehicle / solvent:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Controls

Untreated negative controls:

yes

Remarks:

Milli-Q water (Millipore Corp., Bedford, MA., USA)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Solvents for reference items Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands) DMSO = dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany)

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria

Source: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Merck) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 32.4 – 38.9°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Any variation were evaluated and maintained in the raw data.

All the test were evaluated in triplicate.

Evaluation criteria:

Acceptability of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Data evaluation and statistical procedures

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Water solubility: the substance was soluble at all concentration tested
- Precipitation: no precipitation observed


RANGE-FINDING/SCREENING STUDIES: the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in both tester strains.

In the second mutation experiment, the test item was initially tested up to concentrations of 1600 µg/plate in the strains TA100 and WP2uvrA in the pre-incubation assay. Toxicity was observed in both tester strains.


COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Dose range finding test (direct incorporation test): Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain.
	TA 100					WP2uvrA
	Without S9 mix
Positive control	1061	±	42			1347	±	118
Solvent control	100	±	9			28	±	4
1.7	96	±	8			27	±	6
5.4	88	±	22			22	±	8
17	94	±	16			28	±	4
52	94	±	12			28	±	1
164	98	±	10		n	33	±	4	n
512	73	±	6		m	17	±	3	s
1600	0	±	0		a	0	±	0	a
5000	0	±	0		a NP	0	±	0	a NP
	With S9 mix
Positive control	1456	±	105			402	±	18
Solvent control	106	±	24			32	±	7
1.7	89	±	24			25	±	2
5.4	105	±	25			27	±	7
17	86	±	4			25	±	8
52	90	±	7			30	±	1
164	87	±	8	n		26	±	5		n
512	84	±	16	m		22	±	5		s
1600	0	±	0	a		0	±	0		a
5000	0	±	0	a NP		0	±	0		a NP

NP         No precipitate

a             Bacterial background lawn absent

m           Bacterial background lawn moderately reduced

n            Normal bacterial background lawn

s             Bacterial background lawn slightly reduced

Experiment 1: Mutagenic response in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA 1535							TA 1537						TA 98
	Without S9 mix
Positive control	779		±		26			621	±		80			1513	±	41
Solvent control	8		±		3			4	±		1			26	±	7
5.4	11		±		4			5	±		0			22	±	3
17	12		±		3			7	±		4			16	±	2
52	9		±		6			8	±		3			26	±	14
164	14		±		5		n	9	±		2		n	29	±	3	n
512	8		±		3		s	10	±		5		s	19	±	6	s
1600	0		±		0		a NP	0	±		0		a NP	0	±	0	a NP
	With S9 mix
Positive control	287	±		13				453	±	27				1203	±	34
Solvent control	10	±		2				6	±	1				34	±	8
5.4	8	±		3				6	±	2				27	±	2
17	13	±		6				7	±	4				30	±	4
52	10	±		2				8	±	3				30	±	4
164	11	±		6		n		6	±	1		n		37	±	5	n
512	10	±		0		s		7	±	4		s		40	±	9	s
1600	0	±		0		a NP		0	±	0		a NP		0	±	0	a NP

NP	No precipitate
a	Bacterial background lawn absent
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

Toxicity in the dose range finding test/first experiment ( Direct plate assay)

Strain	Without S9-mix		With S9-mix
Dose         Bacterial                    Revertant              (μg/plate)  background lawn       colonies			Dose         Bacterial                     Revertant (µg/plate)  background lawn        colonies
Dose range finding test
TA100		512       moderate                  -^1 1600     absent                  complete 5000     absent                  complete	512            moderate                       -^1 1600          absent                     complete 5000          absent                      complete
WP2uvrA		512       slight                           -^1 1600     absent                  complete 5000     absent                   complete	512             slight                               -^1 1600          absent                      complete 5000          absent                      complete
First mutation experiment
TA1535		512       slight                         -^1 1600     absent                  complete	512            slight                             -^1 1600          absent                        complete
TA1537		512       slight                         -^1 1600     absent                  complete	512            slight                             -^1 1600          absent                        complete
TA98		512       slight                         -^1 1600     absent                  complete	512            slight                             -^1 1600          absent                        complete

¹         No reduction in the number of revertant colonies less than the minimal value of the historical control data range.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative both in the absence and presence of S9-metabolic activation.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

In the dose range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Toxicity was observed in both tester strains. In the first mutation experiment, the test item was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.

 

In the second mutation experiment, the test item was initially tested up to concentrations of 1600 µg/plate in the strains TA100 and WP2uvrA in the pre-incubation assay. Toxicity was observed in both tester strains. In the second part of the experiment, the test item was tested up to concentrations of 512 µg/plate in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Phosphonium, tetrakis (hydroxymethyl)-sulphate (2:1); polymer with urea did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Phosphonium, tetrakis (hydroxymethyl)-sulfate (2:1); polymer with urea is not mutagenic in the Salmonella typhimurium reverse assay and in Escherichia coli reverse mutation assay.