Reaction products of bis([tetrakis(hydroxyalkyl)phosphonium] sulfate and alkanamine with Urea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-06-14 to 2016-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-09-22

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Test material form:

liquid

Specific details on test material used for the study:

Correction factor of 1.48 will be used at dose formulation preparation.

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 fraction

Test concentrations with justification for top dose:

All concentrations and dose-levels were expressed as active ingredient content of the test item. A correction factor of 1.48 was used.

No. of concentration Concentration Concentration (µg/plate)
of the test item
1 10.54 mg/mL 1581
2 3.333 mg/mL 500
3 1.054 mg/mL 158.1
4 0.3333 mg/mL 50
5 0.1054 mg/mL 15.81
6 0.03333 mg/mL 5
7 0.01054 mg/mL 1.581
8 0.00333 mg/mL 0.5
9 0.001054 mg/mL 0.1581
10 0.000333 mg/mL 0.05

Concentrations 1-7 were examined in the Initial Mutation Test using the plate incorporation method,
Concentrations 1-8 were examined in the Confirmatory Mutation Test using the pre-incubation method.
Concentrations 3-10 were examined in the Complementary Confirmatory Mutation Test (using the pre-incubation method) in Escherichia coli WP2 uvrA strain without metabolic activation.

Vehicle / solvent:

All dilutions in the main tests of test item were made in the testing laboratory using DMSO.
Based on the performed additional trial formulation, the test item formulation using DMSO as vehicle (opalescence formulation without test item precipitate at the concentration of 10.54 mg/ml) was suitable for the test.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and Distilled water

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene for all strain with metabolic activation 4-nitro-1.2-phenylene diamine For TA 98 without metabolic activation

Details on test system and experimental conditions:

Storage
The strains are stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

Procedure for Growing Cultures
The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 µL inoculum were used to inoculate each 50 mL of Nutrient for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37oC in a Gyrotory Water Bath Shaker.

Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10e5, 10e6, 10e7 and 10e8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.

The Typical Composition (g/1000 mL) of Minimal Glucose Agar
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
Distilled water q.s. ad 1000 mL

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000 mg
Distilled water q.s. ad 1000 mL

Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution 947.5 mL

METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd.

Induction of Liver Enzymes
Male Wistar rats (385-450 g, animals were 11 weeks old at the initiation) were treated with phenobarbital (PB) and beta-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Euthanasia was performed by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation date of the induction of liver enzymes used for preparation S9 used in this study was 25 January 2016.

Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 +/- 10ºC. The date of preparation of S9 fraction for this study was 28 January 2016 (CiToxLAB code: E12297).
The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 30.8 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

The S9 Mix
Salt solution for S9 mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 mix (see above) 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

0.2 M Sodium Phosphate Buffer, pH 7.4
Solution A:
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121 °C in an autoclave.

Solution B:
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121oC in an autoclave.

Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL

Rationale for test conditions:

See "Any other information on materials and methods including tables".

Evaluation criteria:

See "Any other information on materials and methods including tables".

Results and discussion

Test results

Test results 1

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

Test results 2

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 3

Species / strain:

S. typhimurium TA 1535

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 4

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

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:

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

The examined concentration range was considered to be adequate as concentrations up to maximum recommended concentration (1581 µg/plate) were examined in the main tests. At least five analyzable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Any other information on results incl. tables

Summary Table of the Preliminary Concentration Range Finding Test

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA 98		TA 100
		-S9	+S9	-S9	+S9
Untreated control	Mean	23.3	44.3	107.7	126.7
	MF	1.04	1.58	1.14	1.23
Distilled water control 50 µL *	Mean	-	-	115.0	-
	MF	-	-	1.22	-
DMSO control 50 µL *	Mean	27.7	30.3	-	121.0
	MF	1.24	1.08	-	1.17
DMSO 150µL control	Mean	22.3	28.0	94.3	103.0
	MF	1.00	1.00	1.00	1.00
5000	Mean	0.0	0.0	0.0	0.0
	MF	0.00	0.00	0.00	0.00
2500	Mean	0.0	0.0	0.0	0.0
	MF	0.00	0.00	0.00	0.00
1000	Mean	0.0	0.0	0.0	0.0
	MF	0.00	0.00	0.00	0.00
316	Mean	34.3	28.3	67.0	67.7
	MF	1.54	1.01	0.71	0.66
100	Mean	29.3	29.7	103.0	162.3
	MF	1.31	1.06	1.09	1.58
31.6	Mean	29.3	31.0	114.7	147.0
	MF	1.31	1.11	1.22	1.43
10	Mean	22.0	31.3	112.7	145.3
	MF	0.99	1.12	1.19	1.41
NPD (4mg)	Mean	375.0	-	-	-
	MF	13.55	-	-	-
2AA (2mg)	Mean	-	2304.0	-	2384.0
	MF	-	75.96	-	19.70
SAZ (2mg)	Mean	-	-	1040.0	-
	MF	-	-	9.04	-

*50µL of Distilled water control and 50µL of DMSO control were used because of positive controls

Summary Table of the Initial Mutation Test (Plate Incorporation Method)

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	18.3	27.7	82.3	124.3	14.0	12.3	6.7	6.0	25.3	38.3
	MF	0.95	1.26	0.86	1.32	0.91	1.37	1.43	0.78	0.90	1.25
Distilled water control 50 µL *	Mean	-	-	95.3	-	11.3	-	-	-	32.7	-
	MF	-	-	0.99	-	0.74	-	-	-	1.17	-
DMSO control 50 µL *	Mean	24.0	25.0	-	127.3	-	8.0	8.0	10.0	-	31.7
	MF	1.24	1.14	-	1.35	-	0.89	1.71	1.30	-	1.03
DMSO 150µL control	Mean	19.3	22.0	96.0	94.3	15.3	9.0	4.7	7.7	28.0	30.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
1581	Mean	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	MF	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
500	Mean	0.0	0.7	1.7	1.3	0.3	0.0	1.0	1.3	4.3	2.7
	MF	0.00	0.03	0.02	0.01	0.02	0.00	0.21	0.17	0.15	0.09
158.1	Mean	27.7	30.3	95.3	101.7	15.7	10.3	10.3	8.7	25.3	26.0
	MF	1.43	1.38	0.99	1.08	1.02	1.15	2.21	1.13	0.90	0.85
50	Mean	24.0	31.3	102.7	111.3	13.0	11.7	8.0	8.0	28.3	25.3
	MF	1.24	1.42	1.07	1.18	0.85	1.30	1.71	1.04	1.01	0.83
15.81	Mean	21.7	29.0	96.3	111.0	15.3	14.3	6.7	7.7	28.3	25.0
	MF	1.12	1.32	1.00	1.18	1.00	1.59	1.43	1.00	1.01	0.82
5	Mean	19.7	25.3	103.0	103.0	16.3	11.3	6.0	12.3	25.7	25.7
	MF	1.02	1.15	1.07	1.09	1.07	1.26	1.29	1.61	0.92	0.84
1.581	Mean	26.3	21.3	83.3	104.0	13.0	8.3	7.7	11.0	23.0	28.0
	MF	1.36	0.97	0.87	1.10	0.85	0.93	1.64	1.43	0.82	0.91
NPD (4mg)	Mean	348.7	-	-	-	-	-	-	-	-	-
	MF	14.53	-	-	-	-	-	-	-	-	-
2AA (2mg)	Mean	-	2165.3	-	2520.7	-	298.0	-	253.0	-	-
	MF	-	86.61	-	19.80	-	37.25	-	25.30	-	-
2AA (50mg)	Mean	-	-	-	-	-	-	-	-	-	264.0
	MF	-	-	-	-	-	-	-	-	-	8.34
SAZ (2mg)	Mean	-	-	1005.3	-	1104.0	-	-	-	-	-
	MF	-	-	10.55	-	97.41	-	-	-	-	-
9AA (50mg)	Mean	-	-	-	-	-	-	429.3	-	-	-
	MF	-	-	-	-	-	-	53.67	-	-	-
MMS (2mL)	Mean	-	-	-	-	-	-	-	-	909.3	-
	MF	-	-	-	-	-	-	-	-	27.84	-

*50µL of Distilled water control and 50µL of DMSO control were used because of positive controls

Summary Table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (Pre-Incubation Method)

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	17.3	20.0	107.0	109.3	14.0	10.0	9.7	8.7	41.3	38.7
	MF	0.87	1.02	1.49	1.26	1.35	0.94	1.26	0.81	0.93	1.23
Distilled water control 50 µL *	Mean	-	-	85.7	-	10.3	-	-	-	41.7	-
	MF	-	-	1.19	-	1.00	-	-	-	0.93	-
DMSO control 50 µL *	Mean	20.0	19.0	-	108.3	-	9.7	7.3	7.7	-	36.3
	MF	1.00	0.97	-	1.25	-	0.91	0.96	0.72	-	1.16
DMSO 150µL control	Mean	20.0	19.7	72.0	87.0	10.3	10.7	7.7	10.7	44.7	31.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
1581	Mean	0.3	0.0	0.0	0.0	0.0	0.0	0.0	0.0	--	0.0
	MF	0.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	--	0.00
500	Mean	2.0	1.3	0.0	22.7	4.3	3.0	1.3	1.7	--	12.3
	MF	0.10	0.07	0.00	0.26	0.42	0.28	0.17	0.16	--	0.39
158.1	Mean	0.0	25.3	39.3	51.7	8.5	7.3	2.3	5.3	4.0	8.7
	MF	0.00	1.29	0.55	0.59	0.82	0.69	0.30	0.50	0.09	0.28
50	Mean	29.3	28.3	98.0	87.7	8.0	7.7	9.3	12.3	13.3	28.7
	MF	1.47	1.44	1.36	1.01	0.77	0.72	1.22	1.16	0.30	0.91
15.81	Mean	21.0	27.7	83.7	93.3	11.7	10.7	10.3	8.7	38.7	39.0
	MF	1.05	1.41	1.16	1.07	1.13	1.00	1.35	0.81	0.87	1.24
5	Mean	21.7	21.0	62.3	104.3	11.0	12.0	5.7	5.3	41.7	38.3
	MF	1.08	1.07	0.87	1.20	1.06	1.13	0.74	0.50	0.93	1.22
1.581	Mean	20.0	26.3	77.0	102.0	17.7	8.3	8.3	7.7	42.3	39.3
	MF	1.00	1.34	1.07	1.17	1.71	0.78	1.09	0.72	0.95	1.26
0.5	Mean	18.3	24.3	74.0	80.7	11.7	10.0	9.0	7.0	42.7	43.3
	MF	0.92	1.24	1.03	0.93	1.13	0.94	1.17	0.66	0.96	1.38
0.1581	Mean	--	--	--	--	--	--	--	--	40.0	--
	MF	--	--	--	--	--	--	--	--	0.90	--
0.05	Mean	--	--	--	--	--	--	--	--	39.0	--
	MF	--	--	--	--	--	--	--	--	0.87	--

*50µL of Distilled water control and 50µL of DMSO control were used because of positive

Summary Table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (Pre-Incubation Method) (continued)

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella thyphimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
NPD (4mg)	Mean	374.7	--	--	--	--	--	--	--	--	--
	MF	18.73	--	--	--	--	--	--	--	--	--
2AA (2mg)	Mean	--	2320.0	--	2069.3	--	264.7	--	230.7	--	--
	MF	--	122.11	--	19.10	--	27.38	--	30.09	--	--
2AA (50mg)	Mean	--	--	--	--	--	--	--	--	--	265.3
	MF	--	--	--	--	--	--	--	--	--	7.30
SAZ (2mg)	Mean	--	--	1126.7	--	1234.0	--	--	--	--	--
	MF	--	--	13.15	--	119.42	--	--	--	--	--
9AA (50mg)	Mean	--	--	--	--	--	--	408.7	--	--	--
	MF	--	--	--	--	--	--	55.73	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1026.7	--
	MF	--	--	--	--	--	--	--	--	24.64	--

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Tetrakis[hydroxymethyl]phosphonium sulphate urea-amine copolymer (Batch Number: 788JD161) had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Tetrakis[hydroxymethyl]phosphonium sulphate urea-amine copolymer was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Mutation Test (Pre-Incubation Method).

Based on the results of a solubility tests, the test item was formulated in

Dimethyl sulfoxide (DMSO). Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate and in the Confirmatory Mutation Test were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate. Examined concentrations in the Complementary Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain without metabolic activation were 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test and in the main tests in all Salmonella typhimurium strains and Escherichia coli WP2 uvrA strain with and without metabolic activation. The effect was excessive in Escherichia coli WP2 uvrA strain, thus a complementary experiment was performed to ensure validity. In the Complementary Confirmatory Mutation Test, the background inhibition was still observed in the examined strain without metabolic activation.

 The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Tetrakis[hydroxymethyl]phosphonium sulphate urea-amine copolymer (Batch Number: 788JD161) had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.