2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

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:

2016-08-29 to 2016-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

EC Number:

230-237-7

EC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Cas Number:

6976-37-0

Molecular formula:

C8H19NO5

IUPAC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Test material form:

liquid

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Remarks:

E.coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix : The complete S9 Mix was freshly prepared. with components: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL + Rat liver homogenate (S9) 100 mL + Salt solution for S9 Mix 400 mL
- volume of S9 mix in the final culture medium: 0.5 mL of the S9 Mix was added to each overlay tube.

Test concentrations with justification for top dose:

First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances.

Vehicle / solvent:

- Vehicle used: ultrapure water for test item, SAZ and MMS; DMSO for NPD, 9AA and 2AA

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine (NPD, TA98 without metabolic activation) and 2-aminoanthracene (2AA, all strains, with metabolic activation)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 ºC
- Exposure duration/duration of treatment: ca. 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; revertant colony numbers

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of the test item stock solutions (50 mg/mL) were 9.39 and 9.18. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was in the range of 7.44 - 7.50.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Precipitation and time of the determination: No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

RANGE-FINDING STUDY: Based on the solubility test, revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

Ames test:
- Signs of toxicity : No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.

HISTORICAL CONTROL DATA: Please refer to table 3.

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)

Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA 98				TA100				TA 1535				TA 1537				WP2 uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17	0.98	20.3	0.94	88.7	0.81	144.7	1.17	9	0.96	11.7	1.09	8.7	1.63	5.3	1	22.3	1.24	24.3	1
DMSO Control	17.3	1	22.3	1	-	-	109.7	1	-	-	9	1	7.7	1	7.7	1	-	-	25.3	1
Ultrapure Water Control	17.3	1	21.7	1	110	1	123.7	1	9.3	1	10.7	1	5.3	1	5.3	1	18	1	24.3	1
5000	17.7	1.02	22	1.02	98.7	0.9	126	1.02	12.7	1.36	14	1.31	5	0.94	4.7	0.88	19.3	1.07	31	1.27
1600	21	1.21	27.3	1.26	95.3	0.87	122	0.99	13	1.39	12.8	1.19	4.7	0.88	8	1.5	16.3	0.91	26	1.07
500	17.3	1	21.3	0.98	107.3	0.98	120.7	0.98	14.3	1.54	13	1.22	7	1.31	5.3	1	20	1.11	30.7	1.26
160	14.7	0.85	26	1.2	117.3	1.07	130.7	1.06	12.7	1.36	13	1.22	5	0.94	6.7	1.25	23.3	1.3	31.7	1.3
50	17.7	1.02	21	0.97	106.7	0.97	136	1.1	11	1.18	10	0.94	4.7	0.88	7.3	1.38	16.7	0.93	23.3	0.96
16	18	1.04	26.7	1.23	100.3	0.91	136	1.1	8.7	0.93	9.3	0.88	4.3	0.81	5	0.94	20.3	1.13	32	1.32
NPD (4 µg/plate)	470	27.12	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg/plate)	-	-	-	-	1549.3	14.08	-	-	979.3	104.93	-	-	-	-	-	-	-	-	-	-
9AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	860	112.17	-	-	-	-	-	-
MMS (2 µL/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	617.3	34.3	-	-
2AA (2 µg/plate)	-	-	1176	52.66	-	-	1008	9.19	-	-	179.3	19.93	-	-	169	22.04	-	-	-	-
2AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	342	13.5

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-incubation Test)

Concentrations (µg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2 uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertauts per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	15.7	1.09	20.3	1.02	74.7	0.86	115	0.97	12.3	1	9.7	1.04	7.3	1.47	6.7	1.17	19	1.04	29.7	0.75
DMSO control	13.3	1	14.7	1	-	-	100.3	1	-	-	10	1	5.3	1	6.3	1	-	-	36	1
Ultrapure Water Control	14.3	1	20	1	86.3	1	118.3	1	12.3	1	9.3	1	5	1	5..7	1	18.3	1	39.7	1
5000	19	1.33	15.7	0.78	98	1.14	99.7	0.84	10.7	0.78	10.3	1.11	8	1.6	7	1.24	25	1.36	39.7	1
1600	15.3	1.07	17..7	0.88	87	1.01	100.3	0.85	14.3	1.05	9	0.96	7	1.4	7.3	1.29	28.7	1.56	36.7	0.92
500	17.7	1.23	15.7	0.78	82	0.95	104	0.88	13	0.95	9	0.96	8	1.6	4.7	0.82	26.7	1.45	33	0.83
160	19.3	1.35	20.3	1.03	95	1.1	103.7	0.88	13	0.95	9.3	1	4	0.8	5.3	0.94	25.7	1.4	29.3	0.74
50	19.7	1.37	17.3	0.87	85.7	0.99	98.3	0.83	18.3	1.34	8	0.86	7	1.4	6	1.06	21	1.15	29.3	0.74
16	15	1.05	15.3	0.77	94	1.09	112.3	0.95	12	0.88	12.7	1.36	7	1.4	6	1.06	23	1.25	28.3	0.71
NPD (4 µg/plate)	293.7	22.03	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg/plate)	-	-	-	-	852	9.87	-	-	968	78.49	-	-	-	-	-	-	-	-	-	-
9AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	598.7	112.25	-	-	-	-	-	-
MMS (2 µL/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	960	52.36	-	-
2AA (2 µg/plate)	-	-	597.3	40.73	-	-	1186.7	11.83	-	-	120.7	12.07	-	-	96	15.16	-	-	-	-
2AA (50 µg/plate)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	165.3	4.59

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine, SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.

Table 3: Historical Control Values for Revertants Plate (for the Period of 2008-2015)

			Bacterial strains
Historical control data of untreated control			TA98	TA100	TA1535	TA1537	E. coli
		Average	21.4	106	10.4	8.1	25.6
	-S9	SD	3.7	27.3	1.5	2.5	2.5
		Minimum	9	65	3	2	11
		Maximum	39	157	23	19	45
			TA98	TA100	TA1535	TA1537	E. coli
		Average	28	117.1	11.9	9	34.3
	+S9	SD	4.2	19.4	1.5	2	5.4
		Minimum	12	75	4	3	18
		Maximum	48	166	24	20	56
			Bacterial strains
Historical control data of DMSO control			TA98	TA100	TA1535	TA1537	E. coli
		Average	20.9	101.4	10.3	7.9	24.9
	-S9	SD	3.5	26.2	1.4	2.5	4.9
		Minimum	10	65	3	2	11
		Maximum	39	150	23	20	44
			TA98	TA100	TA1535	TA1537	E. coli
		Average	27.1	114.7	12	8.8	34.2
	+S9	SD	4	19.3	1.4	2.1	5.2
		Minimum	15	71	4	3	16
		Maximum	48	161	24	20	56
			Bacterial strains
Historical control data of Water control			TA98	TA100	TA1535	TA1537	E. coli
		Average	22.4	105.5	10.4	7.5	26.3
	-S9	SD	3.6	27.6	1.6	2.3	5.9
		Minimum	12	67	3	2	13
		Maximum	36	156	24	15	47
			TA98	TA100	TA1535	TA1537	E. coli
		Average	28	117.4	11.5	8.7	35.2
	+S9	SD	4	19.8	1.4	2.3	5.2
		Minimum	15	83	4	4	18
		Maximum	43	166	22	16	56
			Bacterial strains
Historical control data of positive controls			TA98	TA100	TA1535	TA1537	E. coli
		Average	255.6	958.9	842.1	467.4	712.3
	-S9	SD	30.7	149.9	134	105.7	57.5
		Minimum	123	522	354	109	320
		Maximum	647	1927	1871	1498	1283
			TA98	TA100	TA1535	TA1537	E. coli
		Average	1224.8	1431.9	165.4	148	264.7
	+S9	SD	293.8	339.9	35.1	21.3	74.2
		Minimum	409	581	85	68	141
		Maximum	2587	2923	507	407	487

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA

SD: Standard deviation; DMSO: Dimethyl sulfoxide

Applicant's summary and conclusion

Conclusions:

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 and WP2 uvrA) with and without metabolic activation.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471.

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) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

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

Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:

5000; 1600; 500; 160; 50 and 16 μg/plate.

In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 9.24. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solution (50 mg/mL) were 9.39 and 9.18. The pH of the test item containing overlays was in the range of 7.44 - 7.50 in both experiments. In this study the alkaline test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

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 bacterium tester strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.