2-morpholinoethanesulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-01 to 2017-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-morpholinyl)ethansulfonic acid hydrate

Cas Number:

1266615-59-1

Molecular formula:

C6H13NO4S * xH2O

IUPAC Name:

2-(4-morpholinyl)ethansulfonic acid hydrate

Test material form:

solid: particulate/powder

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: analysis of modal chromosome number
- Cell cycle length, doubling time or proliferation index: cell cycle time was determined in experiment and within the normal range; doubling time: 10-12 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration : DMEM (from Biochrom AG) was used as a basis for complete culture medium with medium DMEM with 5% HS (5% horse serum, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and selection medium (5% horse serum, 1% Penicillin/Streptomycin, 2 µg/mL 6-thioguanine (1 mg/mL))
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen), produced from the livers of male Sprague-Dawley rats
which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 fraction during treatment is 0.4 %.

Test concentrations with justification for top dose:

0.31, 0.63, 1.25, 2.5, 5, 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: no effects on cell viability and good solubility of test substance

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1000000/dish (10 cm diameter)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1: 4h (with and without metabolic activation)
Experiment 2: 24h (without metabolic activation)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): at least 168 h
- Selection time: 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days
- Selection agent: 6-thioguanine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Chi-square test with a significance level of 5%

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity pre-test: highest recommended concentration (10 mM) not cytotoxic, determined by cloning efficiency) and not precipitating after 4h exposure with and without metabolic activation

STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMBA: range: 100 - 461; mean: 2431; sd: 72.2
Without S9 (4h treatment): EMS: range: 71 - 207; mean: 127; sd: 33.4
Without S9 (24h treatment): EMS: range: 35 - 517; mean: 237; sd: 97.0
- Negative (solvent/vehicle) historical control data(number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMEM: range: 4 - 35; mean: 17; sd: 9.3
With S9 (4h treatment): DMSO: range: 2 - 39; mean: 15; sd: 9.2
Without S9 (4h treatment): DMEM: range: 2- 32; mean: 15; sd: 9.0
Without S9 (24h treatment): DMEM: range: 4 - 27; mean: 12; sd: 6.6

Any other information on results incl. tables

Table 1: Mutagenicity in Experiment I with Metabolic Activation

	Conc.	Culture	Cells seeded	Number of colonies per dish					CE mut	MF	MF	Mean
	[mM]		total	I	II	III	IV	V			Per 10E6 cells
Solvent Control Test Item	-	A	2503264	2	8	4	5	1	0.00001	0.00002	20	18
	-	B	2500521	5	3	3	0	4	0.00001	0.00002	15
Solvent Control DMBA		A	2497798	12	4	6	8	6	0.00001	0.00003	34	25
	-	B	2498400	0	1	3	5	4	0.00001	0.00002	17
Positive Control (DMBA)	1.5 µg/mL	A	2499294	34	36	36	37	37	0.00007	0.00020	196	239
		B	2502737	47	65	51	43	50	0.00010	0.00028	282
Test item	10	A	2499351	4	4	3	4	3	0.00001	0.00002	19	26
	10	B	2499539	9	10	5	3	7	0.00001	0.00003	34
Test item	5	A	2496838	2	3	4	2	4	0.00001	0.00002	18	12
	5	B	2501022	0	2	2	1	1	0.00000	0.00001	6
Test Item	2.5	A	2500169	3	6	3	4	7	0.00001	0.00003	26	21
	2.5	B	2495657	2	3	2	5	2	0.00001	0.00002	16
Test Item	1.25	A	2501957	1	0	1	1	1	0.00000	0.00000	4	9
	1.25	B	2501145	4	4	2	1	2	0.00001	0.00001	14
Test Item	0.63	A	2500115	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.63	B	2500176	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
Test item	0.31	A	2498270	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.31	B	2498015	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a

 n/a = not analysed because the OECD 476 guideline requires only 4 concentrations

 

Table 2: Mutagenicity in Experiment I without Metabolic Activation

	Conc.	Culture	Cells seeded	Number of colonies per dish					CE mut	MF	MF	Mean
	[mM]		total	I	II	III	IV	V			Per 10E6 cells
Solvent Control Test Item	-	A	2497286	4	4	5	4	7	0.00001	0.00002	23	26
	-	B	2498413	5	7	6	5	5	0.00001	0.00003	30
Solvent Control DMBA		A	2501274	0	6	2	2	5	0.00001	0.00002	1	11
	-	B	2496879	0	3	1	0	2	0.00000	0.00001	6
Positive Control (DMBA)	300 µg/mL	A	2496689	33	29	27	31	25	0.00006	0.00014	137	145
		B	2500960	26	23	24	31	23	0.00005	0.00015	153
Test item	10	A	2497166	6	8	9	8	7	0.00002	0.00004	35	23
	10	B	2496330	2	3	3	1	1	0.00000	0.00001	10
Test item	5	A	2498978	3	5	4	1	7	0.00001	0.00002	21	21
	5	B	2497623	1	3	6	6	4	0.00001	0.00002	20
Test Item	2.5	A	2500349	3	5	2	3	3	0.00001	0.00002	16	20
	2.5	B	2503188	9	6	5	3	1	0.00001	0.00002	24
Test Item	1.25	A	2499500	4	5	4	2	5	0.00001	0.00002	21	21
	1.25	B	2502812	3	3	3	6	5	0.00001	0.00002	21
Test Item	0.63	A	2500478	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.63	B	2503155	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
Test item	0.31	A	2497970	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.31	B	2499536	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a

Table 3: Mutagenicity in Experiment II without Metabolic Activation

	Conc.	Culture	Cells seeded	Number of colonies per dish					CE mut	MF	MF	Mean
	[mM]		total	I	II	III	IV	V			Per 106 cells
Solvent Control Test Item	-	A	2500933	0	0	3	1	1	0.00000	0.00001	7	11
	-	B	2496490	1	3	2	2	1	0.00000	0.00001	14
Solvent Control DMBA		A	2497077	4	3	0	2	4	0.00001	0.00002	21	25
	-	B	2501950	3	1	3	5	3	0.00001	0.00003	29
Positive Control (DMBA)	300 µg/mL	A	2497340	22	25	23	21	24	0.00005	0.00027	270	280
		B	2503958	22	38	30	35	40	0.00007	0.00029	289
Test item	10	A	2501018	1	2	0	2	0	0.00000	0.00001	7	12
	10	B	2498673	3	3	5	1	1	0.00001	0.00002	17
Test item	5	A	2505206	1	3	2	1	3	0.00000	0.00001	9	18
	5	B	2496744	5	3	3	1	2	0.00001	0.00003	27
Test Item	2.5	A	2499913	2	6	3	6	1	0.00001	0.00003	30	31
	2.5	B	2499050	3	4	6	0	4	0.00001	0.00003	32
Test Item	1.25	A	2501820	1	1	1	0	0	0.00000	0.00000	5	19
	1.25	B	2499000	8	1	3	2	4	0.00001	0.00003	34
Test Item	0.63	A	2498840	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.63	B	2503972	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
Test item	0.31	A	2497800	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
	0.31	B	2498438	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a

 

n/a = not analysed because the OECD 476 guideline requires only 4 concentrations

 

Table 4: Regression Parameters

Treatment	Correlation coefficient |r|	p-value
Exp. I with metabolic activation	0.718	0.282
Exp. I without metabolic activation	0.911	0.089
Exp. II without metabolic activation	0.305	0.695

Table 5: Statistical Significance, pooled replicates (arithmetic means)

Treatment	p-Values
	experiment I		experiment II
	with S9	without S9	without S9
Positive control DMBA	< 0.01	-	-
Positive control EMS	-	< 0.01	< 0.01
Test item 10 mM	0.209	n/c	0.487
Test item 5 mM	n/c	n/c	0.182
Test item 2.5 mM	0.435	n/c	0.002*
Test item 1.25 mM	n/c	n/c	0.139

n.c.: not calculated because the MF is lower than or equal to the solvent control

* = statistically significantly increased in comparison to the solvent control.

 

Table 6: Statistical Significance, individual cultures (Comparison of values of replicate A with corresponding solvent control replicate of A and comparison of values of replicate B with corresponding solvent control replicate of B)

Treatment	Culture	p-Values
		experiment I		experiment II
		with S9	without S9	without S9
Positive control DMBA	A	< 0.01	-	-
	B	< 0.01	-	-
Positive control EMS	A	-	< 0.01	< 0.01
	B	-	< 0.01	< 0.01
Test item 10 mM	A	n/c	0.113	n/c
	B	0.006*	n/c	0.420
Test item 5 mM	A	n/c	n/c	0.430
	B	n/c	n/c	0.042*
Test item 2.5 mM	A	0.313	n/c	< 0.000*
	B	0.490	n/c	0.007*
Test item 1.25 mM	A	n/c	n/c	n/c
	B	n/c	n/c	0.003*

 

n/c: not calculated because the MF is lower than or equal to the solvent control

* = statistically significantly increased in comparison to the solvent control.

 

Table 7: Historical Data of the Solvent Controls

	Number of mutant colonies per 10E6 cells
4 h treatment / with metabolic activation
	Solvent control		Positive control (DMBA)
	DMEM	DMSO
Mean value	16	15	238
Standard deviation	8.8	9.6	74.6
Range	4 - 35	2 - 44	96 - 461
95 % confindence interval	0* - 34	0* - 34	89 - 388
Number of experiments	18	60	41
Study no.: 17050301G865	18	25	239
4 h treatment / with metabolic activation
	Solvent control		Positive control (DMBA)
	DMEM
Mean value	16		136
Standard deviation	10.5		97.7
Range	2 - 48		71 - 294
95 % confindence interval	0* - 37		46 – 227
Number of experiments	52		39
Study no.: 17050301G865	26, 11		145
24 h treatment / with metabolic activation
	Solvent control		Positive control (DMBA)
	DMEM
Mean value	12		232
Standard deviation	6.6		97.7
Range	4 - 27		35 – 517
95 % confindence interval	0* - 25		36 – 427
Number of experiments	40		34
Study no.: 17050301G865	11, 25		280

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions of this study the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic under the conditions of the HPRT assay.

Executive summary:

A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).

The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.

In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.