4-morpholinopropanesulphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA97, TA98, TA100, TA102, TA1535) with and without metabolic activation (reference 7.6.1 -1).

Moreover, based on the results of an analogue approach (read-across), the substance is considered to be not genotoxic in an in vitro micronucleus assay (OECD 487, reference 7.6.1 -2) and in an in vitro mammalian gene mutation assay (OECD 476, reference 7.6.1 -3).

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:

2003-09-30 to 2003-10-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 :
obtained by RCC, Roßdorf
- method of preparation of S9 mix:
producted from the livers of male Wistar rats which were treated with 80 mg Phenobarbital/kg bw intraperitoneally; on the following day, with 80 mg beta-Naphthoflavon/kg bw orally
- concentration or volume of S9 mix and S9 in the final culture medium : 1 mL, 4 %
- quality controls of S9: no data

Test concentrations with justification for top dose:

First experiment: 0.05.; 0.15; 0.5; 1.5; 5.0 mg/plate, where 5 mg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
Second experiment: 1.25; 2.5; 5.0 mg/plate, where 5 mg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances

Vehicle / solvent:

- solvent used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

without metabolic activation, TA97, TA98, TA102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation, TA100, TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation, TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation, TA97, TA 100, TA102 and TA1535

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration: 48 h (both experiments)

DETERMINATION OF CYTOTOXICITY
- Method: titre ratio

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor > 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 97

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

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

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

Key result

Species / strain:

S. typhimurium TA 102

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

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item is soluble in deionised water. A stock solution containing 50 g/L was prepared.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Ames test:
- Signs of toxicity :
No signs of toxicity towards the tested strains could be observed. The determined values for the toxicity control were in the range of the titre. The background lawn was visible and the number of revertant colonies was not reduced.
- Individual plate counts :
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
- Mean number of revertant colonies per plate and standard deviation :
see tables

HISTORICAL CONTROL DATA
- no data reported

Table with results for 1^st Experiment (plate incorporation)

strain		TA97a		TA98		TA100		TA102		TA1535
induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
water	mean	83	66	19	23	144	145	144	131	14	17
	sd	43.3	52.2	11.8	3.5	17	11	28.1	29.1	7.2	5.0
DMSO	mean	78	112	18	21	126	130	123	134	12	11
	sd	66.9	24.5	2.2	4.5	4	26	6.8	12.4	2.8	3.9
pos.contr.	mean	924	560	1038	70	633	754	656	717	590	439
	sd	103	126	221	25	163	256	32	184	76,1	49
	f(I)	11.9	5.0	57.7	3.4	4.4	5.8	5.3	5.4	42.9	40.8
5 mg/pl.	mean	79	116	13	16	136	148	129	141	12	12
	sd	86	78	5	7	28	18	11	13	2	4
	f(I)	0.95	1.75	0.69	0.70	0.94	1.02	0.90	1.07	0.89	0.7
1.5 mg/pl.	mean	61	62	16	13	121	104	101	81	10	13
	sd	50	48	3	4	14	20	11	10	6	2
	f(I)	0.73	0.94	0.83	0.58	0.84	0.72	0,70	0,62	0,73	0,77
0.5 mg/pl.	mean	91	52	14	13	129	83	102	65	14	18
	sd	11	53	3	3	23	25	10	5	1	1
	f(I)	1.10	0.78	0.73	0.59	0.90	0.57	0.71	0.49	1.00	1.03
0.15 mg/pl.	mean	73	59	18	16	71	74	107	57	11	12
	sd	37	39	4	3	14	11	20	10	4	1
	f(I)	0.88	0.90	0.95	0.69	0.49	0.51	0,74	0,43	0,76	0.67
0.05 mg/pl.	mean	90	87	15	16	127	101	117	72	14	16
	sd	17	24	2	5	41	21	11	17	2	4
	f(I)	1.08	1.32	0.75	0.72	0.88	0.70	0.82	0.54	1.02	0.90

Table with results for 2^nd Experiment (pre-incubation)

strain		TA97a		TA98		TA100		TA102		TA1535
induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
water	mean	149	117	10	11	131	120	128	119	10	10
	sd	55.1	67.9	2.1	2.6	22	18	16.8	7.5	2.6	2.2
DMSO	mean	93	118	9	11	93	96	119	116	8	11
	sd	67.8	18.6	2.5	4.1	28	6	36.8	11.9	1.7	2.6
pos.contr.	mean	571	721	604	61	948	575	847	678	1024	223
	sd	275	198	121	6	153	236	62	173	157	11
	f(I)	6.17	6.14	65.3	5.40	7.24	5.98	7.12	5.84	98.9	19.8
5 mg/pl.	mean	93	110	6	8	122	129	131	121	12	9
	sd	44	25	4	1	24	20	20	7	6	2
	f(I)	0.62	0.94	0.64	0.74	0.93	1.08	1.02	1,02	1.2	0.85
2.5 mg/pl.	mean	112	103	7	10	98	120	135	111	11	9
	sd	30	64	3	6	13	9	35	9	2	2
	f(I)	0.75	0.88	0.74	0.95	0.75	1.00	1.05	0.93	1.02	0.85
1.25 mg/pl.	mean	108	71	7	8	97	95	102	94	8	10
	sd	49	70	3	3	9	7	16	7	1	2
	f(I)	0.72	0.60	0.69	0.76	0.74	0.79	0.80	0.79	0.78	0.95

 

 

Conclusions:

The substance was not mutagenic in five strains (TA97, TA98, TA100, TA102, TA1535) with and without metabolic activation.

Executive summary:

The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471 under GLP. The substance was soluble and non-cytotoxic up to 5 mg/plate, i.e. the recommended maximum concentration. The substance was tested in five strains (TA97, TA98, TA100, TA102, TA1535) with and without metabolic activation (S9). In a first experiment, five concentrations (0.05; 0.15; 0.5; 1.5; 5.0 mg/plate) were tested for 48 hours in the plate incorporation method. In the second experiment the concentrations 1.25, 2.5 and 5.0 mg/plate were tested (48 h) in the pre-incubation method. The number of colonies were evaluated by eye. While the positive controls were clearly mutagenic, the substance did not increase the number of colonies. Therefore, the substance is considered to be not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to section 13 for Read-Across Justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to section 13 for Read-Across Justification.

Reason / purpose for cross-reference:

read-across source

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471 under GLP. The substance was soluble and non-cytotoxic up to 5 mg/plate, i.e. the recommended maximum concentration. The substance was tested in five strains (TA97, TA98, TA100, TA102, TA1535) with and without metabolic activation (S9). In a first experiment, five concentrations (0.05; 0.15; 0.5; 1.5; 5.0 mg/plate) were tested for 48 hours in the plate incorporation method. In the second experiment the concentrations 1.25, 2.5 and 5.0 mg/plate were tested (48 h) in the pre-incubation method. The number of colonies were evaluated by eye. While the positive controls were clearly mutagenic, the substance did not increase the number of colonies. Therefore, the substance is considered to be not mutagenic.

An in vitro micronucleus test according to OECD TG 487 and an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD TG 476 form the structurally similar substance CAS 1266615 -59 -1 are available. No experimental data with the test substance is available. Therefore, the studies conducted with the read-across source substance are used to investigate the genotoxicity of the target substance.

Micronucleus test

A study according OECD TG 487 was performed with the source substance CAS 1266615-59-1 to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 100 mM corresponding to 10 mM as highest concentration in the test. In addition, a geometric series of dilutions was prepared from the stock solution.

Two independent and valid experiments were performed. Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. After the culture harvest time, the cells were harvested and slides were prepared. Cytotoxicity and level of micronuclei were determined.

In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected. No cytotoxic effect was detected in any of the tested concentrations in both experiments.

Therefore, the three highest test item concentrations were evaluated for micronuclei.

Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system. In conclusion, under the experimental conditions reported, the substance does not induce the formation of micronuclei in human lymphocytes in vitro.

HPRT test

A study according OECD TG 476 was performed with the source substance CAS 1266615-59 -1 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. 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, amended for the fifteenth time in Regulation (EU) 2020/1182.