Morpholinium sulphamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487 (Draft version)

Deviations:

yes

Remarks:

human peripheral blood lymphocytes

GLP compliance:

yes (incl. QA statement)

Remarks:

The Study was conducted in compliance with Principles of Good Laboratory Practice, Paris 1998, in compliance with the DIRECTIVE 2004/9/EC, the DIRECTIVE 2004/10/EC and with the Slovak Government Act № 67/2010 Coll. and the Slovak Government Decree № 320

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sulphamidic acid

EC Number:

226-218-8

EC Name:

Sulphamidic acid

Cas Number:

5329-14-6

Molecular formula:

H3NO3S

IUPAC Name:

sulfamic acid

Test material form:

other:

Method

Target gene:

Micronuclei

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

Donors: Blood from two different healthy, non-smoking, young (between 31- 34 years of age) people
(always man and women)

Number: For each test replicate two new donors were available.

Source: Department of Haematology and Transfusiology, Šmidke’s street, Bratislava, SLOVAKIA
Treatment: Whole blood treated with an anti-coagulant (heparin), were added to culture medium
containing a mitogen (e.g. phytohemagglutinin) (PHA) and incubated at 37°C.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 MIX

Test concentrations with justification for top dose:

860, 86, 8.6, 0.86, 0.086, 0.0086, 0.00086, 0 µg / mL of blood cultivated

Vehicle / solvent:

Ultrapure water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Thiotepa (CAS: 52-24-4)

Remarks:

-S9 mix (without metabolic activation); concentration: 2 µg.ml -1

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9 mix (with metabolic activation); concentration: 80 µg.ml -1

Details on test system and experimental conditions:

TEST SYSTEM:
Cell used: human peripheral blood lymphocytes
Donors: Blood from two different healthy, non-smoking, young (between 31- 34 years of age) people (always man and women)
Number: For each test replicate two new donors were available.
Source: Department of Haematology and Transfusiology, Šmidke’s street, Bratislava, SLOVAKIA
Treatment: Whole blood treated with an anti-coagulant (heparin), were added to culture medium containing a mitogen (e.g. phytohemagglutinin) (PHA) and incubated at 37°C.
TEST CONDITIONS:
Cytokinesis blocking substance: cytochalasin B
Its concentration and duration of cell exposure: 6,0 µg/mL (30 µg/5 mL), started from 44 hour of each cultivation during remaining 28 hours.
Solvent/vehicle: Ultrapure water without any side effects. Very good solubility of the substance in the water.
Composition of media: RPMI 1640, BOFES, PHAG
CO2 concentration: The concentration of CO2 = 5% in air volume in the culture incubator INCO 2 with
adjustable automatic control.
Volume of vehicle and test substance added: 200 µL (water + substance).
Incubation temperature: 37 °C
Humidity: 90% provided by 500 mL of sterile ultrapure water.
Incubation time: 72 hours (for each test replicate).
Cell density at seeding: The density of lymphocytes in the blood is given approximately at 100000 /mL. After centrifugation about 10 000 cells was spread on slides and then are fixed and count
Type and composition of metabolic activation system: liver enzyme extract from the liver of male white rats, Sprague-Dawley strain after induction of liver enzymes of animals with polychlorinated biphenyl "Delor 103”.
Composition of the activation mixture S9MIX: NADP, G-6-P, sterile distilled H2O,Na2HPO4.12H2O, KH2PO4, KCl, MgSO4, the cooling solution is added to the S9 liver extract.
Negative control: +S9MIX together with -S9MIX blood cultivated in two parallels with the addition of 200 ml ultra-pure H2O.
Positive controls: -S9 mix (without metabolic activation) + Thiotepa (CAS: 52-24-4); concentration: 2 µg/mL
+S9 mix (with metabolic activation) + Cyclophosphamide (CAS: 50-18-0): 80 µg/mL
Methods of slide preparation:
Slides are individually dipped in warm soapy solution for 2-3 hours, then each piece separately washed thoroughly with a brush and rinse with running water. Thus cleaned slides are individually dipped into sulphuric acid, at least 24 hours (or several days). In sulphuric acid glasses are again, individually, rinsed with running water and then distilled water. Cleaned slides are dipped in denatured alcohol, which are stored until use. Before use of alcohol and selected the appropriate polish cloth. Perfect degreasing and cleanliness of the slides is essential for preparing high-quality native specimens and also for colouring techniques.

Evaluation criteria:

1. Micronucleus diameter is greater than the third of nucleus;
2. Colour and structure of the micronucleus is consistent with the colour and structure of the nucleus;
3. Micronucleus distance from the cell nucleus is no more than 3-4 diameters nucleus
4. Micronucleus is located in the cytoplasm of binucleate cells.

Statistics:

Student’s t - test: the arithmetic mean, standard deviation and statistical significance.

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity in range finding test was caused by physical effect of the test substance (high pH). The highest test concentration 8600 µg / ml of blood cultivated was excluded immediately, because total coagulation of blood had occurred.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Data on pH and osmolality of the treatment medium:
Very high concentration were excluded due to extreme coagulation effect on blood. There was also an evidence (very good cell division in the experiments) that very good buffer capacity of the culture media had occurred. (Detail will be specified in the final report).

Definition of what constitutes a micronucleus:
Origination of micronuclei involves an effect of the chemical reaction with chromosomal DNA at mitosis (at division of cell nucleus). Definition of acceptable cells for analysis: Binucleate lymphocytes; micronucleus is located in the cytoplasm of binucleate cell.

Number of cells with micronuclei and number of micronuclei per cell, given separately for each treated and control culture and defining whether from binucleate or mononucleate cells, where appropriate: All micronuclei are in binucleate cells and, in rare cases, less than 1% two or more
micronuclei are located in one binucleate cell.

Dose-response relationship: Very weak, respectively, increasing of doses of the substance does not increase effect sufficiently.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Range finding test – 1st test replicate: Dilution factor = 10, final concentration of the test substance in 1 mL blood cultivated (blood + culture medium)

 

8	8600 µg / mL
7	860 µg / mL
6	86 µg / mL
5	8.6 µg / mL
4	0.86 µg / mL
3	0.086 µg / mL
2	0.0086 µg / mL
1	0.00086 µg / mL

 

Cytotoxicity in range finding test was caused by physical effect of the test substance (low pH value). The highest test concentration (8) was excluded immediately, because total coagulation of blood had occurred. The concentrations from 1 to 7 were included in the range finding test.

 

Range Finding test – 1st test replicate

Number of micronuclei -S9 mix: Without metabolic activation

		Parallel 1		Parallel 2
		M	W	M	W
Substance	Conc. (µg/mL)	Slide1	S.2	S.3	S.4	Average	S.D.	t-value	P-value(%)	SSR
NC	-	4	1	2	5	3,00	1,83	-	-	-
PC	2	11	11	15	17	13,50	3,00	2,99	0,05	*
TSC7	860	2	7	5	4	4,50	2,08	0,54	0,6	N
TSC6	86	3	4	4	2	3,25	0,96	0,12	>	0,6
TSC5	8,6	2	5	3	7	4,25	2,22	0,44	>	0,6
TSC4	0,86	7	7	2	4	5,00	2,45	0,65	0,6	N
TSC3	0,086	6	4	2	6	4,50	1,91	0,57	0,6	N
TSC2	0,0086	4	4	4	5	4,25	0,50	0,66	0,6	N
TSC1	0,00086	3	4	5	4	4,00	0,82	0,50	0,4	N

 

Range Finding test – 1st test replicate

Number of mitoses (MI) -S9 mix: Without metabolic activation

		Parallel 1		Parallel 2
		M	W	M	W
Substance	Conc. (µg/mL)	Slide 1	S.2	S.3	S.4	Average	S.D.	MI(%)
NC	-	42	71	104	38	63,75	30,60	6,4
PC	2	89	68	35	62	63,50	22,25	6,4
TSC7	860	100	106	44	57	76,75	30,87	7,7
TSC6	86	131	108	84	31	88,50	42,87	8,9
TSC5	8,6	108	73	69	50	75,00	24,18	7,5
TSC4	0,86	115	122	65	67	92,25	30,46	9,2
TSC3	0,086	117	59	30	37	60,75	39,48	6,1
TSC2	0,0086	82	56	33	39	52,50	21,95	5,3
TSC1	0,00086	108	121	51	16	74,00	49,19	7,4

 

Dilution factor for the 2nd and 3rd test replicate = 5

From the range - finding test it was concluded that the highest test concentration in both test replicates was 344 µg/mL.

 

3	344 µg/mL
2	68.8 µg/mL
1	3.8 µg/mL

 

Number of micronuclei -S9 mix Without metabolic activation

		2nd test replicate				3rd test replicate (repeated)
		Parallel 1		Parallel 2		Parallel 1		Parallel 2
		M	W	M	W	M	W	M	W
Subst.	Conc. (µg/mL)	Slide 1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	Average	S.D.	t-value	P-value (%)
NC	-	3	5	1	4	1	3	2	2	2,63	1,41
PC	2	19	11	14	22	19	13	17	12	15,88	3,94	3,16	0,01
TSC3	44	2	5	5	1	4	4	3	2	3,25	1,49	0,31	>0,6
TSC2	8,6	6	5	4	1	4	5	2	5	4,00	1,69	0,63	0,6
TSC1	3,8	7	3	4	2	3	6	1	3	3,63	2,00	0,41	>0,6

 

Number of mitoses (MI) -S9 mix Without metabolic activation

		2nd test replicate				3rd test replicate (repeated)
		Parallel 1		Parallel 2		Parallel 1		Parallel 2
		M	W	M	W	M	W	M	W
Subst.	Conc. (µg/mL)	Slide 1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	Average	S.D.	MI(%)
NC	-	88	102	67	108	96	127	78	49	2,63	1,41	8,9
PC	2	65	59	30	70	25	73	49	29	15,88	3,94	5,0
TSC3	344	31	61	51	69	94	79	68	59	3,25	1,49	7,7
TSC2	68,6	22	108	55	99	90	76	64	52	4,00	1,69	8,3
TSC1	13,8	84	57	40	65	79	96	46	52	3,63	2,00	6,5

 

Number of micronuclei +S9 mix With metabolic activation

		2nd test replicate				3rd test replicate (repeated)
		Parallel 1		Parallel 2		Parallel 1		Parallel 2
		M	W	M	W	M	W	M	W
Subst.	Conc. (µg/mL)	Slide 1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	Average	S.D.	t-value	P-value (%)
NC	-	4	3	2	1	3	5	3	2	2,88	1,25
PC	80	14	14	11	12	16	20	12	13	14,00	2,88	3,55	0,01
TSC3	344	4	5	1	3	4	2	4	1	3,00	1,51	0,18	>
TSC2	68,6	4	5	7	6	1	4	1	3	3,88	2,17	0,48	>
TSC1	13,8	6	5	2	4	3	3	1	2	3,25	1,67	0,29	>

 

Number of mitoses (MI) +S9 mix With metabolic activation

		2nd test replicate				3rd test replicate (repeated)
		Parallel 1		Parallel 2		Parallel 1		Parallel 2
		M	W	M	W	M	W	M	W
Subst.	Conc. (µg/mL)	Slide 1	S.2	S.3	S.4	S.5	S.6	S.7	S.8	Average	S.D.	MI(%)
NC	-	34	22	18	20	20	17	30	11	21,50	7,33	2,2
PC	80	19	17	14	16	19	18	12	11	15,75	3,11	1,6
TSC3	344	20	13	15	13	18	15	16	18	16,00	2,51	1,6
TSC2	68,6	27	14	17	22	17	10	10	28	18,13	7,00	1,8
TSC1	13,8	16	17	11	20	13	15	20	14	15,75	3,20	1,6

 

Explanatory notes:

NC -Negative control PC -positive control

MI - mitotic index (number of mitoses) in % * - statistical significant result

** - high statistical significant result *** - very high statistical significant result

S.D. Standard deviation N - statistical not significant result

t - value - calculated t - value from the Student’s test

p - value p-value can be found using a table of values from Student's t-distribution

SSR Statistical significance of result TSC Test substance concentration

M - Man

W - Woman

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance sulphamidic acid analyzed in terms of micronucleus test in vitro did not show mutagenic effects on the target test system.