Ammonium sulphamidate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: June 09, 2011. Experimental Completion Date: July 14, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphamate

IUPAC Name:

Sodium sulphamate

Details on test material:

- Name of test material (as cited in study report): Sodium Sulphamate
- Batch Number: LE12568
- CAS-No.: 13845-18-6
- Identifier: TIS I0442
- Expiration date of the lot/batch: May 06, 2013
- Stability under test conditions: Stable in water
- Storage condition of test material: At room temperature, light protected, moisture protected
- Description: White solid

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 7.8, 13.6, 23.9, 41.8, 73.1, 127.9, 223.9, 391.8*, 685.7*, 1200.0* µg/mL
Experiment II: 127.9, 223.9, 391.8*, 685.7*, 1200.0* µg/mL

Without metabolic activation:
Experiment I: 7.8, 13.6, 23.9, 41.8, 73.1, 127.9, 223.9, 391.8*, 685.7*, 1200.0* µg/mL
Experiment II: 23.9, 41.8, 73.1, 127.9, 223.9, 391.8*, 685.7*, 1200.0* µg/mL

* Evaluated experimental points

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used without metabolic activation.

Migrated to IUCLID6: (EMS)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used with metabolic activation.

Migrated to IUCLID6: (CPA)

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: In each experimental concentration two parallel cultures were analysed.


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Analysis of Metaphase Cells - Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

Evaluation of Results:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
The assay can indicate an aneugenic potential of the test item if:
- the number of induced numerical aberrations is not in the range of the historical control data.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results

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:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Sodium Sulphamate, dissolved in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item.

In each experimental concentration two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.

The highest treatment concentration in this study, 1200.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

No precipitation of the test item in the culture medium was observed at any concentration. No relevant influence on osmolarity or pH value was observed (Exp. I: solvent control: 277 mOsm, pH 7.4 versus 304 mOsm and pH 7.4 at 1200.0 µg/mL; Exp. II: solvent control: 277 mOsm, pH 7.3 versus 298 mOsm and pH 7.3 at 1200.0 µg/mL).

In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices was observed.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. Statistically significant increases were observed in Experiment I after treatment with 391.8 µg/mL (2.0 % aberrant cells, excluding gaps) in the absence of S9 mix and with 685.7 µg/mL (2.0 % aberrant cells, excluding gaps) in the presence of S9 mix. In Experiment II in the presence of S9 mix, statistically significant increases were observed after treatment with 391.8 and 685.7 µg/mL (2.0 % aberrant cells, excluding gaps). These values were in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and are therefore considered as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

In both experiments, either EMS (660 or 825 µg/mL) or CPA (2.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item Sodium Sulphamate did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to the highest required concentration.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosomal aberration study with Sodium Sulphamate

Exp.		Preparation		Test item	Mitotic indices	Aberrant cells
		interval		concentration	in %	in %
				in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
I		22 hrs		Solvent control^1	100.0	0.5	0.0	0.0
				Positive control^2	75.0	8.5	8.0^S	0.5
				391.8	117.6	2.0	2.0^S	0.0
				685.7	111.4	1.5	1.0	0.0
				1200.0	113.3	1.0	1.0	0.0
			Exposure period 22 hrs without S9 mix
II		22 hrs		Solvent control^1	100.0	1.5	1.5	0.0
				Positive control^3	42.2	14.5	14.0^S	1.5
				391.8	90.3	0.0	0.0	0.0
				685.7	100.8	2.0	1.5	0.0
				1200.0	84.8	3.0	2.5	0.0
			Exposure period 4 hrs with S9 mix
I		22 hrs		Solvent control^1	100.0	0.5	0.0	0.0
				Positive control^4	58.5	17.0	16.5^S	2.5
				391.8	86.5	1.0	1.0	0.0
				685.7	89.8	2.5	2.0^S	0.0
				1200.0	95.3	0.0	0.0	0.0
II		22 hrs		Solvent control^1	100.0	0.0	0.0	0.0
				Positive control^5	103.9	9.5	9.5^S	1.0
				391.8	106.5	2.0	2.0^S	0.0
				685.7	115.0	2.5	2.0^S	0.0
				1200.0	107.8	0.5	0.5	0.0

*  Including cells carrying exchanges

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water 10.0 % (v/v)

²     EMS     825.0 µg/mL

³       660.0 µg/mL

⁴   CPA       15.0 µg/mL

⁵   CPA         2.5 µg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Sodium Sulphamate is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.

Executive summary:

Summary

The test item Sodium Sulphamate, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental concentration two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (1200.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473.

In both experiments neither in the absence nor in the presence of S9 mix cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, statistically significant increases were observed in Experiment I after treatment with 391.8 µg/mL (2.0 % aberrant cells, excluding gaps) in the absence of S9 mix and with 685.7 µg/mL (2.0 % aberrant cells, excluding gaps) in the presence of S9 mix. In Experiment II in the presence of S9 mix, statistically significant increases were observed after treatment with 391.8 and 685.7 µg/mL (2.0 % aberrant cells, excluding gaps). These values were in the range of the laboratory’s historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and are therefore considered as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, Sodium Sulphamate is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.