Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 231-871-7 | CAS number: 7773-06-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- 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
- Target gene:
- Not applicable.
- 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. - 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) - 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).
- 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'.
- 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.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- sodium sulphamidate
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
A read across, based on analogue approach, has been performed between ammonium sulphamidate EC 231-871-7 (target chemical) and sodium sulphamidate EC 237-572-8 (source chemical).
The read-across hypothesis, according to Read Across Assessment Framework published by ECHA, is based on the fact that different compounds which have the same type of effect(s). It corresponds to the scenario 2 described as follows:
« This scenario covers the analogue approach for which the read-across hypothesis is based on different compounds which have the same type of effect(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst case assumption. »
1) Chemical structure
The target and source substances share the same anionic structure, i.e. a sulphamidate (formula: –OSO2NH2). They only differ by the positive counter ion: an ammonium ion (NH4+) for the target substance and a sodium ion (Na+) for the source substance. It is well known that usually, the counter ion has no impact on the toxicity profile of the substance. For this reason, the QSARs are classically performed on the “core” of the salt and do not consider the counter ion.
See the structures in attached justification.
2) Kinetics
Ammonium sulphamidate
The substance is highly water soluble, meaning its ions dissolve in water. Following oral administration of ammonium sulfamate to dogs for 5 days, 80 to 84% of the dose was excreted as sulfamic acid in the urine, indicating that ammonium sulfamate is readily absorbed into the bloodstream from the gastrointestinal tract. (Pesticide Active Ingredient Information – EXTOXNET)
Sodium sulphamidate
Absorption of sodium sulphamidate from the gastrointestinal tract is supported by the repeated dose reproductive screening study in rats. The high water solubility and small molecular size of sodium sulphamidate allow absorption through passive diffusion. This would suggest that the gastro-intestinal tract provides a route of absorption, following oral administration, before entering the circulatory system via the blood.
Absorption of sodium sulphamidate may also take place via the skin due to small molecular size and water solubility. Although the substance is not a skin sensitizer there is evidence of mild dermal irritation. Therefore damage to the skin surface may allow for increased penetration of the substance through the skin.
Once absorbed, the substance would be distributed in the serum due to the water solubility.
The results of the repeated dose reproductive screening study would suggest that the most likely route of excretion is the kidney due to the likely systemic distribution and water solubility of the test item. Any test item that is not absorbed will be excreted in the faeces. [ECHA’s registration dossier of sodium sulphamidate].
Conclusion
Both substances are absorbed via oral route and are found excreted in urine. Inhalation exposure is not relevant due to the low vapour pressure of each substance.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
1) Physical and chemical information
The physico-chemical properties were compared between the target and the source substance. Synthron data are in blue and Nalco data (found in ECHA’s registration dossier of sodium sulphamidate) are in green. Published data are in purple and other data are in black (please refer to the comparative table in attached justification).
Both substances share some common physico-chemical properties: white solid appearance, decomposition, high partition coefficient, negligible vapour pressure, absence of surface activity, good water solubility, absence of flammability/explosive and oxidizing properties.
Some physico-chemical differences can be highlighted between the two substances: the different molecular weight is attributed to the counter-ion. The boiling and melting points are slightly different as well as the relative density. The dissociation constants vary due to the different counter-ions which cannot be used for the read-across proposal. In this case, the sulphamidic acid is the most appropriate substance. The pKa values around 1.0 (0.9 or 0.997 or 1.05 as found in the "Handbook of Chemistry and Physics", 85th ed.) all refer to the free acid, sulphamidic acid. Ammonium sulphamate contains as cation the ammonium ion with a pKa of 9.25 ("Handbook of Chemistry and Physics", 85th ed.) Any attempt of coming into the region of pH that is near the pKa of the primary amine group (13.6 ± 0.6) would cause the deprotonation of the ammonium ion and the transformation of the target chemical into the respective alkaline metal salt, for instance sodium sulphamidate. Therefore, the pKa of the primary amine group in the sulphamidate anion reported in the sodium sulphamidate dossier is not relevant for ammonium sulphamidate.
Therefore, both substances share many common physico-chemical features, and the observed differences can be attributed to the different counter-ion.
2) Toxicological and ecotoxicological information
The ammonium ion of the target substance may contribute to the toxicity of ammonium sulphamidate, compared to sodium sulphamidate. However, as both substances are highly water soluble, their ions dissolve in water. Therefore, the ammonium ion is no more a concern.
Please refer to the comparative table in attached justification.
Regarding the toxicity endpoints, some common points are shared by the two substances: low acute toxicity by oral route, no mutagenicity in bacteria, mild to no skin or eye irritation. Some differences occurred in the systemic toxicity study: in the repeated dose toxicity study, the NOAEL are not the same between sodium (NOAEL = 1000 mg/kg bw/d) and ammonium sulphamidate (NOEL = 214.3 mg/kg bw/d). In the reproductive study, NOEL for ammonium sulphamidate was found to be 25 mg/kg bw/d in the literature whereas the NOAEL for sodium sulphamidate is 1000 mg/kg bw/d. However, these differences must be considered with caution as the experimental protocols differ.
As for the ecotoxicity endpoints, both substances seem not to be toxic to fish, based on their LC50 > 100 mg/L (LC50 of at least 650 mg/L).
Last, the environmental fate data on both substances indicate that they are likely to be adsorbed into the soil. Their half-life differ as sodium sulphamidate is very stable (half-life > 1 year) and ammonium sulphamidate may be less stable (half-life of 14 days, based on a published data).
3) Classification proposal
The sodium sulphamidate is not classified in ECHA’s registration dossier. Based on the read-across approach, ammonium sulphamidate would not be classified either.
3. ANALOGUE APPROACH JUSTIFICATION
Based on the available elements, it can be assumed that ammonium and sodium sulphamidate may have close kinetic profiles, physico-chemical, toxicological and ecotoxicological properties. The read-across approach is therefore relevant. - Reason / purpose for cross-reference:
- read-across source
- 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
- Remarks on result:
- other: strain/cell type: human lymphocytes
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Based on the read-across on sodium sulphamidate, ammonium sulphamidate is not expected to induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, ammonium sulphamidate is expected to be non-clastogenic in this chromosome aberration test. - Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental phase of this study was performed between 09 June 2011 and 11 August 2011.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine for Salmonella.
Tryptophan for E.coli - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one (Range-finding Test): 50, 150, 500, 1500 and 5000 µg/plate
Experiment two (Main Test): 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house and was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA): 1 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA): 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA): 2 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene (2AA): 10 µg/plate
- Remarks:
- With S9 mix
- Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With S9 mix
Migrated to IUCLID6: (BP): 5 µg/plate - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA98
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix
Migrated to IUCLID6: (4NQO): 0.2 µg/plate - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1537
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix
Migrated to IUCLID6: (9AA): 80 µg/plate - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA100
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
Migrated to IUCLID6: (ENNG): 3 µg/plate - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of TA1535
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix
Migrated to IUCLID6: (ENNG): 5 µg/plate - Untreated negative controls:
- yes
- Remarks:
- Spontaneous mutation rates of WP2uvrA
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile distilled water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix
Migrated to IUCLID6: (ENNG): 2 µg/plate - Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation in Experiment 2.
DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
NUMBER OF REPLICATIONS: Triplicate plating.
DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn. - Evaluation criteria:
- Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the
following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Standard deviation
Statistical analysis of data as determined by UKEMS Dunnett's t test - Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment
were both shown to be sterile.
COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.
The test item was considered to be non-mutagenic under the conditions of this test. - Executive summary:
Introduction.
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods.
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Sodium sulphamate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.
Results.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 500 µg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 500 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.29 times the concurrent vehicle control.
Conclusion.
The test item, Sodium sulphamate, was considered to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- sodium sulphamidate
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
A read across, based on analogue approach, has been performed between ammonium sulphamidate EC 231-871-7 (target chemical) and sodium sulphamidate EC 237-572-8 (source chemical).
The read-across hypothesis, according to Read Across Assessment Framework published by ECHA, is based on the fact that different compounds which have the same type of effect(s). It corresponds to the scenario 2 described as follows:
« This scenario covers the analogue approach for which the read-across hypothesis is based on different compounds which have the same type of effect(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst case assumption. »
1) Chemical structure
The target and source substances share the same anionic structure, i.e. a sulphamidate (formula: –OSO2NH2). They only differ by the positive counter ion: an ammonium ion (NH4+) for the target substance and a sodium ion (Na+) for the source substance. It is well known that usually, the counter ion has no impact on the toxicity profile of the substance. For this reason, the QSARs are classically performed on the “core” of the salt and do not consider the counter ion.
See the structures in attached justification.
2) Kinetics
Ammonium sulphamidate
The substance is highly water soluble, meaning its ions dissolve in water. Following oral administration of ammonium sulfamate to dogs for 5 days, 80 to 84% of the dose was excreted as sulfamic acid in the urine, indicating that ammonium sulfamate is readily absorbed into the bloodstream from the gastrointestinal tract. (Pesticide Active Ingredient Information – EXTOXNET)
Sodium sulphamidate
Absorption of sodium sulphamidate from the gastrointestinal tract is supported by the repeated dose reproductive screening study in rats. The high water solubility and small molecular size of sodium sulphamidate allow absorption through passive diffusion. This would suggest that the gastro-intestinal tract provides a route of absorption, following oral administration, before entering the circulatory system via the blood.
Absorption of sodium sulphamidate may also take place via the skin due to small molecular size and water solubility. Although the substance is not a skin sensitizer there is evidence of mild dermal irritation. Therefore damage to the skin surface may allow for increased penetration of the substance through the skin.
Once absorbed, the substance would be distributed in the serum due to the water solubility.
The results of the repeated dose reproductive screening study would suggest that the most likely route of excretion is the kidney due to the likely systemic distribution and water solubility of the test item. Any test item that is not absorbed will be excreted in the faeces. [ECHA’s registration dossier of sodium sulphamidate].
Conclusion
Both substances are absorbed via oral route and are found excreted in urine. Inhalation exposure is not relevant due to the low vapour pressure of each substance.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
1) Physical and chemical information
The physico-chemical properties were compared between the target and the source substance. Synthron data are in blue and Nalco data (found in ECHA’s registration dossier of sodium sulphamidate) are in green. Published data are in purple and other data are in black (please refer to the comparative table in attached justification).
Both substances share some common physico-chemical properties: white solid appearance, decomposition, high partition coefficient, negligible vapour pressure, absence of surface activity, good water solubility, absence of flammability/explosive and oxidizing properties.
Some physico-chemical differences can be highlighted between the two substances: the different molecular weight is attributed to the counter-ion. The boiling and melting points are slightly different as well as the relative density. The dissociation constants vary due to the different counter-ions which cannot be used for the read-across proposal. In this case, the sulphamidic acid is the most appropriate substance. The pKa values around 1.0 (0.9 or 0.997 or 1.05 as found in the "Handbook of Chemistry and Physics", 85th ed.) all refer to the free acid, sulphamidic acid. Ammonium sulphamate contains as cation the ammonium ion with a pKa of 9.25 ("Handbook of Chemistry and Physics", 85th ed.) Any attempt of coming into the region of pH that is near the pKa of the primary amine group (13.6 ± 0.6) would cause the deprotonation of the ammonium ion and the transformation of the target chemical into the respective alkaline metal salt, for instance sodium sulphamidate. Therefore, the pKa of the primary amine group in the sulphamidate anion reported in the sodium sulphamidate dossier is not relevant for ammonium sulphamidate.
Therefore, both substances share many common physico-chemical features, and the observed differences can be attributed to the different counter-ion.
2) Toxicological and ecotoxicological information
The ammonium ion of the target substance may contribute to the toxicity of ammonium sulphamidate, compared to sodium sulphamidate. However, as both substances are highly water soluble, their ions dissolve in water. Therefore, the ammonium ion is no more a concern.
Please refer to the comparative table in attached justification.
Regarding the toxicity endpoints, some common points are shared by the two substances: low acute toxicity by oral route, no mutagenicity in bacteria, mild to no skin or eye irritation. Some differences occurred in the systemic toxicity study: in the repeated dose toxicity study, the NOAEL are not the same between sodium (NOAEL = 1000 mg/kg bw/d) and ammonium sulphamidate (NOEL = 214.3 mg/kg bw/d). In the reproductive study, NOEL for ammonium sulphamidate was found to be 25 mg/kg bw/d in the literature whereas the NOAEL for sodium sulphamidate is 1000 mg/kg bw/d. However, these differences must be considered with caution as the experimental protocols differ.
As for the ecotoxicity endpoints, both substances seem not to be toxic to fish, based on their LC50 > 100 mg/L (LC50 of at least 650 mg/L).
Last, the environmental fate data on both substances indicate that they are likely to be adsorbed into the soil. Their half-life differ as sodium sulphamidate is very stable (half-life > 1 year) and ammonium sulphamidate may be less stable (half-life of 14 days, based on a published data).
3) Classification proposal
The sodium sulphamidate is not classified in ECHA’s registration dossier. Based on the read-across approach, ammonium sulphamidate would not be classified either.
3. ANALOGUE APPROACH JUSTIFICATION
Based on the available elements, it can be assumed that ammonium and sodium sulphamidate may have close kinetic profiles, physico-chemical, toxicological and ecotoxicological properties. The read-across approach is therefore relevant. - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Tested up to maximum recommended dose of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Based on the read-across on sodium sulphamidate, ammonium sulphamidate is expected to be non-mutagenic in bacteria.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was performed between 05 July 2011 and 18 October 2011.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster
ovary (CHO) cells. - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Cell Line :
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.
Cell Culture:
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.
Cell Cleansing:
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/betanaphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- The maximum recommended dose was the 10mM concentration of 1200 µg/ml.
A dose range of 4.69 to 1200 µg/ml was used in the preliminary cytotoxicity test.
Mutagenicity Test - Experiment 1
The dose levels of the controls and the test item are given below:
Group Final concentration of test item (µg/ml)
4-hour without S9 0*, 37.5*, 75*,150*, 300*, 600*, 1200*, EMS 500* and 750*
4-hour with S9 (2%) 0*, 37.5*, 75*,150*, 300*, 600*, 1200*, DMBA 0.5* and 1*
Mutagenicity Test - Experiment 2
The dose levels of the controls and the test item are given below:
Group Final concentration of test item (µg/ml)
24-hour without S9 0*, 37.5*, 75*,150*, 300*, 600*, 1200*, EMS 200* and 300*
4-hour with S9 (1%) 0*, 37.5*, 75*,150*, 300*, 600*, 1200*, DMBA 0.5* and 1*
* = Dose levels plated for mutant frequency
EMS = Ethylmethanesulphonate
DMBA = Dimethyl benzanthracene - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Hams F12 cell culture media
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent at the required dose levels suitable for dosing. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hams F12
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Dimethyl benzanthracene (DMBA)
- Remarks:
- With metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hams F12
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation
Migrated to IUCLID6: (EMS) - Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.
DURATION
- Exposure duration: 4 hours (with and without S9), 24 hours (without S9)
- Expression time (cells in growth medium): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: Duplicate cultures
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
OTHER EXAMINATIONS:
Mutant colonies were manually counted and recorded for each flask.
ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic
activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal
survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice
the solubility limit of the test article in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at
least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for
contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.- Evaluation criteria:
- ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the criteria documented in the previous section (Test sytem and conditions) are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria. - Statistics:
- Not performed
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the
conditions of this test.
See any other infoirmation on results incl. tables section for full details. - Remarks on result:
- other: strain/cell type: CHO
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative Non-mutagenic
The test material did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test. - Executive summary:
Introduction.
The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6TG) as the selective agent.
Methods.
Chinese hamster ovary (CHO) cells were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.
The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-
Exposure Group
Final concentration of test item(µg/ml)
4-hour without S9
37.5, 75, 150, 300, 600, 1200
4-hour with S9 (2%)
37.5, 75, 150, 300, 600, 1200
24-hour without S9
37.5, 75, 150, 300, 600, 1200
4-hour with S9 (1%)
37.5, 75, 150, 300, 600, 1200
Results.
The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.
The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.
Conclusion.
The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Remarks:
- sodium sulphamidate
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
A read across, based on analogue approach, has been performed between ammonium sulphamidate EC 231-871-7 (target chemical) and sodium sulphamidate EC 237-572-8 (source chemical).
The read-across hypothesis, according to Read Across Assessment Framework published by ECHA, is based on the fact that different compounds which have the same type of effect(s). It corresponds to the scenario 2 described as follows:
« This scenario covers the analogue approach for which the read-across hypothesis is based on different compounds which have the same type of effect(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst case assumption. »
1) Chemical structure
The target and source substances share the same anionic structure, i.e. a sulphamidate (formula: –OSO2NH2). They only differ by the positive counter ion: an ammonium ion (NH4+) for the target substance and a sodium ion (Na+) for the source substance. It is well known that usually, the counter ion has no impact on the toxicity profile of the substance. For this reason, the QSARs are classically performed on the “core” of the salt and do not consider the counter ion.
See the structures in attached justification.
2) Kinetics
Ammonium sulphamidate
The substance is highly water soluble, meaning its ions dissolve in water. Following oral administration of ammonium sulfamate to dogs for 5 days, 80 to 84% of the dose was excreted as sulfamic acid in the urine, indicating that ammonium sulfamate is readily absorbed into the bloodstream from the gastrointestinal tract. (Pesticide Active Ingredient Information – EXTOXNET)
Sodium sulphamidate
Absorption of sodium sulphamidate from the gastrointestinal tract is supported by the repeated dose reproductive screening study in rats. The high water solubility and small molecular size of sodium sulphamidate allow absorption through passive diffusion. This would suggest that the gastro-intestinal tract provides a route of absorption, following oral administration, before entering the circulatory system via the blood.
Absorption of sodium sulphamidate may also take place via the skin due to small molecular size and water solubility. Although the substance is not a skin sensitizer there is evidence of mild dermal irritation. Therefore damage to the skin surface may allow for increased penetration of the substance through the skin.
Once absorbed, the substance would be distributed in the serum due to the water solubility.
The results of the repeated dose reproductive screening study would suggest that the most likely route of excretion is the kidney due to the likely systemic distribution and water solubility of the test item. Any test item that is not absorbed will be excreted in the faeces. [ECHA’s registration dossier of sodium sulphamidate].
Conclusion
Both substances are absorbed via oral route and are found excreted in urine. Inhalation exposure is not relevant due to the low vapour pressure of each substance.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
1) Physical and chemical information
The physico-chemical properties were compared between the target and the source substance. Synthron data are in blue and Nalco data (found in ECHA’s registration dossier of sodium sulphamidate) are in green. Published data are in purple and other data are in black (please refer to the comparative table in attached justification).
Both substances share some common physico-chemical properties: white solid appearance, decomposition, high partition coefficient, negligible vapour pressure, absence of surface activity, good water solubility, absence of flammability/explosive and oxidizing properties.
Some physico-chemical differences can be highlighted between the two substances: the different molecular weight is attributed to the counter-ion. The boiling and melting points are slightly different as well as the relative density. The dissociation constants vary due to the different counter-ions which cannot be used for the read-across proposal. In this case, the sulphamidic acid is the most appropriate substance. The pKa values around 1.0 (0.9 or 0.997 or 1.05 as found in the "Handbook of Chemistry and Physics", 85th ed.) all refer to the free acid, sulphamidic acid. Ammonium sulphamate contains as cation the ammonium ion with a pKa of 9.25 ("Handbook of Chemistry and Physics", 85th ed.) Any attempt of coming into the region of pH that is near the pKa of the primary amine group (13.6 ± 0.6) would cause the deprotonation of the ammonium ion and the transformation of the target chemical into the respective alkaline metal salt, for instance sodium sulphamidate. Therefore, the pKa of the primary amine group in the sulphamidate anion reported in the sodium sulphamidate dossier is not relevant for ammonium sulphamidate.
Therefore, both substances share many common physico-chemical features, and the observed differences can be attributed to the different counter-ion.
2) Toxicological and ecotoxicological information
The ammonium ion of the target substance may contribute to the toxicity of ammonium sulphamidate, compared to sodium sulphamidate. However, as both substances are highly water soluble, their ions dissolve in water. Therefore, the ammonium ion is no more a concern.
Please refer to the comparative table in attached justification.
Regarding the toxicity endpoints, some common points are shared by the two substances: low acute toxicity by oral route, no mutagenicity in bacteria, mild to no skin or eye irritation. Some differences occurred in the systemic toxicity study: in the repeated dose toxicity study, the NOAEL are not the same between sodium (NOAEL = 1000 mg/kg bw/d) and ammonium sulphamidate (NOEL = 214.3 mg/kg bw/d). In the reproductive study, NOEL for ammonium sulphamidate was found to be 25 mg/kg bw/d in the literature whereas the NOAEL for sodium sulphamidate is 1000 mg/kg bw/d. However, these differences must be considered with caution as the experimental protocols differ.
As for the ecotoxicity endpoints, both substances seem not to be toxic to fish, based on their LC50 > 100 mg/L (LC50 of at least 650 mg/L).
Last, the environmental fate data on both substances indicate that they are likely to be adsorbed into the soil. Their half-life differ as sodium sulphamidate is very stable (half-life > 1 year) and ammonium sulphamidate may be less stable (half-life of 14 days, based on a published data).
3) Classification proposal
The sodium sulphamidate is not classified in ECHA’s registration dossier. Based on the read-across approach, ammonium sulphamidate would not be classified either.
3. ANALOGUE APPROACH JUSTIFICATION
Based on the available elements, it can be assumed that ammonium and sodium sulphamidate may have close kinetic profiles, physico-chemical, toxicological and ecotoxicological properties. The read-across approach is therefore relevant. - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- non-mutagenic
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: strain/cell type: CHO
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Based on the read-across on sodium sulphamidate, ammonium sulphamidate is expected to be non-mutagenic to CHO cells at the HPRT locus.
Referenceopen allclose all
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 control1 |
100.0 |
0.5 |
0.0 |
0.0 |
|
|||
|
|
Positive control2 |
75.0 |
8.5 |
8.0S |
0.5 |
|
|||
|
|
391.8 |
117.6 |
2.0 |
2.0S |
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 control1 |
100.0 |
1.5 |
1.5 |
0.0 |
|
|||
|
|
Positive control3 |
42.2 |
14.5 |
14.0S |
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 control1 |
100.0 |
0.5 |
0.0 |
0.0 |
|
|||
|
|
Positive control4 |
58.5 |
17.0 |
16.5S |
2.5 |
|
|||
|
|
391.8 |
86.5 |
1.0 |
1.0 |
0.0 |
|
|||
|
|
685.7 |
89.8 |
2.5 |
2.0S |
0.0 |
|
|||
|
|
1200.0 |
95.3 |
0.0 |
0.0 |
0.0 |
|
|||
II |
22 hrs |
Solvent control1 |
100.0 |
0.0 |
0.0 |
0.0 |
|
|||
|
|
Positive control5 |
103.9 |
9.5 |
9.5S |
1.0 |
|
|||
|
|
391.8 |
106.5 |
2.0 |
2.0S |
0.0 |
|
|||
|
|
685.7 |
115.0 |
2.5 |
2.0S |
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
Results
Preliminary ToxicityTest
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
The numbers of revertant colonies for the toxicity assay were:
With (+) or without (-) S9-mix |
Strain |
Dose (µg/plate) |
||||||||||
0 |
0.15 |
0.5 |
1.5 |
5 |
15 |
50 |
150 |
500 |
1500 |
5000 |
||
- |
TA100 |
96 |
107 |
92 |
112 |
91 |
107 |
105 |
100 |
110 |
132 |
115 |
+ |
TA100 |
79 |
69 |
86 |
89 |
92 |
83 |
87 |
94 |
94 |
65 |
76 |
- |
WP2uvrA |
35 |
33 |
34 |
39 |
34 |
35 |
51 |
43 |
37 |
46 |
53 |
+ |
WP2uvrA |
37 |
38 |
45 |
41 |
40 |
44 |
44 |
39 |
43 |
42 |
38 |
MutationTest
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4 (see attached background material for Tables and Figures)
A history profile of untreated/vehicle and positive control (reference items) is presented in attached background material (historical profiles).
The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 500 µg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 500 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.29 times the concurrent vehicle control.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Table1 Spontaneous Mutation Rates (Concurrent Negative Controls)
Range-finding Test
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
107 |
|
30 |
|
34 |
|
11 |
|
12 |
|
130 |
(110) |
21 |
(22) |
35 |
(30) |
16 |
(15) |
8 |
(8) |
94 |
|
14 |
|
21 |
|
17 |
|
5 |
|
Main Test
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||
81 |
|
26 |
|
22 |
|
26 |
|
7 |
|
93 |
(87) |
21 |
(24) |
24 |
(26) |
10 |
(18) |
6 |
(7) |
88 |
|
24 |
|
33 |
|
16 |
|
8 |
|
Preliminary Cytotoxicity Test
A dose range of 4.69 to 1200 µg/ml was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in Table 1 (see attached background material). It can be seen that there was a modest reduction in the cloning efficiency (CE) at the maximum dose tested in all three exposure groups. Therefore the maximum dose level selected for both Experiment 1 and 2 was the 10 mM dose level of 1200 µg/ml.
Mutagenicity Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group |
Final concentration of test item (µg/ml) |
4-hour without S9 |
0, 37.5, 75, 150, 300, 600, 1200, EMS 500 and 750 |
4-hour with S9 (2%) |
0, 37.5, 75, 150, 300, 600, 1200, DMBA 0.5 and 1 |
No precipitate of the test item was seen at the end of exposure in either exposure group.
The Day 0 and Day 7 cloning efficiencies are presented in Table 2 and Table 3 (see attached background material). There was no reduction in the Day 0 and Day 7 cloning efficiency in the absence or presence of S9 and it can be seen that there was no marked toxicity with the test item when compared to the vehicle controls. The Day 0 and the Day 7 cloning efficiencies did not achieve 70% but were considered to be acceptable as they did achieve at least 50% and there was no evidence of a positive response. An error was made during the plating of the Day 7 viability plates of the 4-hour exposure group in the presence of S9 which resulted in the 'B' replicates at 150 and 300 µg/ml being lost. The mean percentage control values for Day 7 and the mutant frequency per survivor values for these dose levels were therefore calculated from the 'A' replicate data only. This was considered to be acceptable as there was no indication of increases in mutation frequency at any dose level and this was also confirmed by the results of Experiment 2.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3 (see attached background material). There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6with or without the presence of S9.
Mutagenicity Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group |
Final concentration of test item (µg/ml) |
24-hour without S9 |
0, 37.5, 75, 150, 300, 600, 1200, EMS 200 and 300 |
4-hour with S9 (1%) |
0, 37.5, 75, 150, 300, 600, 1200, DMBA 0.5 and 1 |
No precipitate of the test item was seen at the end of exposure in either exposure group.
The Day 0 and Day 7 cloning efficiencies are presented in Tables 4 and 5 (see attached background material). It can be seen that, as in Experiment 1, there was no reduction in the Day 0 and Day 7 cloning efficiency in the absence or presence of S9 and no toxicity of the test item was observed when compared to the vehicle controls. The Day 0 counts for the 24-hour exposure group did not achieve 70% but were considered to be acceptable as they did achieve the 50% minimum required.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 4 and 5 (see attached background material). There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6with or without the presence of S9.
It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6mutants per viable cell in both Experiment 1 and Experiment 2, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.
Additional information
Ames Test:
No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 500 µg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 500 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.29 times the concurrent vehicle control.
The test item, Sodium sulphamate, was considered to be non-mutagenic under the conditions of this test.
Chromosome Aberration Test in Human Lymphocytes:
Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
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.
CHO HPRT Forward Mutation Assay:
The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.
The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.
Short
description of key information:
Three
in-vitro studies were conducted on the substance:
-
Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli
-
Chromosome aberration test in human lymphocytes
-
CHO HPRT forward mutation assay.
All
3 studies gave negative results.
Endpoint
Conclusion: No
adverse effect observed (negative)
Based on the read-across on sodium sulphamidate, ammonium sulphamidate is expected to be non mutagenic in bacteria or CHO cells, nor clastogenic in human lymphocytes.
Justification for classification or non-classification
Based on negative results in the three following in-vitro studies, in substance is not classified for mutagencity.
- Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli:
Sodium sulphamate, was considered to be non-mutagenic under the conditions of this test.
- Chromosome aberration test in human lymphocytes:
Sodium Sulphamate is considered to be non-clastogenic in this chromosome aberration test, when tested up to the highest required concentration.
- CHO HPRT forward mutation assay:
Sodium sulphamidate was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.
Based on the read-across on sodium sulphamidate, ammonium sulphamidate is not expected to be classified for Genotoxicity according to GHS/CLP criteria.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
