Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471): negative

Gene mutation study in mammalian cells (OECD TG 476): negative

Genetic tox in vitro DNA damage and/or repair study : negative

Chromosome abberation in mammalian cells (OECD TG 474): negative

Link to relevant study records

Referenceopen allclose all

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:
26 June 2000 - 10 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed under GLP and according to standard protocol.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Cited as Directive 2000/32/EC, B.13/14
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine synthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene) -
The Salmonella typhimurium strains were regularly checked to confirm their histidinerequirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Range-finding : 333, 1000, 3330, 5000 µg/plate.

Experiment 1:
without S-9 mix:3, 10, 33, 100, 200, 333, 1000 and 3330 µg/plate
with S-9 mix: 3, 10, 33, 100, 333, 666, 1000, 3330, 5000 µg/plate

Experiment 2:
without S-9 mix: 10, 50, 100, 150 and 200 µg/plate
with S-9 mix: 100, 200, 300, 400 and 500 µg/plate
Vehicle / solvent:
Milli-Q water
Remarks:
see below "any other information on tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method:other: growth inhibition. Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Halamid pharma grade used in the subsequent mutation assay was the level at which the test substance inhibited bacterial growth.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a. The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b. The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c. The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Statistics:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
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:
other: All tester strains at 333 ug/plate and higher
Vehicle controls validity:
not valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
All tester strains at 333 ug/plate and higher
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no effects
- Precipitation: no precipitation
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
To determine the toxicity of Halamid pharma grade, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. At and above 333 µg/plate reduction of the bacterial background lawn and the number of colonies was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: comparable

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Conclusions:
Interpretation of results: negative
Based on the results of this study it is concluded that Halamid pharma grade is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Executive summary:

Halamid pharma grade was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. In the dose range finding test, Halamid pharma grade was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. Halamid pharma grade did not precipitate on the plates. In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence and presence of S9 - mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence of S9 -mix and at dose levels of 1000 µg/plate and upwards in the presence of S9 -mix. In the first mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 666 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In the second mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 500 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains Halamid pharma grade did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that Halamid pharma grade is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Study was performed pre-GLP according to Ames et al. (1975). Duplicate plates were used and no independent repeat was performed. Only average results are shown.
Principles of method if other than guideline:
Method: other: Ames et al ,Mut Res. 31 (1975), pp. 347-364
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine synthesis gene
Species / strain / cell type:
other: Method according to Ames et al ,Mut Res. 31 (1975), pp. 347-364
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0.2 , 2, 20, 200 and 1000 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
IUCLID4 Type: Ames test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 1000 µg/plate
Remarks on result:
other: other: Method according to Ames et al ,Mut Res. 31 (1975), pp. 347-364
Remarks:
Migrated from field 'Test system'.

GENOTOXIC EFFECTS: 
- With metabolic activation: No adverse effects measured
- Without metabolic activation: No adverse effects measured
FREQUENCY OF EFFECTS: -
PRECIPITATION CONCENTRATION: not described
MITOTIC INDEX: not determined
CYTOTOXIC CONCENTRATION: at concentrations of 100 ug/plate 
for both with and without S9 activation
TEST-SPECIFIC CONFOUNDING FACTORS: -
STATISTICAL RESULTS: not described

Conclusions:
Interpretation of results: negative

The substance Tosylchloramide sodium, trihydrate did not display a significantly increased number of his+ revertant colonies in any of the five strains, with or without addition of S9 mix, at any concentration, and thus displays no mutagenic action.
Executive summary:

Tosylchloramide sodium, trihydrate was provided by Veip bv for testing in respect of mutagenicity. The test was to be carried out in an Ames in-vitro bacteria test. The strains of Salmonella typhimurium developed and described by B. Ames TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were used for the in-vitro test of mutagenicity according to: B.N. Ames et al., Mut. Res. 3 1 (1975) 347-64. The substance Halamid did not display a significantly increased number of his+ revertant colonies in any of the five strains, with or without addition of S9 mix, at any concentration, and thus displays no mutagenic action.

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:
07 April 1998 - 25 May 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed under GLP and according to standard guideline.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
F10 complete culture medium: F10 complete culture medium consisted o f Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and penicillinlstreptomycin (50 U/ml and 50 µg/ml respectively).
Cell culture conditions: L5178Y mouse lymphoma cells were cultured in F10 complete culture medium. Cell density was preferably kept below 7 x 105 cells/ml .
Exposition medium: Cells were exposed to Halamid for 3 hours in F10 culture medium, buffered with 20 mM HEPES.
Selective medium: Selective medium consisted of F10 complete culture medium, supplemented with 10% ( v/v ) horse serum and 5 µg/ml TFT (Sigma).
Non-selective medium: Non-selective medium consisted of F10 complete culture medium, supplemented with 10% horse serum.
Environmental conditions: All incubations were carried out in a humid atmosphere (80-95%) containing 5% CO2 in air in the dark at 37°C. The temperature and CO2-percentage were monitored during the experiment.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, before study
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Range finding study, with and without metabolic activation: 0, 33, 100, 333, 1000, 3330, 5000 µg/ml
Experiment 1 without metabolic activation: 0, 6.3, 12.5, 25, 50 µg/ml
Experiment 1 with metabolic activation: 0, 3.4, 6.7, 12.5, 18.8 µg/ml
Experiment 2 without metabolic activation: 0, 20, 30, 40, 50 µg/ml
Experiment 2 with metabolic activation: 0, 10, 15, 50, 25 µg/ml
Vehicle / solvent:
dimethylsulphoxide (DMSO)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulphonate (EMS) without metabolic activation or Dimethylnitrosamine (DMN) with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: none
- Exposure duration: 3 hours
- Expression time (cells in growth medium): To determine the cloning efficiency the cells were cultured in 2x 96 wells plate with 1 cell/well for 9 days. For expression of the mutant phenotype, the remaining cells were cultured for 3 days after the 3 hours exposure time. The cells were cultured at
least every other day in order to maintain log phase growth. During this culture period at least 4 x 10E6 cells (if possible) were subcultured. Three days after the start of the treatment with Halamid the cells were plated for determination of the CE (CE3) and the mutant frequency (MF).
- Selection time (if incubation with a selection agent): The microtiter plates for CE3 and MF were incubated at 37°C in humified air with 5% CO2 for 11 or 12 days.
After this incubation period the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide, Sigma) to each well. The plates for the CE3 and MF were scored with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): TFT-selection

NUMBER OF REPLICATIONS:For determination of the CE3 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 X 96-well microtiter plates/concentration) in non selective medium.
For determination of the MF a total number of 5.76 x 10E5 cells concentration were plated in three 96-well micratiter plates, each well containing 2000 cells in selective medium (TFT-selection).
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls was ≥ 50%.
b) At feast at three of the four doses of the test substance, an acceptable number of surviving cells (lo6) could be analysed for expression of the
TK mutation.
c) The spontaneous mutant frequency in the untreated or solvent control was < 10 per 10E5 clonable cells.
d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant fat least three-fold) increases in the mutant
frequencies.
e) In the absence of S9-mix the selected dose range had to include a clearly toxic concentration.
Statistics:
The calculation of the mutation frequency was determined, as follows: MF = {-In P(0)/2000)/CE3. The mutant frequency was expressed as the number of mutants per 10E5 surviving cells.

No formal hypothesis testing was done.
A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and b) The results were reproducible in an independently repeated test.
A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b ) The results were confirmed in an independently repeated test.
The preceding criteria were not absolute and other modifying factors might
enter into the final evaluation decision.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see remarks below.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Experiment 1
In the absence of S-9mix, after 3 hours treatment the cell count of the highest test substance concentration was reduced by 61% compared to the
cell count of the sofvent control. The cloning efficiency o f the remaining cells directly after treatment showed a reduction of 57% compared to the
solvent control. In the presence of S-9 mix, after 3 hours treatment the cell count of the highest test substance concentration was not reduced even at the highest tested dose. The cloning efficiency of the remaining cells was comparable to the solvent control even at the highest tested dose.

Experiment 2
In the absence of S9-mix, after 3 hours treatment the cell count of the test substance concentration of 50 µg/ml was reduced by 53% compared to
the cell count of the solvent control. The cloning efficiency of the remaining cells after treatment showed a reduction of 77% compared to the solvent
control. In the presence of S-9 mix, after 3 hours treatment the cell count of the highest test substance concentration was not reduced even at the highest tested dose. The cloning efficiency of the remaining cells was comparable to the solvent control even at the highest tested dose.

Evaluation of the mutagenicity
The spontaneous mutant frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range (0.7- and 5.8 x 10E5 (mean 3.0 x 10E5) in the absence of S-9 mix) and (1.0- and 6.6 x 10E5 (mean 2.9 x 10E5) in the presence of S-9 mix); for n=66 and 63 respectively). Mutant frequencies induced by positive control chemicals were increased by 15- and 10-fold for EMS in the first and second experiment respectively, and by 15- and 9-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection o f a mutagenic response and that the metabolic activation system (S-9 mix) functioned properly. In the absence of S9 metabolic activation, Halamid showed a 2.9-fold increase in the mutant frequency at the TK-locus in the first experiment. However, this increase was observed in one experiment only and no dose relationship was observed. Furthermore, the increase was within the historical control data range. Therefore, this increase was considered not to be biologically relevant and Halamid is considered not to be mutagenic in the absence of S-9 mix. In the presence o f S-9 mix, no dose level could be obtained with any toxicity i n the cell numbers or in the cloning efficiency after 3 hours treatment compared to the solvent control in neither experiment. Hence, the range between the dose levels chosen for both experiments was very small. The concentrations 18.8 and 25 µg/ml, in the first and second experiment respectively, in which no toxicity was observed and the concentrations 25 and 30 µg/ml in the first and second experiment respectively, in which total cell death was observed were only separated by a factor of 1.3 or 1.2 in the first and second experiment respectively. Therefore, this borderline toxicity between the dose level with no toxicity and the dose level with total cell death is a test substance related effect and the test is considered to be acceptable. In the presence of 8% (v/v) S9 metabolic activation no significant increase in the mutant frequency at the TK-locus was observed in neither experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1: without S-9 mix enough the dose level of 100 µg/mll was too toxic for further testing. In the presence of S9 metabolic activation, the

dose levels of 25, 37.5 and 50 µg/ml were too toxic for further testing.

Experiment 2: withou S-9 mix 60 µg/ml was too toxic for further testing. With S-9 mix 30 µg/ml was too toxic for further testing.

Conclusions:
Interpretation of results: negative
It is concluded that Tosylchloramide sodium, trihydrate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

This report describes the effects of Tosylchloramide sodium, trihydrate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. Tosylchloramide sodium, trihydrate was tested up to concentrations of 50 µg/ml in the absence o f S9 -mix in two experiments. Tosylchloramide sodium, trihydrate was tested up to concentrations of 18.8 and 25 µg/ml in the presence of 8% (v/v) S9 -fraction, in the first and second experiment respectively. In the absence of S9 metabolic activation, Tosylchloramide sodium, trihydrate showed a 2.9- fold increase in the mutant frequency at the TK-locus in the first experiment. However, this increase was observed in one experiment only and no dose relationship was observed. Furthermore, the increase was within the historical control data range. Therefore, this increase was considered not to be biologically relevant and Tosylchloramide sodium, trihydrate is considered not to be mutagenic in the absence o f S9-mix. In the presence of 8% (v/v) S9 metabolic activation no significant increase in the mutant frequency at the TK- locus was observed in neither experiment. Mutant frequencies induced by positive control chemicals were increased by 15- and 10-fold for EMS in the first and second experiment respectively, and by 15- and 9 -fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9 -mix) functioned properly. It is concluded that Tosylchloramide sodium, trihydrate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 March 1980 - 24 March 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not performed according to a standard guideline or under GLP but inhouse quality assurance was in place. Limited reporting.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method: other: DNA Repair Test With Escherichia coli pol A+(W3110) - and pol A-(p3478)
GLP compliance:
no
Remarks:
inhouse quality assurance was in place
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
other: Escherichia coli pol A+ (W3110) and pol A- (p3478)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0.5 - 5000 µg/plate
Details on test system and experimental conditions:
DNA damage and repair assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 500 µg/plate and higher
Remarks on result:
other: other: Escherichia coli pol A+ (W3110) and pol A- (p3478)
Remarks:
Migrated from field 'Test system'.

GENOTOXIC EFFECTS: 
- With metabolic activation: No adverse effects measured
- Without metabolic activation: No adverse effects measured


FREQUENCY OF EFFECTS: -


PRECIPITATION CONCENTRATION: no precipitation observed at
test concentrations


MITOTIC INDEX: not determined


CYTOTOXIC CONCENTRATION: at concentrations of 500 ug/plate
and higher for both with and without S9 activation


TEST-SPECIFIC CONFOUNDING FACTORS: -


STATISTICAL RESULTS: not described

Conclusions:
Interpretation of results: negative

The test compound Tosylchloramide sodium, trihydrate did not exhibit a DNA-modifying effect in any of the assays conducted in this evaluation and is considered as not genotoxic under these test conditions.
Executive summary:

Under the conditions of a DNA Repair Test With Escherichia coli pol A+(W3110) - and pol A-(p3478) the test compound Tosylchloramide sodium, trihydrate did not exhibit a DNA-modifying effect in any of the assays conducted in this evaluation and is considered as not genotoxic under these test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian Erythrocyte Micronucleus Test (OECD TG 474): negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 1991 - 26 July 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Study performed accoring to method comparable to OECD 474 and under GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Method: other: TSCA 40 CFR 792, ASTM standard No. E 1263-88
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc, USA
- Age at study initiation: birth date 19 Marcg 1991
- Weight at study initiation: males 20.0-26.0 grams, females 18.0-23.0 grams
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: mice were housed 10 to a cage during acclimatisation, 3 to a cage during the range finding and 5 to a cage during the main study. polycarbonate cages with hardwood-chup bedding were used throughout the study.
- Diet (e.g. ad libitum): ad libitum, purina certified rodent chow
- Water (e.g. ad libitum): ad libitum, UV purified deionized tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-24.4
- Humidity (%): 26-75
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 23 April 1991 - 26 July 1991
Route of administration:
oral: gavage
Vehicle:
Name: Sterile deionized water.
Purity: Water analysis is on file in Building 205, Room 39.
Autoclave date: May 6, 1991.
Expiration date: May 6, 1992.
Supplier: SRI International, 333 Ravenswood Avenue Menlo Park, CA 94025.
Storage conditions: Stored at room temperature (about 20.6 ºC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was mixed well in glass vials with sterile deionized water immediatly before dose administration. The volume of the test solution administered was 10 ml/kg body weight.
Duration of treatment / exposure:
On 2 consecutive days the mice were treated and half of the animals was sacrificed approximately 24 hours and the other half 48 hours after the final dose.
Frequency of treatment:
Once on two consecutive days.
Post exposure period:
24 or 48 hours
Remarks:
Doses / Concentrations:
Range finding study: 300, 600, 1200 2500 and 5000 mg/kg body weight.Definitive test : 300, 600 and 1200 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
Range finding study: 3
Definitive test: 10
Control animals:
yes, concurrent vehicle
Positive control(s):
Urethane
- Justification for choice of positive control(s): known clastogenic agent
- Route of administration: oral intubation
- Doses / concentrations: 300 mg/kg day
Tissues and cell types examined:
Polychromatic erythrocytes (PCEs) and red blood cells (RBCs) from peripheral blood smears and also from bonemarrow smears.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study

DETAILS OF SLIDE PREPARATION:
Peripheral blood:
Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 µl of blood into a capillary tube. The sample was transferred to 3 clean, prelabeled microscope slides per mouse, spread, air-dried, fixed in absolute methanol for 5 minutes and stored until staining. Two of the 3 prepared slides were coded. Both coded slides from each test animal were visually examined, and the slide with the most uniform preparation was stained with acridine orange. unstained slides were filed for future use in case extras were needed.

Bone marrow:
Bone marrow smears were analyzed in both the range finding and definitive assays. One femur from each mouse was removed and flushed gently with 0.2 ml of fetal bovine serum (FBS) into 0.5 ml of FBS in a 1.5 ml polycarbonate tube. Cells were concentrated by centrifugation, then resuspended in a volume of supernatant, approximately equal to the pellet volumes. The sample was transferred to 3 clean, prelabeled microscope slides per animal, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Two of the 3 prepared slides were coded. Both coded slides from each test animal were visually examined, and the slide with the most uniform preparation was stained with acridine orange. Unstained slideswere filed for future use in case extras were needed.

METHOD OF ANALYSIS: Peripheral blood smears and bone marrow smears were evaluated using epifluorescence microscopy. In the range-finding assay, peripheral blood smears and bone marrow smears were analyzed for the number of RNA-positive (polychromatic) erythrocytes in at least 1000 and 200 erythrocytes, respectively, per animal. In the definitive assay, two parameters were determined in teh bone marrow smears: (1) the number of micronucleated RNA-positive erythrocytes in at least 1000 RNA-positive erythrocytes per animal, which provides an index of chromosomal damage; and (2) the number of RNA-positive erythrocytes in at least 200 erythrocytes per animal, which provides and index of cytotoxicty to the nucleated erythrocyte precursors.
Evaluation criteria:
The data from this assay were considered valid if (1) the frequency of micronucleated cells in the vehicle control group was within the normal historical range, (2) administration of the positive control substance resulted in a statistically significant elevation of micronucleated cells, and (3) there were at least 3 surviving animals of each sex with a ratio of RNA-positive erythrocytes to total erythrocytes greater than or equal to 0.1 in two or more dose groups.

Positive: The test substance is considered unequivocally positive if the incidence of micronucleated RNA-contaning erythrocytes in 2 different dose groups is significantly higher than in the vehicle control group (p < 0.05) in either (1) two different dose groups from one experiment or (2) a single dose if confirmed by a separate experiment. A positive dose-related increase in the incidence of micronucleated cells is also considered a positive response.

Negative: The test substance is considered unequivocally negative if the criteria for a positive or inconclusive response are not met.

Inconclusive. The results are considered inconclusive if there is reason to believe that the concentrations of the test substance selected for evaluation were inappropriate (e.g., excessive toxicity) or if a statistically significant elevation in the frequency of micronucleated RNA-containing erythrocytes is observed in only one treatment group and the dose-response trend is not significant.

Statistics:
The data were analyzed according to sex. The ratio of micronucleated RNAcontaining erythrocytes (i.e., micronucleated PCEs) to RNA-positive erythrocytes and the RNA-positive erythrocytes as a percentage of total erythrocytes were calculated for each animal. The statistical significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskal-Wallis analysis of variance on ranks. In experiments where the frequencies of micronucleated cells are determined by scoring 1000 cells per animal, data are not expected to be distributed normally. Such data were analyzed using the Cochran-Armitage test for trends in binomial proportions (to determine a significant dose response relationship) and a test for equality of binomial proportions (to determine wether values for individual dose groups were statistically different from those for controls. These tests and the rationale for each are discussed in the ASTM Standard guide for conduct of Micronucleus Assays in Mammalian Bone es (ASTM Committee, 1988) and in Margolin et al. (1983). Animal mean body weights were analyzed using the Student's t-test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

MORTALITY: Range-finding test :all animals at 2500 and 500 mg/kg/day died within the assay,and one animal at 1200 mg/kg/day. 
Definitive test : one male and one female at 1200 mg/kg/day died.
CLINICAL SIGNS: Range-finding test : Rough fur, humped backs, labored breathing, slow movement, moribundity.  Definitive test: rough fur and humped backs for both sexes. 

NECROPSY FINDINGS: Range-finding test : RNA positive polychromatic erythrocytes (PCE) counts were lower in all doses as compared to control.
Definitive test : All treatment groups (0.3 % male and female) had average micronucleus count approximately equal to those of the vehicle control group (0.26% male, 0.29% female) 
BODY WEIGHT CHANGES: Range-finding test : no significant change.
Definitive test : no significant change.
FOOD AND WATER CONSUMPTION CHANGES: not described
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: not described
GENOTOXIC EFFECTS: No induction of increased incidences of micronuclei in the bone marrow erythrocytes of male and female mice up to the MTD of 1200 mg/kg day 
NOAEL (NOEL) (C) / LOAEL (LOEL) (C): 1200 mg/kg day, > 1200
mg/kg/day
MUTANT/ABERRATION/mPCE/ POLYPLOIDY FREQUENCY: not described 
STATISTICAL RESULTS: No treatment group had a statistically higher (p<0.05) micronucleus frequency than the control group. Kruskal-Wallis analysis were employed when data distribution was normal, otherwise the Cochran-Armitage test
was used.

Conclusions:
Interpretation of results: negative
At dosages up to and including the MTD of 1200 mg/kg/day, Tosylchloramide sodium did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss- Webster mice. Therefore, Tosylchloramide sodium was considered non-genotoxic under these test conditions.
Executive summary:

The genotoxic potential of orally administered Tosylchloramide sodium to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice. In the range-finding assay, 3 mice per sex received orally administered, Tosylchloramide sodium in sterile deionized water at dosage levels of 300,600,1200,2500, or 5000 mg/kg body weight/day to determine a maximum tolerated dose (MTD) that would be used to set dosages for the definitive study. A control group consisting of 3 male and 3 female mice received sterile deionized water only. All mice were dosed for 2 consecutive days and were appropriately observed from the start of dosing to euthanasia. Bone marrow and peripheral blood samples were obtained 48 hours after the administration of the last dose from all mice surviving the dosing regimen. These samples were evaluated for evidence of cytotoxcity reflected in the frequency of RNA-positive (polychromatic) erythrocytes (PCEs) among total erythrocytes (RBCs).

All mice receiving Tosylchloramide sodium at dosages of 2500 and 5000 mg/kg day and one mouse at the 1200 mg/kg day died during the range finding assay. adverse clinical observation reported for the 2500 and 5000 mg/kg day dosage groups included rough fur, humped backs, labored breathing, slow movement and moribundity. Erythrocytes in both bone marrow and peripheral red blood cell (RBC) pools from mice surviving to sacrifice were examined, and the frequency of PCEs among total RBCs was determined. In bone marrow, PCE ratios observed in surviving miceat all dosages of Tosylchloramide sodium were lower than the control values, but the difference was not statistically significant. from the mortality rate observed at 2500 and 5000 mg/kg day, an MTD of 1200 mg/kg day was determined for Tosylchloramide sodium.

In the definitive assay, 10 mice per sex per dosage group were dosed by gavage with Tosylchloramide sodium in sterile deionized water at dosage levels of 300, 600, or 1200 mg/kg day for 2 consecutive days. Five mice per sex per dosage group were sacrificed 21 hours after teh final dose and the same number 48 hours after the final dose; all were evaluated for cytotoxicity and micronucleus formation in the bone marrow erythrocytes. A steri;e deionized water vehicle control group (10 mice per sex) and a urethane control group (10 male mice only) were treated similarly and evaluated concurrently with the test groups.

One male mouse and 1 female mouse dosed with 1200 mg/kg/day of Tosylchloramide sodium died during the definitive assay. In the 600 and 1200 mg/kg/day Tosylchloramide sodium dosage groups, clinical signs of toxicity included rough fur and humped backs for both sexes. All Tosylchloramide sodium-treated groups, when compared with the sterile deionized water control groups, had average micronucleus counts approximately equal to that of the negative control groups. Background micronucleus incidences in bone marrow erythrocytes of male and female mice treated with sterile deionized water alone averaged 0.30% and 0.30%, respectively. No Tosylchloramide sodium-treated group had a micronucleus frequencies statistically higher than the control group at p < 0.05. The urethane positlve control group had micronucleus frequencies approximately 11 -fold greater than that of background at the 24 -hour sampling time.

At dosages up to and including the MTD of 1200 mg/kg/day, Tosylchloramide sodium did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss- Webster mice. Therefore, Tosylchloramide sodium was considered non-genotoxic under these test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames (OECD TG 471)

Halamid pharma grade was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. In the dose range finding test, Halamid pharma grade was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. Halamid pharma grade did not precipitate on the plates. In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence and presence of S9 - mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence of S9 -mix and at dose levels of 1000 µg/plate and upwards in the presence of S9 -mix. In the first mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 666 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In the second mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 500 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains Halamid pharma grade did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that Halamid pharma grade is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Gene mutation study in mammalian cells (OECD TG 476)

This report describes the effects of Halamid on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. Halamid was tested up to concentrations of 50 µg/ml in the absence o f S9 -mix in two experiments. Halamid was tested up to concentrations of 18.8 and 25 µg/ml in the presence of 8% (v/v) S9 -fraction, in the first and second experiment respectively. In the absence of S9 metabolic activation, Halamid showed a 2.9- fold increase in the mutant frequency at the TK-locus in the first experiment. However, this increase was observed in one experiment only and no dose relationship was observed. Furthermore, the increase was within the historical control data range. Therefore, this increase was considered not to be biologically relevant and Halamid is considered not to be mutagenic in the absence o f S9-mix. In the presence of 8% (v/v) S9 metabolic activation no significant increase in the mutant frequency at the TK- locus was observed in neither experiment. Mutant frequencies induced by positive control chemicals were increased by 15- and 10-fold for EMS in the first and second experiment respectively, and by 15- and 9 -fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9 -mix) functioned properly. It is concluded that Halamid is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

 

Genetic tox in vitro DNA damage and/or repair study

Under the conditions of a DNA Repair Test With Escherichia coli pol A+(W3110) - and pol A-(p3478) the test compound Halamid did not exhibit a DNA-modifying effect in any of the assays conducted in this evaluation and is considered as not genotoxic under these test conditions.

 

Mammalian Erythrocyte Micronucleus Test (OECD TG 474)

The genotoxic potential of orally administered Chloramine T to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice. In the range-finding assay, 3 mice per sex received orally administered, Chloramine T in sterile deionized water at dosage levels of 300,600,1200,2500, or 5000 mg/kg body weight/day to determine a maximum tolerated dose (MTD) that would be used to set dosages for the definitive study. A control group consisting of 3 male and 3 female mice received sterile deionized water only. All mice were dosed for 2 consecutive days and were appropriately observed from the start of dosing to euthanasia. Bone marrow and peripheral blood samples were obtained 48 hours after the administration of the last dose from all mice surviving the dosing regimen. These samples were evaluated for evidence of cytotoxcity reflected in the frequency of RNA-positive (polychromatic) erythrocytes (PCEs) among total erythrocytes (RBCs). All mice receiving Chloramine T at dosages of 2500 and 5000 mg/kg day and one mouse at the 1200 mg/kg day died during the range finding assay. adverse clinical observation reported for the 2500 and 5000 mg/kg day dosage groups included rough fur, humped backs, labored breathing, slow movement and moribundity. Erythrocytes in both bone marrow and peripheral red blood cell (RBC) pools from mice surviving to sacrifice were examined, and the frequency of PCEs among total RBCs was determined. In bone marrow, PCE ratios observed in surviving miceat all dosages of Chloramine T were lower than the control values, but the difference was not statistically significant. from the mortality rate observed at 2500 and 5000 mg/kg day, an MTD of 1200 mg/kg day was determined for Chloramine T. In the definitive assay, 10 mice per sex per dosage group were dosed by gavage with Chloramine T in sterile deionized water at dosage levels of 300, 600, or 1200 mg/kg day for 2 consecutive days. Five mice per sex per dosage group were sacrificed 21 hours after teh final dose and the same number 48 hours after the final dose; all were evaluated for cytotoxicity and micronucleus formation in the bone marrow erythrocytes. A steri;e deionized water vehicle control group (10 mice per sex) and a urethane control group (10 male mice only) were treated similarly and evaluated concurrently with the test groups. One male mouse and 1 female mouse dosed with 1200 mg/kg/day of Chloramine T died during the definitive assay. In the 600 and 1200 mg/kg/day Chloramine T dosage groups, clinical signs of toxicity included rough fur and humped backs for both sexes. All Chloramine T-treated groups, when compared with the sterile deionized water control groups, had average micronucleus counts approximately equal to that of the negative control groups. Background micronucleus incidences in bone marrow erythrocytes of male and female mice treated with sterile deionized water alone averaged 0.30% and 0.30%, respectively. No Chloramine T-treated group had a micronucleus frequencies statistically higher than the control group at p < 0.05. The urethane positlve control group had micronucleus frequencies approximately 11 -fold greater than that of background at the 24 -hour sampling time. At dosages up to and including the MTD of 1200 mg/kg/day, Chloramine T did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss- Webster mice. Therefore, Chloramine T was considered non-genotoxic under these test conditions.

Justification for classification or non-classification

Based on the available data, Tosylchloramide sodium does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).