Tosylchloramide sodium

Genetic toxicity: in vivo

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tosylchloramide sodium

EC Number:

204-854-7

EC Name:

Tosylchloramide sodium

Cas Number:

127-65-1

Molecular formula:

(C7H4SO2NCl)Na

IUPAC Name:

sodium chloro(4-methylbenzenesulfonyl)azanide

Details on test material:

Name: Chloramine T.
Lot no.: 0299103520059.
Purity: 100.8%
Appearance and physical state at room temperature: White crystalline powder
Storage conditions: Stored in a secondary container at room temperature (66ºF)

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.