Monalazone disodium

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Monalazone disodium is an organic compound, which has the chemical formula of (C7H4ClNO4S)Na2 and molecular weight 279.60 g/mol. The compound is a solid white powder with a decomposition point at 242ºC °C.
In this document read-across is employed from two source substances (Sodium N-chlorobenzenesulphonamide a.ka. Chloramine B and Tosylchloramide sodium a.k.a. Chloramine T) to a target substance. The target and source substances are mono-constituent substances.
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological and environmental fate properties. This prediction is supported by the fact that the substances are structurally similar
The studies conducted on the source substances have been performed according to the current guidelines and in compliance with GLP. The read-across data on the source substances are used as key studies and are considered as valid for the target substance.
Therefore, read-across from toxicokinetic information and the toxicological studies (repeated dose toxicity (oral), study on fertility and reproduction toxicity) on the source substances are considered as an appropriate adaptation to the standard information requirements of Annex VIII of the REACH regulation.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Target substance chemical formula Min% (w/w) Max% (w/w) Typical conc. % (w/w)
Monalazone disodium (C7H4ClNO4S)Na2 98.0 100.0 99.0
No identified impurities

Source substances
Sodium N-chlorobenzenesulphonamide (C6H6ClNO2S)Na


3. ANALOGUE APPROACH JUSTIFICATION
The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
Experimental data obtained with the source and target substances indicate that the substances has moderate or low oral (LD50 > 300 mg/kg bw) and low dermal (LD50 > 2000 mg/kg bw) acute toxicity. Furthermore, the substances are corrosive/irritating to skin and eye, and sensitising to skin/respiratory tract and concluded to be negative for genetic toxicity. There is no toxicological information available on the target substance for repeated dose toxicity or reproduction toxicity endpoints. The similar structure and similar physico-chemical / available toxicological properties of the target and source substances suggest similar systemic toxicity profiles after repeated exposure, and thus support the proposed read-across.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.


4. DATA MATRIX
See Other Assessment Report in IUCLID Section 13.2

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Monalazone disodium is an organic compound, which has the chemical formula of (C7H4ClNO4S)Na2 and molecular weight 279.60 g/mol. The compound is a solid white powder with a decomposition point at 242ºC °C.
In this document read-across is employed from two source substances (Sodium N-chlorobenzenesulphonamide a.ka. Chloramine B and Tosylchloramide sodium a.k.a. Chloramine T) to a target substance. The target and source substances are mono-constituent substances.
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological and environmental fate properties. This prediction is supported by the fact that the substances are structurally similar
The studies conducted on the source substances have been performed according to the current guidelines and in compliance with GLP. The read-across data on the source substances are used as key studies and are considered as valid for the target substance.
Therefore, read-across from toxicokinetic information and the toxicological studies (repeated dose toxicity (oral), study on fertility and reproduction toxicity) on the source substances are considered as an appropriate adaptation to the standard information requirements of Annex VIII of the REACH regulation.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Target substance chemical formula Min% (w/w) Max% (w/w) Typical conc. % (w/w)
Monalazone disodium (C7H4ClNO4S)Na2 98.0 100.0 99.0
No identified impurities

Source substances
Sodium N-chlorobenzenesulphonamide (C6H6ClNO2S)Na


3. ANALOGUE APPROACH JUSTIFICATION
The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
Experimental data obtained with the source and target substances indicate that the substances has moderate or low oral (LD50 > 300 mg/kg bw) and low dermal (LD50 > 2000 mg/kg bw) acute toxicity. Furthermore, the substances are corrosive/irritating to skin and eye, and sensitising to skin/respiratory tract and concluded to be negative for genetic toxicity. There is no toxicological information available on the target substance for repeated dose toxicity or reproduction toxicity endpoints. The similar structure and similar physico-chemical / available toxicological properties of the target and source substances suggest similar systemic toxicity profiles after repeated exposure, and thus support the proposed read-across.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.


4. DATA MATRIX
See APPENDIX of the CSR

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Chloramin B; sodium N-chlorobenzenesulphonamide trihydrate
- Substance type: Biocide
- Physical state: White to light yellow powder
- Analytical purity: 98%
- Impurities (identity and concentrations): 0.6% NaOH.
- Composition of test material, percentage of components: 77% CAS 127-52-6, 22.4% Water (incl. trihydrate), 0.6% NaOH.
- Isomers composition: Not applicable.
- Purity test date: No data.
- Lot/batch No.: 13102006
- Expiration date of the lot/batch: 10/2011
- Stability under test conditions: Test substance is in a water solution at defined laboratory conditions homogenously dissolved and stable at least for 120 minutes. (study No. 41/06/8)
- Storage condition of test material: Stable (cfr. section 4.18)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o. Koleč u Kladna
- Age at study initiation: 6-7 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: SPF (Specified Pathogen Free) animal house of CETA , 2 rats of the same sex in one plastic cage (40x25x20 cm) containing terilised clean shavings of soft wood.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and lice in SPF breeding (ST 1 BERGMAN) (producer: Mill Kocanda, Jesenice by Prague ); diet was sterilized before using.
- Water (e.g. ad libitum): Free access to drinking water (ad libitum); water quality corresponded to Regulation No. 252/2004/Czech Coll. Of Law, Health Ministry. Water was sterilized before using.
- Acclimation period: Minimally 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14-2-2007 To: 25-5-2007 main groups;20-6-2007 satellite groups

Route of administration:

oral: gavage

Vehicle:

other: water for injections

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance solution in water for injection) was prepared daily just before administration; these solutions were mixed for 10 minutes by magnetic stirrer. The procedure was based on the results of analyses of test substance application form homogeneity and stability. These results showed that the test substance at defined laboratory conditions is homogenously dissolved in water and the solution is stable at least for 120 minutes.
The vehicle control group was administered by water for injection in the same volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical report includes results of testing homogeneity and stability of a water solution of the test substance (an application form for " Repeated Dose 90-day Oral Toxicity Study"). The test purpose was to find out whether the application form is homogenous and stable during the application process.
Homogeneity of the application form was checked by determination of a concentration (an area peak) of Chloramin B in three places of suspension (at the bottom, in the middle and at the surface).
Stability of the application form was checked by analyses of the application form in the interval of 120 min (at the time 0 min, 30 min, 60 min and 120 min).
The measurement was performed on two concentration levels (250 mg/100 mL and 4000 mg/100 mL). The application form was prepared in a way that is conformable with "Repeated Dose 90-day Oral Toxicity Study" (the similar equipment: container, mixing stirrer). The analyses were performed by HPCL.
From the results of analyses follows that the test substance is in a water solution at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) homogenously dissolved and stable at least for 120 minutes.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily (8.00 -10.00 a.m.)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Dose / conc.:

180 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10
6 (satellite group)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The doses for the 90-day study were chosen with respect to the results of the Dose-range finding study and Acute toxicity study.
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and immediately before euthanasia too.

FOOD CONSUMPTION: Yes
- Food consumption for animal / day was calculated of average values of each group

FOOD EFFICIENCY:
- Calculation of food conversion in %: weight increment/food consumption x 100: Yes

WATER CONSUMPTION): Yes
- Time schedule for examinations: Twice a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of study and at the last week of administration
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91th (main group); 119th (satellite group) day of study
- Anaesthetic used for blood collection: Yes (light ether narcosis )
- Animals fasted: Yes
- How many animals: 10/group/sex (main study); 6/group/sex (satellite)
- Parameters checked:
Total erythrocytes count
Mean corpuscular volume
Haematocrit
Haemoglobin concentration
Total leucocytes count
Total platelets count
Partial thromboplastin time
Prothrombin time
Granulocytes
Lymphocytes
Monocytes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: : 91th (main group); 119th (satellite group) day of study
- Animals fasted: Yes approximately 18 hours before blood collection, but they were supplied by drinking water ad libitum
- How many animals: 10/group/sex (main study); 6/group/sex (satellite)
- Parameters examined:
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main group); 118th (satellite group) day of study
- Metabolism cages used for collection of urine: Yes, for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.
- Animals fasted: No data
- Parameters examined:
Volume
Colour
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrites
Leucocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At last week of administration or recovery
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity (on auditory, visual, proprioceptive stimuli and papillary reflex) / grip strength (pectoral legs; pelvis legs; all four legs; expressed in Newtons) / motor activity.

- BEHAVIOURAL ASSESSMENTS: Before the first application and the weekly.
In the cage: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behavior
Out the cage: reaction to handling, elasticity of the skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.
- Organs for consequent histopathological examination were taken out and stored in containers with fixative (neutral 4% formaldehyde).

BIOMETRY OF ORGANS: Yes
- absolute and relative weights of liver, kidneys, adrenals, tested or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart)

HISTOPATHOLOGY: Yes
- Tissue specimens fixed in 4% neutral formaldehyde were processed by routine paraffin technique and stained by hematoxyline-eosine.
- Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

Statistics:

The ANOVA test – Analysis if Variance (QC. Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians and averages.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no unscheduled deaths during all the study. Immediately after the administration of the top dose level reversible changes in health status (piloerection) and clinical changes (excited behaviour) were observed in both sexes.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during all the study. Immediately after the administration of the top dose level reversible changes in health status (piloerection) and clinical changes (excited behaviour) were observed in both sexes.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reversible decrease in weight increments in both sexes ( more marked in males) in all dose levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reversible decrease of food consumption and food conversion in both sexes.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Decreased food conversion was observed in males and females. The effect of the test substance was reversible.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Effect on leukocyte differential count in both sexes not dose dependent: portion of lymphocytes increased and portion of monocytes and granulocytes decreased. In females of the highest dose level also increased number of leucocytes and platelets.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes high dose level: increased urea in both sexes, increased chloride ions (M), decreased AST, creatinine and potassium ions (M), increased bilirubin (irriversible) and ALT (F) and decreased creatinine and sodium ions (F).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased volume and increased specific gravity (M + F) at (medium and) high dose level; increased pH in males of high dose recovery group.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increase in absolute and relative weights of kidney and liver in high dosed males and females; partly reversible in female or irriversible in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Kidney and liver: change of colour and marked structure in mid and high dosed males; reversible.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose males: hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli; increased incidence of histiocytosis of lymph nodes; involution of thymus. All treated females: increased incidence of hydrometra of uterus.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during all the study.
Immediately after the administration of the top dose level reversible changes in health status (piloerrection) and clinical changes (excited behaviour) were observed in both sexes.

BODY WEIGHT AND WEIGHT GAIN
Negative effect of the test substance on organism of animals was manifested especially by lower weight increments. It was caused by decrease in food intake and decreased food conversion. Weight difference in comparison with control animals was marked in males and females of the highest dose level but in lower dose levels weight increments were decreased too. The effect of the test substance on weight increment was reversible –in recovery period food intake and conversion of satellite treated animals were same or higher compared to satellite control so that weight increments of satellite treated animals were mostly higher than in satellite control animals. Nevertheless satellite treated animals were not able to compensate body weight difference during recovery period.


FOOD EFFICIENCY
Decreased food conversion was observed in males and females. The effect of the test substance was reversible.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects were detected.

HAEMATOLOGY
Haematological examination showed an effect on leukocyte differential count in both sexes without dependence on dose level. Portion of lymphocytes was increased and portion of monocytes and granulocytes was decreased. In females of the highest dose level also the increase of number of leucocytes and platelets was observed.


CLINICAL CHEMISTRY
During biochemical examination of blood the statistically significant changes were detected at the highest dose level: increased value of urea in both sexes, increased value of chloride ions in males, decreased value of AST, creatinine and potassium ions in males, increased value of bilirubin and ALT in females and decreased value of creatinine and sodium ions in females. Increase of bilirubin was irreversible.

URINALYSIS
Statistically significantly decreased volume of urine was detected in males especially at highest dose level. It was connected with increase of specific gravity of urine. Increased value of pH of urine was recorded in satellite males of the dose level 180 mg/kg bw/day and females of the dose levels 60 and 180 mg/kg bw/day. Decreased volume of urine accompanied by higher specific gravity of urine occurred also in females of the dose level 180 mg/kg bw/day.

NEUROBEHAVIOUR (Functional observation)
No treatment-related effects were detected.

ORGAN WEIGHTS
Decrease in absolute weight dependent on dose level was detected in epididymides, thymus and spleen of all dose levels in males. Statistically significant changes against control were found: increase of absolute weight of kidneys, decrease of absolute weight of epididymides, thymus, spleen and pituitary gland at the highest dose level and decrease of absolute weight of spleen at the middle dose level in males. The effects were reversible.
Increase of absolute weight of liver and kidneys, dependent on dose level, and decrease of absolute weight of brain dependent on dose level, were observed in females at all dose levels. Statistically significant increase of absolute weight in liver and kidneys and decrease of absolute brain weight in comparison with control were recorded at the highest dose level in females. The effect was partly reversible.
Increase of relative weight of liver, kidneys, brain and heart dependent on dose level was registered at all dose levels in males. Statistically significant changes against control were described: increase of relative weight of liver and kidneys at the middle and the highest dose level in males and increase of relative weight of adrenal glands, testes, brain and heart at the highest dose level in males. The effect was irreversible.
Increase of relative weight of liver, kidneys, adrenal glands and heart dependent on dose level was registered in females of all dose levels. Statistically significant change against control were recorded at the highest dosed females: increase of relative weight of liver, kidneys, adrenal glands and heart. The effect was partly reversible.


GROSS PATHOLOGY
Kidneys: change of colour in 0-0-3-5 males, marked structure in 0-0-1-5 males
Liver: change of colour in 0-1-6-3 males and marked structure in 0-2-5-3 males.
No relevant changes in recovery goups.

HISTOPATHOLOGY: NON-NEOPLASTIC
The most important histological affections were diagnosed in kidneys of males of the highest dose level –hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli. In the same treated group also the increase of incidence of histiocytosis of lymph nodes and involution of thymus was registered.
In females the increased incidence of hydrometra of uterus was found out in all treated groups irrespective of dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: oral gavage

Key result

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: oral gavage

Key result

Critical effects observed:

not specified

Table 1. RESULTS OF HISTOPATHOLOGICAL FINDINGS IN MALES

Organs/Diagnosis	MALE
Dose (mg.kg^-1.day^-1)	0	20	60	180	0S	180S
Number of examined animals	10	10	10	10	6	6
Without histopathological changes	0	0	2	0	0	0
Heart: pres. valvular endocardiosis	2	0	0	0	0	0
Heart: valvular endocardiosis	1	0	0	0	4	0
Kidney: pres. mesangial cell proliferation	2	0	0	0	0	0
Kidney: mesangial cell proliferation	0	0	0	10	0	0
Kidney: hyaline droplets	0	0	3	10	0	0
Kidney: hyaline casts	0	0	1	1	0	0
Kidney: proteinuria	1	2	1	3	0	1
Kidney: presumptive glomerulonephrosis	1	0	0	1	0	0
Kidney: glomerulonephrosis 1^stdegree	1	0	0	0	0	1
Liver: focal inflammation	0	1	0	0	1	1
Liver: extramedullary haemopoiesis	1	1	0	0	0	0
Liver: pres. oval cell proliferation	1	1	0	0	0	0
Lymph nodes: presumptive atrophy of cortex	2	0	1	0	0	0
Lymph nodes: mild atrophy of cortex	8	10	4	10	6	6
Lymph nodes: lymphostasis	1	0	1	0	0	0
Lymph nodes: histiocytosis	0	0	0	8	0	0
Prostate gland: interstitial inflammation	3	3	1	1	0	0
Prostate gland: oedema of interstitium	1	1	0	0	1	1
Prostate gland: pres. hyperplasia of epithelium	4	3	0	0	1	2
Prostate gland: hyperplasia of epithelium	3	4	0	0	0	1
Prostate gland: alveolar dilatation	0	0	0	0	3	5
Spleen: atrophy of PALS	0	1	1	0	0	0
Stomach: pres. atrophy of phundal glands	0	0	1	0	0	0
Stomach: atrophy of phundal glands	0	0	2	1	2	1
Stomach: erosion	1	0	0	0	0	0
Stomach: oedema of submucosa	1	0	0	1	1	0
Stomach: inflammation	1	0	0	1	0	0
Testes: dystrophy of germinal epithelium	0	0	1	1	0	0
Testes: pres. atrophy of germinal epithelium	0	3	0	2	0	0
Testes: atrophy of germinal epithelium	0	0	1	2	1	0
Thymus: involution	7	5	1	10	6	6
Thymus: haemorrhages	7	8	2	0	4	1
Thymus: tubular structures	0	0	0	0	1	1
Thymus: hyperaemia of medulla	6	0	0	0	0	0
Thymus : hyperplasia of stroma of epithelium	0	0	0	0	0	0
Thyroid gland: pres. inspissation of colloid	0	0	0	0	2	0

Table 2. RESULTS OF HISTOPATHOLOGICAL FINDINGS IN FEMALES

Organs/Diagnosis	FEMALE
Dose (mg.kg^-1.day^-1)	0	20	60	180	0S	180S
Number of examined animals	10	10	10	10	6	6
Without histopathological changes	0	0	0	0	0	0
Heart: pres. valvular endocardiosis	1	1	1	3	1	1
Heart: valvular endocardiosis	0	1	0	1	0	1
Kidney: pres. mesangial cell proliferation	0	4	0	0	0	0
Kidney: mesangial cell proliferation	0	0	0	0	0	0
Kidney: hyaline droplets	0	0	0	0	0	0
Kidney: hyaline casts	0	0	0	0	0	0
Kidney: proteinuria	0	0	0	0	0	0
Kidney: presumptive glomerulonephrosis	0	0	0	0	0	0
Kidney: glomerulonephrosis 1^stdegree	0	0	0	0	0	0
Liver: focal inflammation	0	0	1	0	0	0
Liver: extramedullary haemopoiesis	0	2	0	0	0	0
Liver: oval cell proliferation	0	2	0	0	0	0
Lymph nodes: pres. atrophy of cortex	0	0	1	4	0	0
Lymph nodes: mild atrophy of cortex	10	10	6	0	5	6
Lymph nodes: lymphostasis	1	1	0	0	0	0
Lymph nodes: histiocytosis	0	0	0	0	0	0
Ovaries: persistent corpus luteum	7	7	8	9	3	2
Ovaries: hyperplasia of interstitial glands	10	9	6	7	4	4
Spleen: atrophy of PALS	0	4	2	0	0	1
Stomach: pres. atrophy of phundal glands	0	0	0	0	0	0
Stomach: atrophy of phundal glands	0	0	0	1	0	1
Stomach: erosion	2	0	0	0	0	0
Stomach: oedema of submucosa	1	0	0	1	0	0
Stomach: inflammation	2	0	0	2	0	0
Thymus: involution	4	7	2	0	6	6
Thymus: haemorrhages	3	4	4	0	3	2
Thymus: tubular structures	0	2	0	0	0	4
Thymus: hyperaemia of medulla	0	4	0	0	3	2
Thymus : hyperplasia of stroma of epithelium	0	2	0	0	0	0
Thyroid gland: pres. inspissation of colloid	0	0	0	0	0	0
Uterus: hydrometra	1	5	5	4	3	3

Executive summary:

The oral administration of Chloramin B trihydrate to rats by gavage for period of 90 consecutive days at dose levels 20, 60, 180 mg/kg bw/day produced no significant changes in functional observations, ophthalmological examination and did not cause mortality.

Overall assessment of results showed, that the test substance, after 90-day oral application caused reversible decrease in body weight increments in both sexes (more marked in males) in all dose levels. It was connected with reversible decrease of food consumption and food conversion in both sexes.

Reversible changes of health status (piloerection) and clinical changes immediately after application (excited behavior) were recorded in both sexes of the highest dose level.

Haematological examination showed an effect on leukocyte differential count in both sexes without dependence on dose level. Portion of lymphocytes was increased and portion of monocytes and granulocytes was decreased. In females of the highest dose level also the increase of number of leucocytes and platelets was observed.

During biochemical examination of blood the statistically significant changes were detected at the highest dose level: increased value of urea in both sexes, increased value of chloride ions in males, decreased value of AST, creatinine and potassium ions in males, increased value of bilirubin and ALT in females and decreased value of creatinine and sodium ions in females. Increase of bilirubin was irreversible.

Statistically significantly decreased volume of urine was detected in males especially at highest dose level. It was connected with increase of specific gravity of urine. Increased value of pH of urine was recorded in satellite males of the dose level 180 mg/kg bw/day and females of the dose levels 60 and 180 mg/kg bw/day. Decreased volume of urine accompanied by higher specific gravity of urine occurred also in females of the dose level 180 mg/kg bw/day.

The most important histological affections were diagnosed in kidneys of males of the highest dose level –hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli. In the same treated group also the increase of incidence of histiocytosis of lymph nodes and involution of thymus was registered.

In females the increased incidence of hydrometra of uterus was found out in all treated groups irrespective of dose level.

The LOAEL (Lowest Observed Adverse Effect Level) for males and females was established as 60 mg/kg bw/day. The changes however were mild and did not indicate specific target organ toxicity. The NOAEL (No Observed Adverse Effect Level) for males and females is 20 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium N-chlorobenzenesulphonamide

EC Number:

204-847-9

EC Name:

Sodium N-chlorobenzenesulphonamide

Cas Number:

127-52-6

Molecular formula:

C6H6ClNO2S.Na

IUPAC Name:

sodium (benzenesulfonyl)(chloro)azanide

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Name of test material (as cited in study report): Chloramin B; sodium N-chlorobenzenesulphonamide trihydrate
- Substance type: Biocide
- Physical state: White to light yellow powder
- Analytical purity: 98%
- Impurities (identity and concentrations): 0.6% NaOH.
- Composition of test material, percentage of components: 77% CAS 127-52-6, 22.4% Water (incl. trihydrate), 0.6% NaOH.
- Isomers composition: Not applicable.
- Purity test date: No data.
- Lot/batch No.: 13102006
- Expiration date of the lot/batch: 10/2011
- Stability under test conditions: Test substance is in a water solution at defined laboratory conditions homogenously dissolved and stable at least for 120 minutes. (study No. 41/06/8)
- Storage condition of test material: Stable (cfr. section 4.18)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o. Koleč u Kladna
- Age at study initiation: 6-7 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: SPF (Specified Pathogen Free) animal house of CETA , 2 rats of the same sex in one plastic cage (40x25x20 cm) containing terilised clean shavings of soft wood.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and lice in SPF breeding (ST 1 BERGMAN) (producer: Mill Kocanda, Jesenice by Prague ); diet was sterilized before using.
- Water (e.g. ad libitum): Free access to drinking water (ad libitum); water quality corresponded to Regulation No. 252/2004/Czech Coll. Of Law, Health Ministry. Water was sterilized before using.
- Acclimation period: Minimally 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14-2-2007 To: 25-5-2007 main groups;20-6-2007 satellite groups

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: water for injections

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance solution in water for injection) was prepared daily just before administration; these solutions were mixed for 10 minutes by magnetic stirrer. The procedure was based on the results of analyses of test substance application form homogeneity and stability. These results showed that the test substance at defined laboratory conditions is homogenously dissolved in water and the solution is stable at least for 120 minutes.
The vehicle control group was administered by water for injection in the same volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical report includes results of testing homogeneity and stability of a water solution of the test substance (an application form for " Repeated Dose 90-day Oral Toxicity Study"). The test purpose was to find out whether the application form is homogenous and stable during the application process.
Homogeneity of the application form was checked by determination of a concentration (an area peak) of Chloramin B in three places of suspension (at the bottom, in the middle and at the surface).
Stability of the application form was checked by analyses of the application form in the interval of 120 min (at the time 0 min, 30 min, 60 min and 120 min).
The measurement was performed on two concentration levels (250 mg/100 mL and 4000 mg/100 mL). The application form was prepared in a way that is conformable with "Repeated Dose 90-day Oral Toxicity Study" (the similar equipment: container, mixing stirrer). The analyses were performed by HPCL.
From the results of analyses follows that the test substance is in a water solution at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) homogenously dissolved and stable at least for 120 minutes.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily (8.00 -10.00 a.m.)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

180 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10
6 (satellite group)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The doses for the 90-day study were chosen with respect to the results of the Dose-range finding study and Acute toxicity study.
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and immediately before euthanasia too.

FOOD CONSUMPTION: Yes
- Food consumption for animal / day was calculated of average values of each group

FOOD EFFICIENCY:
- Calculation of food conversion in %: weight increment/food consumption x 100: Yes

WATER CONSUMPTION): Yes
- Time schedule for examinations: Twice a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the 1st week of study and at the last week of administration
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91th (main group); 119th (satellite group) day of study
- Anaesthetic used for blood collection: Yes (light ether narcosis )
- Animals fasted: Yes
- How many animals: 10/group/sex (main study); 6/group/sex (satellite)
- Parameters checked:
Total erythrocytes count
Mean corpuscular volume
Haematocrit
Haemoglobin concentration
Total leucocytes count
Total platelets count
Partial thromboplastin time
Prothrombin time
Granulocytes
Lymphocytes
Monocytes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: : 91th (main group); 119th (satellite group) day of study
- Animals fasted: Yes approximately 18 hours before blood collection, but they were supplied by drinking water ad libitum
- How many animals: 10/group/sex (main study); 6/group/sex (satellite)
- Parameters examined:
Glucose
Cholesterol, total
Urea
Bilirubin, total
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Calcium
Phosphorus
Protein, total
Protein, albumin
Creatinine
Sodium
Potassium
Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main group); 118th (satellite group) day of study
- Metabolism cages used for collection of urine: Yes, for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.
- Animals fasted: No data
- Parameters examined:
Volume
Colour
Odour
Glucose
Protein
Bilirubin
Urobilinogen
pH
Specific gravity
Blood
Ketones
Nitrites
Leucocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At last week of administration or recovery
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity (on auditory, visual, proprioceptive stimuli and papillary reflex) / grip strength (pectoral legs; pelvis legs; all four legs; expressed in Newtons) / motor activity.

- BEHAVIOURAL ASSESSMENTS: Before the first application and the weekly.
In the cage: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behavior
Out the cage: reaction to handling, elasticity of the skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.
- Organs for consequent histopathological examination were taken out and stored in containers with fixative (neutral 4% formaldehyde).

BIOMETRY OF ORGANS: Yes
- absolute and relative weights of liver, kidneys, adrenals, tested or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart)

HISTOPATHOLOGY: Yes
- Tissue specimens fixed in 4% neutral formaldehyde were processed by routine paraffin technique and stained by hematoxyline-eosine.
- Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

Statistics:

The ANOVA test – Analysis if Variance (QC. Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians and averages.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no unscheduled deaths during all the study. Immediately after the administration of the top dose level reversible changes in health status (piloerection) and clinical changes (excited behaviour) were observed in both sexes.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during all the study. Immediately after the administration of the top dose level reversible changes in health status (piloerection) and clinical changes (excited behaviour) were observed in both sexes.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reversible decrease in weight increments in both sexes ( more marked in males) in all dose levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reversible decrease of food consumption and food conversion in both sexes.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Decreased food conversion was observed in males and females. The effect of the test substance was reversible.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Effect on leukocyte differential count in both sexes not dose dependent: portion of lymphocytes increased and portion of monocytes and granulocytes decreased. In females of the highest dose level also increased number of leucocytes and platelets.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes high dose level: increased urea in both sexes, increased chloride ions (M), decreased AST, creatinine and potassium ions (M), increased bilirubin (irriversible) and ALT (F) and decreased creatinine and sodium ions (F).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased volume and increased specific gravity (M + F) at (medium and) high dose level; increased pH in males of high dose recovery group.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increase in absolute and relative weights of kidney and liver in high dosed males and females; partly reversible in female or irriversible in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Kidney and liver: change of colour and marked structure in mid and high dosed males; reversible.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose males: hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli; increased incidence of histiocytosis of lymph nodes; involution of thymus. All treated females: increased incidence of hydrometra of uterus.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during all the study.
Immediately after the administration of the top dose level reversible changes in health status (piloerrection) and clinical changes (excited behaviour) were observed in both sexes.

BODY WEIGHT AND WEIGHT GAIN
Negative effect of the test substance on organism of animals was manifested especially by lower weight increments. It was caused by decrease in food intake and decreased food conversion. Weight difference in comparison with control animals was marked in males and females of the highest dose level but in lower dose levels weight increments were decreased too. The effect of the test substance on weight increment was reversible –in recovery period food intake and conversion of satellite treated animals were same or higher compared to satellite control so that weight increments of satellite treated animals were mostly higher than in satellite control animals. Nevertheless satellite treated animals were not able to compensate body weight difference during recovery period.


FOOD EFFICIENCY
Decreased food conversion was observed in males and females. The effect of the test substance was reversible.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects were detected.

HAEMATOLOGY
Haematological examination showed an effect on leukocyte differential count in both sexes without dependence on dose level. Portion of lymphocytes was increased and portion of monocytes and granulocytes was decreased. In females of the highest dose level also the increase of number of leucocytes and platelets was observed.


CLINICAL CHEMISTRY
During biochemical examination of blood the statistically significant changes were detected at the highest dose level: increased value of urea in both sexes, increased value of chloride ions in males, decreased value of AST, creatinine and potassium ions in males, increased value of bilirubin and ALT in females and decreased value of creatinine and sodium ions in females. Increase of bilirubin was irreversible.

URINALYSIS
Statistically significantly decreased volume of urine was detected in males especially at highest dose level. It was connected with increase of specific gravity of urine. Increased value of pH of urine was recorded in satellite males of the dose level 180 mg/kg bw/day and females of the dose levels 60 and 180 mg/kg bw/day. Decreased volume of urine accompanied by higher specific gravity of urine occurred also in females of the dose level 180 mg/kg bw/day.

NEUROBEHAVIOUR (Functional observation)
No treatment-related effects were detected.

ORGAN WEIGHTS
Decrease in absolute weight dependent on dose level was detected in epididymides, thymus and spleen of all dose levels in males. Statistically significant changes against control were found: increase of absolute weight of kidneys, decrease of absolute weight of epididymides, thymus, spleen and pituitary gland at the highest dose level and decrease of absolute weight of spleen at the middle dose level in males. The effects were reversible.
Increase of absolute weight of liver and kidneys, dependent on dose level, and decrease of absolute weight of brain dependent on dose level, were observed in females at all dose levels. Statistically significant increase of absolute weight in liver and kidneys and decrease of absolute brain weight in comparison with control were recorded at the highest dose level in females. The effect was partly reversible.
Increase of relative weight of liver, kidneys, brain and heart dependent on dose level was registered at all dose levels in males. Statistically significant changes against control were described: increase of relative weight of liver and kidneys at the middle and the highest dose level in males and increase of relative weight of adrenal glands, testes, brain and heart at the highest dose level in males. The effect was irreversible.
Increase of relative weight of liver, kidneys, adrenal glands and heart dependent on dose level was registered in females of all dose levels. Statistically significant change against control were recorded at the highest dosed females: increase of relative weight of liver, kidneys, adrenal glands and heart. The effect was partly reversible.


GROSS PATHOLOGY
Kidneys: change of colour in 0-0-3-5 males, marked structure in 0-0-1-5 males
Liver: change of colour in 0-1-6-3 males and marked structure in 0-2-5-3 males.
No relevant changes in recovery goups.

HISTOPATHOLOGY: NON-NEOPLASTIC
The most important histological affections were diagnosed in kidneys of males of the highest dose level –hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli. In the same treated group also the increase of incidence of histiocytosis of lymph nodes and involution of thymus was registered.
In females the increased incidence of hydrometra of uterus was found out in all treated groups irrespective of dose level.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: oral gavage

2

Key result

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: oral gavage

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. RESULTS OF HISTOPATHOLOGICAL FINDINGS IN MALES

Organs/Diagnosis	MALE
Dose (mg.kg^-1.day^-1)	0	20	60	180	0S	180S
Number of examined animals	10	10	10	10	6	6
Without histopathological changes	0	0	2	0	0	0
Heart: pres. valvular endocardiosis	2	0	0	0	0	0
Heart: valvular endocardiosis	1	0	0	0	4	0
Kidney: pres. mesangial cell proliferation	2	0	0	0	0	0
Kidney: mesangial cell proliferation	0	0	0	10	0	0
Kidney: hyaline droplets	0	0	3	10	0	0
Kidney: hyaline casts	0	0	1	1	0	0
Kidney: proteinuria	1	2	1	3	0	1
Kidney: presumptive glomerulonephrosis	1	0	0	1	0	0
Kidney: glomerulonephrosis 1^stdegree	1	0	0	0	0	1
Liver: focal inflammation	0	1	0	0	1	1
Liver: extramedullary haemopoiesis	1	1	0	0	0	0
Liver: pres. oval cell proliferation	1	1	0	0	0	0
Lymph nodes: presumptive atrophy of cortex	2	0	1	0	0	0
Lymph nodes: mild atrophy of cortex	8	10	4	10	6	6
Lymph nodes: lymphostasis	1	0	1	0	0	0
Lymph nodes: histiocytosis	0	0	0	8	0	0
Prostate gland: interstitial inflammation	3	3	1	1	0	0
Prostate gland: oedema of interstitium	1	1	0	0	1	1
Prostate gland: pres. hyperplasia of epithelium	4	3	0	0	1	2
Prostate gland: hyperplasia of epithelium	3	4	0	0	0	1
Prostate gland: alveolar dilatation	0	0	0	0	3	5
Spleen: atrophy of PALS	0	1	1	0	0	0
Stomach: pres. atrophy of phundal glands	0	0	1	0	0	0
Stomach: atrophy of phundal glands	0	0	2	1	2	1
Stomach: erosion	1	0	0	0	0	0
Stomach: oedema of submucosa	1	0	0	1	1	0
Stomach: inflammation	1	0	0	1	0	0
Testes: dystrophy of germinal epithelium	0	0	1	1	0	0
Testes: pres. atrophy of germinal epithelium	0	3	0	2	0	0
Testes: atrophy of germinal epithelium	0	0	1	2	1	0
Thymus: involution	7	5	1	10	6	6
Thymus: haemorrhages	7	8	2	0	4	1
Thymus: tubular structures	0	0	0	0	1	1
Thymus: hyperaemia of medulla	6	0	0	0	0	0
Thymus : hyperplasia of stroma of epithelium	0	0	0	0	0	0
Thyroid gland: pres. inspissation of colloid	0	0	0	0	2	0

Table 2. RESULTS OF HISTOPATHOLOGICAL FINDINGS IN FEMALES

Organs/Diagnosis	FEMALE
Dose (mg.kg^-1.day^-1)	0	20	60	180	0S	180S
Number of examined animals	10	10	10	10	6	6
Without histopathological changes	0	0	0	0	0	0
Heart: pres. valvular endocardiosis	1	1	1	3	1	1
Heart: valvular endocardiosis	0	1	0	1	0	1
Kidney: pres. mesangial cell proliferation	0	4	0	0	0	0
Kidney: mesangial cell proliferation	0	0	0	0	0	0
Kidney: hyaline droplets	0	0	0	0	0	0
Kidney: hyaline casts	0	0	0	0	0	0
Kidney: proteinuria	0	0	0	0	0	0
Kidney: presumptive glomerulonephrosis	0	0	0	0	0	0
Kidney: glomerulonephrosis 1^stdegree	0	0	0	0	0	0
Liver: focal inflammation	0	0	1	0	0	0
Liver: extramedullary haemopoiesis	0	2	0	0	0	0
Liver: oval cell proliferation	0	2	0	0	0	0
Lymph nodes: pres. atrophy of cortex	0	0	1	4	0	0
Lymph nodes: mild atrophy of cortex	10	10	6	0	5	6
Lymph nodes: lymphostasis	1	1	0	0	0	0
Lymph nodes: histiocytosis	0	0	0	0	0	0
Ovaries: persistent corpus luteum	7	7	8	9	3	2
Ovaries: hyperplasia of interstitial glands	10	9	6	7	4	4
Spleen: atrophy of PALS	0	4	2	0	0	1
Stomach: pres. atrophy of phundal glands	0	0	0	0	0	0
Stomach: atrophy of phundal glands	0	0	0	1	0	1
Stomach: erosion	2	0	0	0	0	0
Stomach: oedema of submucosa	1	0	0	1	0	0
Stomach: inflammation	2	0	0	2	0	0
Thymus: involution	4	7	2	0	6	6
Thymus: haemorrhages	3	4	4	0	3	2
Thymus: tubular structures	0	2	0	0	0	4
Thymus: hyperaemia of medulla	0	4	0	0	3	2
Thymus : hyperplasia of stroma of epithelium	0	2	0	0	0	0
Thyroid gland: pres. inspissation of colloid	0	0	0	0	0	0
Uterus: hydrometra	1	5	5	4	3	3

Applicant's summary and conclusion

Executive summary:

The oral administration of Chloramin B trihydrate to rats by gavage for period of 90 consecutive days at dose levels 20, 60, 180 mg/kg bw/day produced no significant changes in functional observations, ophthalmological examination and did not cause mortality.

Overall assessment of results showed, that the test substance, after 90-day oral application caused reversible decrease in body weight increments in both sexes (more marked in males) in all dose levels. It was connected with reversible decrease of food consumption and food conversion in both sexes.

Reversible changes of health status (piloerection) and clinical changes immediately after application (excited behavior) were recorded in both sexes of the highest dose level.

Haematological examination showed an effect on leukocyte differential count in both sexes without dependence on dose level. Portion of lymphocytes was increased and portion of monocytes and granulocytes was decreased. In females of the highest dose level also the increase of number of leucocytes and platelets was observed.

During biochemical examination of blood the statistically significant changes were detected at the highest dose level: increased value of urea in both sexes, increased value of chloride ions in males, decreased value of AST, creatinine and potassium ions in males, increased value of bilirubin and ALT in females and decreased value of creatinine and sodium ions in females. Increase of bilirubin was irreversible.

Statistically significantly decreased volume of urine was detected in males especially at highest dose level. It was connected with increase of specific gravity of urine. Increased value of pH of urine was recorded in satellite males of the dose level 180 mg/kg bw/day and females of the dose levels 60 and 180 mg/kg bw/day. Decreased volume of urine accompanied by higher specific gravity of urine occurred also in females of the dose level 180 mg/kg bw/day.

The most important histological affections were diagnosed in kidneys of males of the highest dose level –hyaline droplets in renal tubules and mesangial cell proliferation in renal glomeruli. In the same treated group also the increase of incidence of histiocytosis of lymph nodes and involution of thymus was registered.

In females the increased incidence of hydrometra of uterus was found out in all treated groups irrespective of dose level.

The LOAEL (Lowest Observed Adverse Effect Level) for males and females was established as 60 mg/kg bw/day. The changes however were mild and did not indicate specific target organ toxicity. The NOAEL (No Observed Adverse Effect Level) for males and females is 20 mg/kg bw/day.