Monalazone disodium

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

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) Typicalconc. % (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:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

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) Typicalconc. % (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 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Chloramin B; Sodium N-chlorobenzenesulphonamide
- Substance type: Biocide
- Physical state: White to light yellow powder with a faint odor of chlorine.
- Analytical purity: 99.8 %
- Impurities (identity and concentrations): 0.2% NaOH.
- Composition of test material, percentage of components: 81.3% CAS 127-52-6 (98% pure), 18.5% Water (in form of hydrate), 0.2% NaOH. 27% active chlorine.
- Isomers composition: Not applicable.
- Purity test date: August 25, 2011
- Lot/batch No.: 863/11.
- Expiration date of the lot/batch: 24.1.2013.
- Stability under test conditions: Stable
- Storage condition of test material: Stable; at room temperature, protected from sunlight.

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Facility Löhndorf, 24601Löhndorf/Post Wankendorf, Germany
- Age at study initiation: 4 - 6 months
- Weight at study initiation: 1.70 - 2.81 kg
- Housing: The dams were kept separately in breeding cages with wire floors (with an area of approx. 0.2 m2).
- Diet (e.g. ad libitum): Commercial ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ,offered daily ad libitum. Food residue was removed and weighed. In addition, a restricted feeding control group 5 was employed consisting of untreated dams which were offered the food amount monitored for the high dose dams.
- Water (e.g. ad libitum): Tap water (in drinking bottles) was offered ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20°C ±3 °C
- Humidity (%): 55% ± 5%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 4, 2011 (first administration preliminary exp.)To: August 9, 2011

Route of administration:

oral: gavage

Vehicle:

physiological saline

Remarks:

water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle (water for injection) to the appropriate concentrations. The dose levels administered in this test refer to the supplied product.
The test item-vehicle mixtures were freshly prepared every day.
The daily dose was adjusted to the animal’s actual body weight daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of the Sodium N-chlorobenzenesulphonamide – Chloramine B trihydrate concentration in the test item vehicle- mixtures revealed that the formulations used for the administration were correctly prepared and measured actual concentrations ranged from 98.7% to 104.4% of the nominal values. The results were well within the admissible limits of 90% to 110%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes , sexually mature (‘proved’) male rabbits of the same breed served as partners.
- Proof of pregnancy: Successful copulation was ascertained by observation. The day of copulation was considered as day 0 of gestation. Only animals with positive signs of copulation were included in the study.

Duration of treatment / exposure:

Day 6 to 28 of gestation

Frequency of treatment:

once daily

Duration of test:

Treatment period Day 6 to 28 of gestation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6 non-pregnant females for the preliminary experiment
120 animals in groups 1 - 5 :
- Treated animals (vehicle control and restricted feeding control incl.): 24 females per group
- Evaluated animals: 20 dams with evaluable litters

Control animals:

yes, concurrent vehicle

other: A restricted feeding control group 5 was employed consisting of untreated dams which were offered the food amount monitored for the high dose dams.

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on the results of a preliminary experiment. In this dose-range-finding study, sodium N-chlorobenzenesulphonamide-Chloramine B was administered to non-pregnant female rabbits by gavage at dose levels of 90 or 180 mg/kg bw/day once daily for 7 days. None of the animals died prematurely. A slight reduction of body weight, marked reduction of food consumption and a reduced discharge of faeces were noted – in relation to the dose – for both treatment groups ( 90 – 180 mg/kg bw/day).
Dose levels of 10, 30 or 90 mg/kg bw/day , administered by gavage once daily from the 6th to 28th day of pregnancy were selected in agreement with the sponsor for the present embryotoxicity study.

- Rationale for animal assignment (if not random):
The animals were randomly assigned to their respective groups according to their mating day i.e. in a cyclic way following the listing of successful copulation.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
- Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rabbit was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time in the morning.
- The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29).

FOOD & DRINK WATER CONSUMPTION: Yes
- The quantity of food consumed by each rabbit was recorded.
- Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.
- The relative food consumption (g/kg bw/day) was calculated.
- Daily monitoring by visual appraisal of the drinking water consumption was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
The ovaries and uteri were removed; the uteri (in toto) were weighed.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and size of resorptions: Yes
- Other: macroscopic inspection (gross evaluation) of the placenta for example for focal indurations; number of fetuses (alive and dead); sex and viability of the fetuses; weight of fetuses

Fetal examinations:

- External examinations: Yes: all per litter
Foetuses were inspected externally for damages, especially for malformations.

- Soft tissue examinations: Yes: all per litter
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities of soft tissue.

- Skeletal examinations: Yes: all per litter
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).

- Head examinations: Yes: half per litter
The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head structures.
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.

Statistics:

For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test. When the variances were homogeneous, the DUNNETT test (p ≤ 0.05 or p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT’s t-test was carried out, limits of significance were p ≤ 0.05 or p ≤ 0.01.
For the comparison of classification measurements (for example malformation-, resorption-, retardation-, and variation rate) the FISHER’s exact test (n < 100) or chi2-test with YATES’ correction for continuity (n ≥ 100) (p ≤ 0.05 or p ≤ 0.01) was employed.
All data were evaluated statistically in this manner.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reports number of decimal places. Hence, deviations to the last decimal place up to ± 1 may occur caused by rounding.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient slight or moderate reductions were noted for the body weight and food intake of the dams treated with 30 mg Sodium N-chlorobenzenesulphonamide – Chloramine B /kg bw/day. Distinct reductions of body weight and food consumption, especially during the first days of treatment, were noted at 90 mg/kg bw/day. Similar reductions of body weight and food intake were noted in the untreated dams with restricted feeding.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

he severely materno-toxic dose level of 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw /day resulted in developmental retardations in the form of reduced placental and fetal body weights compared to the control.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A dose level of 30 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw /day resulted in increased incidences of total skeletal retardations, in particular sternebra(e) not ossified.
No test item related malformations were noted during external, internal or skeletal examination (according to DAWSON) of the fetuses or soft tissue examination of the fetal heads (according to WILSON) at any tested dose level, not even at materno-toxic dose levels.
No embryotoxic changes were noted for the restricted feeding group.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

The test item Sodium N-chlorobenzenesulphonamide – Chloramine B possessed no teratogenic properties.
Sodium N-chlorobenzenesulphonamide – Chloramine B revealed slight embryotoxicity at materno-toxic dose levels (30 or 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw/day).

Executive summary:

Sodium N-chlorobenzenesulphonamide – Chloramine B (trihydrate) was administered to female rabbits at dose levels of 10, 30 and 90 mg/kg bw/day orally, by gavage from the 6th to 28th day of pregnancy. In addition, a restricted feeding control was employed consisting of untreated dams which were offered the food amount consumed by the high dose dams. Under the present test conditions, the no-observed-effect level (NOEL) was 10 mg/kg bw/day for the dams. Transient slight or moderate reductions were noted for the body weight and food intake of the dams treated with 30 mg /kg bw/day. Distinct reductions of body weight and food consumption, especially during the first days of treatment, were noted at 90 mg/kg bw/day. Similar reductions of body weight and food intake were noted in the untreated dams with restricted feeding.

The no-observed-effect level (NOEL) for the fetal organism also was 10 mg Sodium N-chlorobenzenesulphonamide-Chloramine B (trihydrate)/ kg bw /day. A dose level of 30 mg/ kg bw /day resulted in increased incidences of total skeletal retardations, in particular sternebra(e) not ossified. The severely materno-toxic dose level of 90 mg/kg bw /day resulted in developmental retardations in the form of reduced placental and fetal body weights compared to the control. Skeletal examination (according to DAWSON) revealed increased fetal incidences for variations of the sternum (fusions) and total skeletal variations as well as retardations of the sternum and lumbar vertebral bodies (incomplete or missing ossification) and total skeletal retardations.

No test item related malformations were noted during external, internal or skeletal examination (according to DAWSON) of the fetuses or soft tissue examination of the fetal heads (according to WILSON) at any tested dose level, not even at materno-toxic dose levels.

No embryotoxic changes were noted for the restricted feeding group. In conclusion, the test item possessed no teratogenic properties.
Chloramine B trihydrate revealed slight embryotoxicity at materno-toxic dose levels (30 or 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw/day).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

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
- Substance type: Biocide
- Physical state: White to light yellow powder with a faint odor of chlorine.
- Analytical purity: 99.8 %
- Impurities (identity and concentrations): 0.2% NaOH.
- Composition of test material, percentage of components: 81.3% CAS 127-52-6 (98% pure), 18.5% Water (in form of hydrate), 0.2% NaOH. 27% active chlorine.
- Isomers composition: Not applicable.
- Purity test date: August 25, 2011
- Lot/batch No.: 863/11.
- Expiration date of the lot/batch: 24.1.2013.
- Stability under test conditions: Stable
- Storage condition of test material: Stable; at room temperature, protected from sunlight.

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Facility Löhndorf, 24601Löhndorf/Post Wankendorf, Germany
- Age at study initiation: 4 - 6 months
- Weight at study initiation: 1.70 - 2.81 kg
- Housing: The dams were kept separately in breeding cages with wire floors (with an area of approx. 0.2 m2).
- Diet (e.g. ad libitum): Commercial ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ,offered daily ad libitum. Food residue was removed and weighed. In addition, a restricted feeding control group 5 was employed consisting of untreated dams which were offered the food amount monitored for the high dose dams.
- Water (e.g. ad libitum): Tap water (in drinking bottles) was offered ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20°C ±3 °C
- Humidity (%): 55% ± 5%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 4, 2011 (first administration preliminary exp.)To: August 9, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

physiological saline

Remarks:

water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle (water for injection) to the appropriate concentrations. The dose levels administered in this test refer to the supplied product.
The test item-vehicle mixtures were freshly prepared every day.
The daily dose was adjusted to the animal’s actual body weight daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of the Sodium N-chlorobenzenesulphonamide – Chloramine B trihydrate concentration in the test item vehicle- mixtures revealed that the formulations used for the administration were correctly prepared and measured actual concentrations ranged from 98.7% to 104.4% of the nominal values. The results were well within the admissible limits of 90% to 110%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes , sexually mature (‘proved’) male rabbits of the same breed served as partners.
- Proof of pregnancy: Successful copulation was ascertained by observation. The day of copulation was considered as day 0 of gestation. Only animals with positive signs of copulation were included in the study.

Duration of treatment / exposure:

Day 6 to 28 of gestation

Frequency of treatment:

once daily

Duration of test:

Treatment period Day 6 to 28 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

6 non-pregnant females for the preliminary experiment
120 animals in groups 1 - 5 :
- Treated animals (vehicle control and restricted feeding control incl.): 24 females per group
- Evaluated animals: 20 dams with evaluable litters

Control animals:

yes, concurrent vehicle

other: A restricted feeding control group 5 was employed consisting of untreated dams which were offered the food amount monitored for the high dose dams.

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on the results of a preliminary experiment. In this dose-range-finding study, sodium N-chlorobenzenesulphonamide-Chloramine B was administered to non-pregnant female rabbits by gavage at dose levels of 90 or 180 mg/kg bw/day once daily for 7 days. None of the animals died prematurely. A slight reduction of body weight, marked reduction of food consumption and a reduced discharge of faeces were noted – in relation to the dose – for both treatment groups ( 90 – 180 mg/kg bw/day).
Dose levels of 10, 30 or 90 mg/kg bw/day , administered by gavage once daily from the 6th to 28th day of pregnancy were selected in agreement with the sponsor for the present embryotoxicity study.

- Rationale for animal assignment (if not random):
The animals were randomly assigned to their respective groups according to their mating day i.e. in a cyclic way following the listing of successful copulation.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
- Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rabbit was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time in the morning.
- The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29).

FOOD & DRINK WATER CONSUMPTION: Yes
- The quantity of food consumed by each rabbit was recorded.
- Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.
- The relative food consumption (g/kg bw/day) was calculated.
- Daily monitoring by visual appraisal of the drinking water consumption was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
The ovaries and uteri were removed; the uteri (in toto) were weighed.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and size of resorptions: Yes
- Other: macroscopic inspection (gross evaluation) of the placenta for example for focal indurations; number of fetuses (alive and dead); sex and viability of the fetuses; weight of fetuses

Fetal examinations:

- External examinations: Yes: all per litter
Foetuses were inspected externally for damages, especially for malformations.

- Soft tissue examinations: Yes: all per litter
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities of soft tissue.

- Skeletal examinations: Yes: all per litter
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).

- Head examinations: Yes: half per litter
The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head structures.
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.

Statistics:

For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test. When the variances were homogeneous, the DUNNETT test (p ≤ 0.05 or p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT’s t-test was carried out, limits of significance were p ≤ 0.05 or p ≤ 0.01.
For the comparison of classification measurements (for example malformation-, resorption-, retardation-, and variation rate) the FISHER’s exact test (n < 100) or chi2-test with YATES’ correction for continuity (n ≥ 100) (p ≤ 0.05 or p ≤ 0.01) was employed.
All data were evaluated statistically in this manner.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reports number of decimal places. Hence, deviations to the last decimal place up to ± 1 may occur caused by rounding.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Transient slight or moderate reductions were noted for the body weight and food intake of the dams treated with 30 mg Sodium N-chlorobenzenesulphonamide – Chloramine B /kg bw/day. Distinct reductions of body weight and food consumption, especially during the first days of treatment, were noted at 90 mg/kg bw/day. Similar reductions of body weight and food intake were noted in the untreated dams with restricted feeding.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

he severely materno-toxic dose level of 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw /day resulted in developmental retardations in the form of reduced placental and fetal body weights compared to the control.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A dose level of 30 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw /day resulted in increased incidences of total skeletal retardations, in particular sternebra(e) not ossified.
No test item related malformations were noted during external, internal or skeletal examination (according to DAWSON) of the fetuses or soft tissue examination of the fetal heads (according to WILSON) at any tested dose level, not even at materno-toxic dose levels.
No embryotoxic changes were noted for the restricted feeding group.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Key result

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test item Sodium N-chlorobenzenesulphonamide – Chloramine B possessed no teratogenic properties.
Sodium N-chlorobenzenesulphonamide – Chloramine B revealed slight embryotoxicity at materno-toxic dose levels (30 or 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw/day).

Executive summary:

Sodium N-chlorobenzenesulphonamide – Chloramine B (trihydrate) was administered to female rabbits at dose levels of 10, 30 and 90 mg/kg bw/day orally, by gavage from the 6th to 28th day of pregnancy. In addition, a restricted feeding control was employed consisting of untreated dams which were offered the food amount consumed by the high dose dams. Under the present test conditions, the no-observed-effect level (NOEL) was 10 mg/kg bw/day for the dams. Transient slight or moderate reductions were noted for the body weight and food intake of the dams treated with 30 mg /kg bw/day. Distinct reductions of body weight and food consumption, especially during the first days of treatment, were noted at 90 mg/kg bw/day. Similar reductions of body weight and food intake were noted in the untreated dams with restricted feeding.

The no-observed-effect level (NOEL) for the fetal organism also was 10 mg Sodium N-chlorobenzenesulphonamide-Chloramine B (trihydrate)/ kg bw /day. A dose level of 30 mg/ kg bw /day resulted in increased incidences of total skeletal retardations, in particular sternebra(e) not ossified. The severely materno-toxic dose level of 90 mg/kg bw /day resulted in developmental retardations in the form of reduced placental and fetal body weights compared to the control. Skeletal examination (according to DAWSON) revealed increased fetal incidences for variations of the sternum (fusions) and total skeletal variations as well as retardations of the sternum and lumbar vertebral bodies (incomplete or missing ossification) and total skeletal retardations.

No test item related malformations were noted during external, internal or skeletal examination (according to DAWSON) of the fetuses or soft tissue examination of the fetal heads (according to WILSON) at any tested dose level, not even at materno-toxic dose levels.

No embryotoxic changes were noted for the restricted feeding group. In conclusion, the test item possessed no teratogenic properties.
Chloramine B trihydrate revealed slight embryotoxicity at materno-toxic dose levels (30 or 90 mg Sodium N-chlorobenzenesulphonamide – Chloramine B/ kg bw/day).