Sodium sulphamidate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2011 and 08 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the studywas conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphamate

IUPAC Name:

Sodium sulphamate

Details on test material:

Sponsor's identification:Sodium sulphamateIdentifier:TIS 10442Description:White solidChemical name:Sodium SulphamateCAS number:13845-18-6Purity:99.67%Batch number: LE12568Label:Sodium sulfamate (freeze dried)Date received :06 May 2011Storage conditions:Room temperature in the dark and over silica gelExpiry date:06 May 2013

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal HusbandryA sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from a reputable supplier. On receipt the animals were examined for signs of ill-health or injury.  The animals were acclimatised for a total of twelve days during which time their health status was assessed.  A total of eighty animals (forty males and forty females) were accepted into the study.  At the start of treatment the males weighed 299 to 371g, the females weighed 190 to 233g, and were approximately twelve weeks old.Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.  During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group.  Following evidence of successful mating, the males were returned to their original cages.  Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.The animals were allowed free access to food and water. Certificates of analysis of the batches of diet used are given in the attached Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage.  The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.  Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for mated females during gestation and lactation.The animals were housed in a single air-conditioned room within the Laboratories Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

water

Details on exposure:

Preparation of Test ItemFor the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were previously determined. Results from a previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.Samples of each test item formulation were taken and analysed for concentration of Sodium sulphamate at Harlan Laboratories Ltd. Analytical Services. The method used for analysis of formulations and the results obtained are given in the attached Appendix 26. The results indicate that the prepared formulations were within ± 6% of the nominal concentration

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Sodium sulphamate, in the test item formulations was determined by ion chromatography (IC) using an external standard. The test item gave a chromatographic profile consisting of a single peak. The results indicate that the prepared formulations were within ± 6% of the nominal concentration For full details please see attached Appendix 26 - Chemical Analysis of Test Item Formulations, Methods and Results

Duration of treatment / exposure:

The oral administration of the test substance to rats for a period of up to fifty-four consecutive days

Frequency of treatment:

Daily

Details on study schedule:

Procedure:The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.  Control animals were treated in an identical manner with 5 ml/kg bw/day of Distilled water.The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.Chronological Sequence of Study:i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).  The first day of dosing was designated as Day 1 of the study.ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.  Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42.  Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.ix) Blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum.  At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:100 mg/kg bw/day (20 mg/ml)Basis:actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:300 mg/kg bw/day (60 mg/ml)Basis:actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:1000 mg/kg bw/day (200 mg/ml)Basis:actual ingested

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

ObservationsClinical Observations:All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week.  Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).  All observations were recorded.Functional Observations:Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity.  Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli. Behavioural Assessments:Detailed individual clinical observations were performed for each animal using a purpose built arena.  The following parameters were observed:GaitHyper/HypothermiaTremorsSkin colourTwitchesRespirationConvulsionsPalpebral closureBizarre/Abnormal/Stereotypic behaviourUrinationSalivationDefecationPilo-erectionTransfer arousalExophthalmiaTail elevationLachrymation This test was developed from the methods used by Irwin (1968) and Moser et al (1988).  Functional Performance Tests:Motor Activity:  Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity.  Animals were randomly allocated to the activity monitors.  The tests were performed at approximately the same time each day, under similar laboratory conditions.  The evaluation period was thirty minutes for each animal.  The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).Forelimb/Hindlimb Grip Strength.: An automated meter was used.  Each animal was allowed to grip the proximal metal bar of the meter with its forepaws.  The animal was pulled by the base of the tail until its grip was broken.  The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar.  The animal was pulled by the base of the tail until its grip was broken.  A record of the force required to break the grip for each animal was made.  Three consecutive trials were performed for each animal.  The assessment was developed from the method employed by Meyer et al (1979).Sensory Reactivity:Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.  This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).  The following parameters were observed:Grasp responseTouch escapeVocalisationPupil reflexToe pinchBlink reflex Tail pinchStartle reflex Finger approach Normal range data for the functional performance test parameters are presented in attached Addendum 6.Body Weight:Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident.  Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.  Normal range data for body weight changes in pregnant and lactating females are presented in attached Addendum 3.Food Consumption:During the maturation period, weekly food consumption was recorded for each cage of adults.  This was continued for males after the mating phase.  For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.  For females with live litters, food consumption was recorded on Days 1 and 4 post partum.  Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase.  Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.Normal range data for pregnant and lactating females are presented in the attached Addendum 3.Water Consumption:Water intake was measured daily throughout the study (with the exception of the mating phase).Reproduction Screening:Normal range data for reproductive parameters and offspring are presented in the attached Addendum 3 and attached Addendum 4.Mating:Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days.  Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina.  A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.  The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.Pregnancy and Parturition:Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition.  Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays.  The following was recorded for each female:i) Date of pairingii) Date of matingiii) Date and time of observed start of parturitioniv) Date and time of observed completion of parturitionLaboratory Investigations:Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).  Blood samples were obtained from the lateral tail vein.  Where necessary repeat samples were taken by cardiac puncture at termination.  Animals were not fasted prior to sampling.  The methods used for haematological and blood chemical investigations are given in the attached Addendum 2 and normal ranges are shown in the attached Addendum 5.

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: During histopathology, the male testes and epididymides were examined for spermatocoel granuloma formation.

Litter observations:

Litter DataOn completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.For each litter the following was recorded:i)Number of offspring bornii)Number of offspring alive recorded daily and reported on Days 1 and 4 post partum iii)Sex of offspring on Days 1 and 4 post partumiv)Clinical condition of offspring from birth to Day 5 post partumv)Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)Physical DevelopmentAll live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

PathologyAdult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.  Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum.  Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.  Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.  This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (offspring):

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Due to the nature & quantity of this data please see section “any other results including tables”

Reproductive indices:

Mating Performance and Fertility:The following parameters were calculated from the individual data during the mating period of the parental generation. i) Pre-coital Interval Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating. ii) Fertility Indices For each group the following were calculated: Mating Index (%) = (Number of animals paired ÷ Number of animals mated) x 100 Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100 Gestation and Parturition Data :The following parameters were calculated for individual data during the gestation and parturition period of the parental generation. i) Gestation Length Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. ii) Parturition Index The following was calculated for each group: Parturition Index (%) = (Number of pregnant females ÷ Number of females delivering live offspring) x 100

Offspring viability indices:

Litter Responses:The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age). i) Implantation Losses (%) Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows: % pre – implantation loss = [(Number of corpora lutea - Number of Corpora Lutea) ÷ Number of implantation sites] x 100 % post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100 ii) Live Birth and Viability Indices The following indices were calculated for each litter as follows: Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100 Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100 iii) Sex Ratio (% males) Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula: (Number of male offspring ÷ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Mortality:One female treated with 100 mg/kg bw/day died during the bleeding procedure. The exact cause of death of this animal could not be established microscopically therefore, in the absence of similar findings in animals treated with 1000 mg/kg bw/day this death was considered to be incidental. There were no further unscheduled deaths.Clinical Observations:A summary incidence of clinical observations is given in attached Table 2. Individual data are given in attached Appendix 1.There were no clinical signs of toxicity detected in any treated animal.One male treated with 1000 mg/kg bw/day showed an isolated incident of increased salivation on Day 28. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation is considered not to be of toxicological importance. Functional Observations:Summary incidence of behavioural assessment observations are given in attached Table 3 and group mean behavioural assessment scores are given in Table 4. Group mean functional test values and standard deviations are given in attached Table 5 (statistically significant differences are indicated).Individual values are given in attached Appendices 2 to 5. Group mean sensory reactivity assessment scores are given in attached Table 6. Individual responses are given in attached Appendix 6.Behavioural Assessments:Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.Functional Performance Tests:There were no toxicologically significant changes in functional performance.Females from all treatment groups showed a statistically significant reduction in overall activity. The majority of individual values were within the normal range for rats of the strain and age used and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in mean fore limb grip strength and mean hind limb grip strength. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.Sensory Reactivity Assessments:There were no treatment-related changes in sensory reactivity.Body Weight:Group mean body weights, body weight changes and standard deviations are given in attached Table 7 and attached Table 8 (statistically significant differences are indicated). Group mean body weight data are presented graphically in attached Figure 1 and attached Figure 2. Individual data are given in attached Appendix 7 and attached Appendix 8.Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first and final weeks of treatment. A slight reduction in overall body weight gain was also evident in these males. No toxicologically significant effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.Females treated with 1000 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 2 of maturation. A slight reduction in body weight gain was evident in these females during the first week of treatment. This may have influenced the increase seen during Week 2 and in the absence of any effect on absoulute or percent gain between Days 1 and 15 the increase cannot be regarded as an adverse effect of treatment and as such, the intergroup difference was considered to be of no toxicological importance. Food Consumption and Food Efficiency:Group mean food consumptions are given in attached Table 9 and are presented graphically in attached Figure 3 and attached Figure 4. Individual and group mean food consumptions for females following mating and during lactation are presented in attached Appendix 9.Food efficiencies for males and females during the pre-mating phase are given in attached Table 10.Males treated with 1000 mg/kg bw/day showed a slight reduction in overall food consumption when compared to controls. A slight reduction in food efficiency was also evident in these males during the first and final weeks of treatment. No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.Water Consumption:Group mean water consumptions are given in attached Table 11. Individual and group mean water consumptions for females following mating and lactation are presented in attached Appendix 10.There was no adverse effect on water consumption throughout the treatment period.Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in water consumption during the first week of gestation. In the absence of any associated changes the isolated intergroup difference was considered not to be of toxicological importance.Reproductive Performance:Mating:A summary of adult performance is given in Table 1. Group values and summary incidence for mating performance are presented in Table 12. Individual data are given in Appendix 11.There were no treatment-related effects on mating performance.Fertility:A summary of adult performance is given in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 13 and 14. Individual data are given in Appendices 11 to 13.No treatment-related effects on fertility were detected for treated animals when compared to controls.One female treated with 300 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs however, the male partner for this female showed bilateral spermatic granulomas in the epididymides (marked severity on right side) and a marked diffuse tubular atrophy in the left testis. These lesions were considered to be the reason for the non pregnancy.Gestation Length:A summary incidence of gestation lengths is given in Table 12. Individual gestation lengths are given in Appendix 11.No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22 to 23½ days.Haematology:Group mean values and standard deviations for test and control group animals are given in attached Table 18 (statistically significant differences are indicated). Individual data are given in attached Appendix 17 and attached Appendix 18.Females treated with 1000 mg/kg bw/day showed statistically significant increases in haemoglobin, haematocrit and clotting time. No such effects were detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant reduction in neutrophil count. The majority of individual values were within the normal range for rats of the strain and age used and as such the intergroup differences were considered not to be of toxicological importance. Blood Chemistry:Group mean values and standard deviations for test and control group animals are given in attached Table 19 (statistically significant differences are indicated). Individual data are given in attached Appendices 19 and 20.There were no toxicologically significant effects detected in the blood chemical parameters examined.Males treated with 300 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio. In isolation and in the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.Organ Weights:Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in attached Table 20 (statistically significant differences are indicated). Individual data are given in attached Appendix 21 and attached Appendix 22.Females treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight.No such effects were detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.Females treated with 1000 and 300 mg/kg bw/day showed statistically significant reductions in absolute and relative adrenal and ovary weight. Females treated with 1000 mg/kg bw/day also showed a statistically significant reduction in absolute and relative thyroid/parathyroid weight. In the absence of true dose related responses or any associated histology correlates the intergroup differences were considered not to be of toxicological significance. Necropsy:A summary incidence of necropsy findings is given in attached Tables 22. Individual data are given in attached Appendix 24. AdultsThere were no toxicologically significant macroscopic abnormalities detected.One male treated with 300 mg/kg bw/day had an enlarged right caudate lobe of the epididymis together with a mass containing green fluid and flaccid testes. One female treated with 100 mg/kg bw/day had increased pelvic space in both kidneys. In the absence of similar effects detected in 1000 mg/kg bw/day animals the intergroup differences were considered not to be of toxicological importance.The female treated with 100 mg/kg bw/day that died during the bleeding procedure had reddened lungs at necropsy. Histopathology:A complete histopathology report is presented in attached Appendix 25.The following microscopic findings were detected:Spleen: minimal increased hematopoesis was detected in animals of either sex treated with 1000 mg/kg bw/day. Bone Marrow: increased incidence and mean severity of erythroid hyperplasia was evident in animals of either sex treated with 1000 mg/kg bw/day. This finding was associated with missing adipocytes in females treated with 1000 mg/kg bw/day.Kidneys: minimal, mainly focal and multifocal occurring basophilic tubules were evident in females treated with 1000 mg/kg bw/day.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

2

Key result

Dose descriptor:

NOEL

Remarks:

reproductive/developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive/developmental toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Litter Response:In total all females from the control and 1000 mg/kg bw/day dose groups and nine females from the 100 and 300 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.Offspring Litter Size and Viability:Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in attached Tables 13 to 15. Individual data are given in attached Appendices 12 to 14.No toxicologically significant effects were detected.No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.Offspring Growth and Development:Group values for total litter weights, offspring body weight and body weight change, surface righting reflex and a summary incidence of clinical signs are given in attached Tables 13, 16 and 17. Individual values and observations are given in attached Appendices 12, 15 and 16.No toxicologically significant effects were detected.There were no significant differences in litter, offspring weights or surface righting reflex. Statistical analysis of the data did not reveal any significant intergroup differences.No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, no milk in stomach, cold, weak, pale and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.NecropsyA summary incidence of necropsy findings is given in attached Tables 21. Individual data are given in attached Appendix 23. OffspringNo treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 1 000 other: mg/kg bw/day for treated parental animals, F1 offspring were not treated

Based on:

other: Test material received by parental animals

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: No test item related finding in F1 offspring reared to day 4pp from treated parental animals

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Evaluation of Data

Treatment of Data

Data were processed to give group mean values and standard deviations where appropriate.

Data shown in the appendices are frequently rounded values for presentation purposes.  Group mean values are generally calculated using unrounded values therefore is it not always possible to calculate the exact group values from the individual values presented in the appendices.

For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.

For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Statistical Analysis

The following parameters were subjected to statistical analysis:

Quantitative functional performance data

Body weight and body weight change

Food consumption during gestation and lactation

Pre-coital interval and gestation length

Litter size and litter weights

Sex ratio

Corpora lutea and implantation sites

Implantation losses and viability indices

Offspring body weight and body weight change

Offspring surface righting

Haematology, blood chemistry, adult absolute and body weight-relative organ weights

The following statistical procedures were used:

Data for males and females prior to pairing, and functional performance test data, where appropriate, were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test.  Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.

Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (P) were calculated as follows:

P<0.001 ***

P<0.01 **

P<0.05 *

p ≥0.05 (not significant)

Applicant's summary and conclusion

Conclusions:

The oral administration of Sodium sulphamate to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.

One female treated with 100 mg/kg bw/day died during the bleeding procedure. There were no further unscheduled deaths.

Clinical Observations.

No clinical signs of toxicity were detected in any treated animal.

Behavioural Assessment.

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Body Weight.

Males treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the first and final week of treatment. A slight reduction in overall body weight gain was also evident in these males.  

No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption.Males treated with 1000 mg/kg bw/day showed a slight reduction in overall food consumption. A slight reduction in food efficiency was also evident in these males during the first and final weeks of treatment.

No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Water Consumption.

No adverse effect on water consumption was detected.

Reproductive Performance:

Mating.

There were no treatment-related effects on mating for treated animals.

Fertility.

There were no treatment-related effects on conception rates for treated animals.

Gestation Lengths.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability

.Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls. There were no intergroup differences in sex ratio.

Offspring Growth and Development.

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. No effect on surface righting reflex was detected. 

Offspring Observations.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Laboratory Investigations:

Haematology.

Females treated with 1000 mg/kg bw/day showed an increase in haemoglobin, haematocrit and clotting time when compared to control animals. No such effects were detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Blood Chemistry.

There were no toxicologically significant effects detected in the blood chemical parameters measured.

Pathology:

Necropsy.

No toxicologically significant macroscopic abnormalities were detected.

Organ Weights.

Females treated with 1000 mg/kg bw/day showed an increase in kidney weight both absolute and relative to terminal body weight.

No such effects were detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Histopathology.

The following microscopic findings were detected:

Spleen:

minimal increased hematopoesis was detected in animals of either sex treated with 1000 mg/kg bw/day.

Bone Marrow:

increased incidence and mean severity of erythroid hyperplasia was evident in animals of either sex treated with 1000 mg/kg bw/day. This finding was associated with missing adipocytes in females treated with 1000 mg/kg bw/day.

Kidneys:

minimal, mainly focal and multifocal occurring basophilic tubules were evident in females treated with 1000 mg/kg bw/day.

Conclusion.

The oral administration of Sodium sulphamate to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 1000 mg/kg bw/day.