A reaction mixture containing the following components;A. Tetralithium [2(or 3), 9 (or 10), 16 (or 17), 23 (or 24)-tetrakis (3-sulfonato-propylsulfonyl) phthalocyaninato]cupurate (II)B. Trilithium [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl]tris (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)C.Dilithium bis [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] bis (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)D. Lithium tris [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)E. Tetrakis [3-(2-hydroxpropylsulfamoyl) proppylsulfonyl]phthalocyaninatocupurate (II)In the ratio A:B:C:D:E 6.25 : 25 : 37.5 : 25 : 6.25

Toxicity to reproduction

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 08 March 2011 and 04 November 2011. The in-life phase of the study was conducted between 22 March 2011 (first day of treatment) and 06 May 2011 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 study was conducted under GLP conditions.

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 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The study was also designed to be compatible with the requirements of 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A 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 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 323 to 392g, the females weighed 187 to 223g, and were approximately 12 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, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. 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. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. was used. Certificates of analysis of the batches of diet used are given in 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. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd. UK 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:

For 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 determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results 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 S194055 at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in attached Appendix 17. The results indicate that the prepared formulations were within ± 5% 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 S194055 in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Duration of treatment / exposure:

Up to forty five consecutive days.

Frequency of treatment:

Daily.

Details on study schedule:

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 Studyi)Groups of up to 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)On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.iii)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.iv)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.v)The surviving males from each dose group were killed and examined macroscopically on Day 43.vi)At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:0 mg/kg bw/dayBasis:actual ingested0 mg/ml

Doses / concentrations 2

Remarks:

Doses / Concentrations:100 mg/kg bw/dayBasis:actual ingested20 mg/ml

Doses / concentrations 3

Remarks:

Doses / Concentrations:300 mg/kg bw/dayBasis:actual ingested60 mg/ml

Doses / concentrations 4

Remarks:

Doses / Concentrations:1000 mg/kg bw/dayBasis:actual ingested200 mg/ml

No. of animals per sex per dose:

10 animals per sex per dose (including control)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on previous toxicity data.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

ObservationsClinical ObservationsAll animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon 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 and public holidays (except for females during parturition where applicable). All observations were recorded.Body WeightIndividual body weights were recorded on Day 1 (prior to dosing) and then weekly for all surviving 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 2.Food ConsumptionDuring the maturation period, weekly food consumption was recorded for each cage of adults until pairing. 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 during the lactation period (Days 1-4). Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males 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 attached Addendum 2.Water ConsumptionWater intake was observed daily by visual inspection of water bottles for any overt changes. A possible treatment-related effect on water intake was detected during Week 1 of treatment, therefore daily gravimetric measurements were initiated from Day 8 onwards.Reproduction PerformanceNormal range data for reproductive parameters and offspring are presented in attached Addendum 2 and Addendum 3.MatingAnimals 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 ParturitionEach 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 parturitionLitter 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.

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:

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:

no effects observed

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)

MortalityOne male treated with 1000 mg/kg bw/day was killed in extremis on Day 14. There were no further unscheduled deaths attributed to test item toxicity.One control male was killed in extremis on Day 9. In the absence of treatment with the test item the cause of death for this animal is likely to be related to the process of gavage administration and as such is not considered to be of any toxicological significance.Clinical ObservationsA summary incidence of clinical observations is given in attached Table 2. Individual clinical observations are presented in attached Appendix 1. Prior to being killed in extremis the high dose male showed hunched posture, lethargy, pilo-erection, pallor of the extremities, dehydration and respiratory pattern changes. Prior to being killed in extremis the control male was observed to have hunched posture, pilo-erection, lethargy, pallor of the extremities and a reduced respiratory rate. Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day with sporadic episodes noted in the 300 mg/kg bw/day males and an isolated incidence also noted in one 100 mg/kg bw/day male. Wet bedding was detected in cages of animals of either sex treated with 1000 mg/kg bw/day. Blue coloured faeces, staining that was observed in the cages or fur staining by the test item is considered to be a result of the excretion of the test item and/or its metabolites and as such is not considered to be toxicologically significant.Body WeightGroup mean body weights, body weight changes and standard deviations for males and group mean body weights, body weight changes and standard deviations during the pre-mating, gestation and lactation phases for females are given in attached Table 3 and Table 4 (statistically significant differences are indicated). Group mean body weights are presented graphically in attached Figure 1 and Figure 2. Individual data are given in attached Appendix 2 and Appendix 3.Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (P<0.05) in body weight gain during the lactation period in comparison to controls. A statistically significant reduction in body weight gain was evident in males treated with 1000 mg/kg bw/day during Weeks 1 and 2 of treatment (P<0.05, P<0.01 respectively) with an increase noted in Week 3 (P<0.05), in comparison to controls. There was no adverse effect detected in overall body weight gain between the treatment and control animals. In the absence of an effect on dietary intake or supporting histopathological correlates this finding is considered to be of no toxicological significance.Food Consumption and Food EfficiencyGroup mean food consumptions for males and group mean food consumptions during the pre-mating, gestation and lactation phases for females are given in attached Table 5 and are presented graphically in attached Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in attached Appendix 4. Food efficiency for males and for females during maturation is presented in attached Table 6. No adverse effect on dietary intake or food efficiency was detected for treated animals, in comparison to controls.Water ConsumptionGroup mean water consumptions are given in attached Table 7 (statistically significant differences are indicated). Individual daily water consumptions for females following mating and lactation are presented in attached Appendix 5.A marked increase in water intake was evident in animals of either sex treated with 1000 mg/kg bw/day. An increase in water intake was also evident in animals of either sex treated with 300 and 100 mg/kg bw/day albeit to a lesser extent in comparison to controls.NecropsyNo adverse macroscopic abnormalities were detected in the treatment or control animals at terminal kill. The control male killed in extremis had a yellow, fluid filled mass approximately 20 x 20mm in size in the thoracic region. This finding is considered to be a result of involuntary injury which occurred during the administration of the control item and as such is not considered to be of any toxicologicalsignificance. Blue coloured staining and blue coloured contents of a number of internal tissues was evident throughout the treatment animals of either sex. Such findings are consistent with the administration of a coloured test item and/or its metabolites and as such are not considered to be of any toxicological importance. There were no abnormalities detected in the remaining animals.Organ WeightsGroup mean absolute and body weight relative male reproductive organ weights and standard deviations for test and control group animals are presented in attached Table 14. Individual data are given in attached Appendix 12 and Appendix 13.No treatment-related effect on organ weight measurement was detected for treated animals, in comparison to controls.HistopathologyA complete histopathology report is presented in attached Appendix 16.MORTALITIES AND NON-PREGNANT ANIMALSNo microscopic findings were found in the tissues examined of animals 4 and 63 killed in extremis which could have contributed to their poor condition therefore the causes of their condition were not established.Females 75, 77 and 80 treated with 1000 mg/kg bw/day were all found to be non-pregnant and this was confirmed by an absence of histopathological signs of pregnancy for each of these animals. These non-pregnant animals were paired respectively with males 65, 67 and 70. There were no morphological abnormalities in the non-pregnant females and paired males that were considered to have resulted in impaired reproductive performance. A total litter loss was seen for female 79 treated with 1000 mg/kg bw/day.TREATMENT-RELATED HISTOPATHOLOGYThe following treatment-related changes were observed in the kidney:Tubular basophilia, although found in untreated animals, is considered to represent a degenerative/regenerative change but the severity seen in the treated animals in this study is usually reversible. Interstitial mononuclear cells are an indication of an inflammatory response in the interstitium and the slightly higher incidence and severity in the treated animals was considered to be related to the test item.Corticomedullary mineralization is seen in the untreated female rat but factors affecting the composition of tubular filtrate can cause a higher severity as seen in some of the treated animals. Dilatation of the cortical tubular epithelium is usually adverse and non-reversible. It can be considered as one of the first stages of degenerative kidney disease.Tubular inflammatory cells in the lumen indicate an inflammatory process somewhere along the length of the tubule and are not seen in the untreated laboratory rats. Interstitial fibrosis is an indication of an inflammatory response which has taken place and in which connective tissue has taken the place of normal tissue where the latter has been compromised. OTHER HISTOPATHOLOGYNo treatment related findings were detected in the reproductive organs and the few other findings are those commonly found in the untreated laboratory rat of this strain and age and were considered to be of no toxicological importance.MatingA summary of adult performance is presented in attached Table 1. Group values and summary indices for mating performance are presented in attached Table 8. Individual data are given in attached Appendix 6.There were no intergroup differences in mating performance or gestation lengths.FertilityGroup values for fertility, litter data and implantation losses are given in attached Tables 8 to 10. Individual data are given in attached Appendices 6 to 8.A reduction in fertility was evident in the 1000 mg/kg bw/day females with seven out of ten females achieving pregnancy. Pregnancy was achieved for all control, 100 and 300 mg/kg bw/day females. All pregnant females went on to give birth resulting in live litters.Gestation LengthA summary incidence of gestation lengths is given in attached Table 8. Individual lengths are given in attached Appendix 6.There was no adverse effect on gestation lengths between treatment and control females. Females from all treatment groups went on to give birth following 22 to 23.5 days of gestation.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive/developmental toxicity

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Resulted in toxicologically significant systemic effects at all treatment levels. A ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore not established.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

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 ResponseIn total ten females from the control, 100 and 300 mg/kg bw/day and six females from the 1000 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. One 1000 mg/kg bw/day female gave birth but went on to have a total litter loss on Day 2 of lactation. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.Offspring Litter Size and ViabilityGroup mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in attached Tables 9 to 11. Individual data are given in attached Appendices 7 to 9.Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (P<0.01) in offspring viability in comparison to controls.No such effect was detected in the 300 or 100 mg/kg bw/day dose groups. There were no adverse differences in the number of corpora lutea or implantation sites from treated females when compared to controls, and pre-implantation losses from treated animals were comparable to controls. No obvious effect on littler size and sex ratio were detected from treatment animals, in comparison to controls. An increase in the number of corpora lutea was evident for 100 mg/kg bw/day females in comparison to controls. In the absence of a dose-related response this finding is considered to be of no toxicological significance.Offspring Growth and DevelopmentGroup mean values for total litter weights, offspring body weights and body weight changes, surface righting reflex and a summary incidence of clinical signs are given in attached Tables 9, 12 and 13. Individual values and observations are given in attached Appendices 7, 10 and 11.No adverse effect on total litter weights, offspring body weight development or surface righting reflex was detected in treatment animals, in comparison to controls.NecropsyA summary incidence of necropsy findings for offspring is given in attached Table 15. Individual data are given in attached Appendix 14 .The observations detected in the control, low and intermediate offspring were considered to be incidental and of no toxicological importance. The findings detected in three litters from the 1000 mg/kg bw/day dose group including pups found dead and missing are considered to be related to maternal toxicity.At terminal kill one female treated with 300 mg/kg bw/day produced a litter with one pup which showed a reddened left testis. In addition, five males and four female offspring from one 1000 mg/kg bw/day litter were observed with blue external staining.The following interim death animals showed autolytic changes; four male and two female pups from one high dose litter, two females from one intermediate dose litter and two pups of either sex from one control litter. An additional interim death female pup from the 300 mg/kg bw/day group was observed to have no milk in its stomach whilst a further interim death male pup from the 100 mg/kg bw/day group had no abnormalities detected.There were no abnormalities detected in the remaining animals.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This is for the reproduction and offspring growth

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

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 non-rounded 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:

Body weight and body weight change

Food consumption for females during gestation and lactation

Water consumption for females 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

Adult absolute and body weight relative organ weights (Males)

The following statistical procedures were used:

Data for males and females prior to pairing, where appropriate, quantitative data 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’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 nonhomogeneity 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 also 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 *

p³0.05 (not significant)

In addition, histopathological findings were analysed (excluding any decedent, non-mated females and females not producing a pregnancy/litter) using Armitage Cochran analysis, and/or Fisher’s exact test.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test sustance to rats for a period of up to forty-five consecutive days at dose levels of up to 1000 mg/kg bw/day, resulted in toxicologically significant systemic effects at all treatment levels. A ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore not established. The effects identified in the reproductive parameters measured at 1000 mg/kg bw/day are considered to be a result of adult/maternal toxicity. A ‘No Observed Effect Level’ (NOEL) for reproduction was therefore considered to be 300 mg/kg bw/day.

Executive summary:

Introduction.

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with the requirements of 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 forty five consecutive days (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, body weight change, dietary intake 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.

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

Results.

Adult Responses:

Mortality.One male treated with 1000 mg/kg bw/day was killed in extremis on Day 14.

There were no further unscheduled deaths attributed to test item toxicity.

One control male was killed in extremis on Day 9. In the absence of treatment with the test item the cause of death for this animal is likely to be related to the process of gavage administration and as such is not considered to be of any toxicological significance.

Clinical Observations.Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day and males treated with 300 and 100 mg/kg bw/day. Wet bedding was detected in cages of animals of either sex treated with 1000 mg/kg bw/day.

Body Weight.Females treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the lactation period in comparison to controls.

No such effect was detected in females treated with 300 or 100 mg/kg bw/day.

Food Consumption and Food Efficiency.No adverse effect on dietary intake or food efficiency was detected for treated animals, in comparison to controls.

Water Consumption.A marked increase in water intake was evident in animals of either sex treated with 1000 mg/kg bw/day. An increase in water intake was also evident in animals of either sex treated with 300 and 100 mg/kg bw/day albeit to a lesser extent in comparison to controls.

Reproductive Screening:

Mating, Gestation and Fertility.There were no intergroup differences in mating performance or gestation lengths.

Pregnancy was achieved for all control, 100 and 300 mg/kg bw/day females and seven 1000 mg/kg bw/day females.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.There was no treatment-related difference on litter size or sex ratio for treated females in comparison to controls.

A reduction in viability was evident in the 1000 mg/kg bw/day litters in comparison to controls.

No such effect was detected in the 300 or 100 mg/kg bw/day treatment groups.

Offspring Growth and Development.There was no adverse effect on offspring growth, body weight development or surface righting reflex detected for treatment animals, in comparison to controls.

Offspring Observations.The incidental findings detected in the control, low and intermediate dose groups were considered to be unrelated to treatment. The findings detected in three litters from the 1000 mg/kg bw/day dose group including pups found dead and missing are considered to be related to maternal toxicity.

Pathology:

Necropsy.The incidental signs detected in the adult and offspring were considered to be of no toxicological importance.

Uterine Examination.From evaluation of the corpora lutea and implantation data, three females treated with 1000 mg/kg bw/day showed no corpora lutea or implantation sites, therefore were never pregnant.

Organ Weights.No adverse effect on organ weight measurement was detected for treated animals in comparison to controls.

Histopathology.Macroscopic examination of the tissues revealed the following kidney effects:

An increased incidence and severity of tubular basophilia was detected in animals of either sex from all treated groups. Interstitial mononuclear cells were seen in animals of either sex treated with 300 and 1000 mg/kg bw/day. An increased severity of corticomedullary mineralisation was evident in females from all treated groups. Tubular dilatation was seen in three females treated with 100 mg/kg bw/day, one 300 mg/kg bw/day male and animals of either sex treated with 1000 mg/kg bw/day. Tubular inflammatory cells were noted in animals of either sex treated with 1000 mg/kg bw/day. Interstitial fibrosis was evident in the 300 mg/kg bw/day females and animals of either sex treated with 1000 mg/kg bw/day.

There were no treatment-related changes in the reproductive organs examined.

No microscopic abnormalities were detected which could have contributed to the poor condition of the two animals killed in extremis.

Conclusion.The oral administration of S194055 to rats for a period of up to forty-five consecutive days at dose levels of up to 1000 mg/kg bw/day, resulted in toxicologically significant systemic effects at all treatment levels. A ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore not established.

The effects identified in the reproductive parameters measured at 1000 mg/kg bw/day are considered to be a result of adult/maternal toxicity. A ‘No Observed Effect Level’ (NOEL) for reproduction was therefore considered to be 300 mg/kg bw/day.