Reaction products of tetraethylenepentamine and maleic anhydride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-30 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study performed according to OECD guideline 422, in compliance with GLP. No deviations were noted. Only a draft report is available. A final report will be added to the dossier as soon as available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): XTA 823 (reaction product of tetraethylene pentamine and maleic anhydride)- Substance type: yellow slightly viscous liquid- Physical state: liquid- Analytical purity: 49.6%- Purity test date: no data- Lot/batch No.: BLW0010057- Expiration date of the lot/batch: 2015-12-01- Stability under test conditions: no data- Storage condition of test material: approximately 4°C, in the dark; formulated and used at ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: 48 male and 48 female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. - Age at start of treatment: approximately 12 weeks- Weight at start of treatment: 296 to 347 grams for males, 195 to 231 grams for females- Fasting period before study: no data- Housing: all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing 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. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.- Diet (e.g. ad libitum): ad libitum, pelleted diet - Water (e.g. ad libitum): ad libitum, mains drinking water from polycarbonate bottles attached to the cage- Acclimation period: seven days, during which time their health status was assessed.ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 +/- 3°C- Humidity (%): 50 +/- 20%- Air changes (per hr): at least 15 - Photoperiod (hrs dark / hrs light): 12/12, low intensity fluorescent lightingIN-LIFE DATES: From: 2015-04-01 To:2015-05-16

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:- 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 (by visual inspection) of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty days when stored at approximately 4 °C, in the dark. Formulations were therefore prepared approximately fortnightly, aliquoted and stored as above before use. - The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals. VEHICLE- Concentration in vehicle: 0, 40.3, 121.0, 403.2 mg/mg test item (equivlalent to 0, 20, 60, 200 mg/ml active ingredient)- Amount of vehicle (if gavage): 5 ml/kg- Lot/batch no. (if required): no data- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1/1- Length of cohabitation: maximum of 14 days- Proof of pregnancy: 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)- After successful mating each pregnant female was caged (how): mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. - Any other deviations from standard protocol: due to the unscheduled death of male 60 from the intermediate dose group, female 72 from this dose group was paired on day 16 with male 49 from the same dose group; this male had successfully paired with another female from this dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test item formulation were taken and analyzed for concentration of the substance at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The test item concentration in the test samples was determined by high performance liquid chromatography - mass spectrometry (HPLC-MS) using an external standard technique.

Duration of treatment / exposure:

42 days (females), 43 or 44 days (males)

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

active ingredient

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

active ingredient

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

active ingredient

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

active ingredient

No. of animals per sex per dose:

12 males and 12 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were chosen in consultation with the Sponsor, based on the available data from a 14 day dose range-finding study in the rat (Study Number: 41403876). A dose level of 1000 mg/kg bw/day was considered a suitable high dose for this study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).- All animals were examined for overt signs of toxicity, ill-health and behavioral change. All observations were recorded. DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: prior to the start of treatment and at weekly intervals thereafter. 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.- All animals were observed for signs of functional/behavioral toxicity in a purpose built arena.- The following parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophtalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.BODY WEIGHT: Yes- Time schedule for examinations: individual body weights were recorded on day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on days 0, 7, 14 and 20 post coitum, and on days 1 and 4 post partum. Body weights were also recorded at terminal kill.FOOD CONSUMPTION: Yes- During the pre-pairing 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 consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No dataWATER CONSUMPTION: Yes- Water intake was observed daily by visual inspection of water bottles for any overt changes. OTHER:FOOD EFFICIENCY- 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-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation. HAEMATOLOGY- Time schedule for collection of blood: prior to termination (day 42 for males and day 4 post partum for females)- Blood samples were collected from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination.- Anaesthetic used for blood collection: no data- Animals fasted: no- How many animals: five males and five females selected from each test and control group- Blood was collected into tubes containing potassium EDTA anti-coagulant.- The following parameters were examined: hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration), total leukocyte count, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count (methylene blue stained slides were prepared but reticulocytes were not assessed), prothrombin time, activated partial thromboplastin timeCLINICAL CHEMISTRY- Time schedule for collection of blood: prior to termination (day 42 for males and day 4 post partum for females)- Blood samples were collected from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination.- Animals fasted: no - How many animals: five males and five females selected from each test and control group- Blood was collected into tubes containing potassium EDTA anti-coagulant.- The following parameters were examined: urea, glucose, total protein, albumin, albumin/globulin (A/G) ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids.NEUROBEHAVIOURAL EXAMINATION- Time schedule for examinations: prior to the start of treatment and at weekly intervals thereafter. 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.- Dose groups that were examined: all animals- 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 on each occasion (at least two hours after dosing), 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).

Sperm parameters (parental animals):

- testis weight, epididymis weight- Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

PARAMETERS EXAMINEDThe following parameters were examined in offspring:- Number of offspring born - Number of offspring alive recorded daily and reported on days 1 and 4 post partum - Sex of offspring on days 1 and 4 post partum - Clinical condition of offspring from birth to day 5 post partum - Individual offspring weights on days 1 and 4 post partum (litter weights were calculated retrospectively from this data) - All live offspring were assessed for surface righting reflex on day 1 post partum.GROSS EXAMINATION OF DEAD PUPS:- yes, for external and internal abnormalities; any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes- All surviving adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on day 43 or 44. Adult littering females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on day 26 post coitum. - All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.- 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). The corpora lutea were also counted. ORGAN WEIGHTS:- The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: adrenals, brain, epididypides, heart, kidneys, liver, ovaries, pituitary (post fixation), prostate, seminal vesicles, spleen, testes, thymus, thyroid (weighed post fixation with parathyroid), uterus (weighed with cervix).- The following tissues were weighed from all remaining animals: epididymides, ovaries pituitary (post fixation), prostate, seminal vesicles, testes, uterus (weighed with cervix).HISTOPATHOLOGY: Yes- Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (in Modified Davidson's fluid), esophagus, eyes (in Davidson's fluid), gross lesions, heart, ileum (including Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi) (inflated to approximately norma inspiratory volume with buffered 10% formalin before immersion in fixative), lumph nodes (mandibular and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes (in Modified Davidson's fluid), thymus, urinary bladder, uterus/cervix, vagina.- The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. - The following tissues from the remaining control and 1000 mg/kg bw/day animals and animals which did not achive a pregnancy were also processed: coagulating gland, epididymides, gross lesions, mammary gland, ovaries, pituitary, prostate, seminal vesicles, testes, uterus/cervix, vagina.- In addition, sections of testes from all control and 1000 mg/kg bw/day males and males not siring a pregnancy were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

GROSS PATHOLOGY:- Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent at day 5 post partum.- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.ORGAN WEIGHTS:- The following tissues were weighed: epididymides, ovaries pituitary (post fixation), prostate, seminal vesicles, testes, uterus (weighed with cervix).HISTOPATHOLOGY:- The following tissues were preserved: coagulating gland, epididimydes, gross lesios, mammary gland, ovaries, pituitary, prostate, seminal vesicles, testes, uterus/cervix, vagina.

Statistics:

Statistical analysis was performed on: grip strength, motor activity, body weight, body weight change, food consumption during gestation and lactation, pre-coital interval, gestation length, litter size, litter weight, sex ratio, corpora lutea, implantation sites, implantation losses, viability indices, offspring body weight, offspring body weight change, offspring surface righting, hematology, blood chemistry, absolute organ weights, body weight-relative organ weights. Data were analyzed using the decision tree from the Provantis(TM) Tables and Statistics Module as detailed as follows: where appropriate, data transformations were performed using the most suitable method. Homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test (parametric data) or the Shirley Test (nonparametric data). If no dose response was found but data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of homogeneity of the data using Bartlett's test. Where considered appropriate, analysis of the data was applied incorporating analysis of variance (ANOVA) (parametric) followed by pair-wise comparisons using Dunnett's test (if significantnt) or the Kruskall-Wallis test (nonparametric) followed by the Mann-Whitney U test.

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 mated/Number of animals paired)x100; Pregnancy Index (%) = Number of pregnant females/Number of animals mated)x100 Gestation and Parturition Data:- The following parameters were calculated from 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 females delivering live offspring/Number of pregnant females)x100

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 implantation sites)/Number of corpora lutea)x100 Post–implantation loss (%) = ((Number of implantation sites) -Total number of offspring born)/Number of implantation sites)x100 ii. Live Birth and Viability Indices: the following indices were calculated for each litter as follows: Live Birth Index (%) = (Number of offspring alive on day 1/Number of offspring born)x100 Viability Index (%) = (Number of offspring alive on day 4/Number of oofspring alive on day 1)x100 iii. Sex Ratio (% males): sex ratio was calculated for each litter value on days 1 and 4 post partum, using the following formula: (Number of male offspring/Total number of offspring)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

- There were no clinical observations related to treatment with the test item at any dose level. - One male receiving the test item (active ingredient) at a dose level of 100 mg/kg bw/day showed clinical signs of noisy respiration between days 23 to 24 and 32 to 41 of dosing. None of the remaining animals on the study, which survived until the scheduled necropsy, showed any clinical signs and as such this observation was deemed unlikely to be related to treatment with the test item.- There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level. Male 60 given 300 mg/kg bw/day, the early decedent, showed signs of noisy respiration and decreased respiratory rate during behavioral assessment on day 7; these findings were considered likely to be associated with possible prior dosing trauma.

Mortality:

no mortality observed

Description (incidence):

- One male (animal no. 60) treated with the substance (active ingredient) at a dose level of 300 mg/kg bw/day was killed in extremis before dosing on day 9 due to deteriorating clinical condition. Clinical signs of noisy respiration, decreased respiratory rate, hunched posture and/or lethargy had been apparent for this male since day 7 (animal not dosed on this day) and there was an overall body weight loss of approximately 18% relative to day 1. At necropsy, macroscopic findings were confined to gaseous distension of duodenum, jejunum and ileum. Histopathology evaluation of the selected tissues revealed evidence of inflammation in the lungs which may have contributed to the clinical signs and the distribution and type of change suggested there may have been a technical problem with dosing. There were no further unscheduled deaths during the study and the death of this animal was considered unrelated to treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Throughout the dosing period, there was no adverse effect of treatment with the test item on body weight development in animals of either sex. - At 1000 mg/kg bw/day (active ingredient), males showed slightly lower group mean body weight gains than controls over the first two weeks of dosing albeit without achieving statistical significance. Group mean body weight gain for males treated with 300 mg/kg bw/day over the first week of dosing was also lower than controls but this was mainly due to male 60 (an early decedent) showing a body weight loss of 40g over this period. Subsequent weekly body weight gains for these animals were generally comparable with controls and any initial effect on body weight development was deemed to be of no toxicological significance At 100 mg/kg bw/day, group mean weight gains for males were similar to controls throughout the dosing period. - A high degree of inter-individual variation was apparent for the females during the pre-pairing phase of the study, but overall there was considered to be no effect of treatment with test item on body weight performance for these animals during this period. At all dose levels, group mean body weight gains in females during late gestation (days 14 to 20) were slightly lower than controls albeit without any dose-dependence or attaining statistical significance. It is worth noting, however, that female 92 from the high dose group, which showed a total litter loss on day 2 of lactation, had a small litter size at birth (two male pups, only one of which was born viable), and excluding gestation body weights for this animal resulted in comparable body weight gains between the high dose females and control females over this period, and this finding was therefore considered to be of no toxicological significance. During lactation phase of the study, group mean body weight gains were comparable across all dose groups including controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Throughout the dosing period, there was no adverse effect of treatment with the test item on dietary intake or food conversion efficiency in animals of either sex. - Over the first week of dosing, group mean dietary intake for males given 300 or 1000 mg/kg bw/day (active ingredient) was marginally lower than controls. Subsequent food consumption for these animals was generally comparable with controls and as such any initial effect on food intake was deemed to be of no toxicological importance. Food consumption for males treated with 100 mg/kg bw/day and females from all test item-treated dose group remained comparable with the respective controls throughout the treatment period.

Description (incidence and severity):

- There was no effect of treatment with the test item on food conversion efficiency in animals of either sex receiving the test item. Any minor differences were deemed to be reflective of intergroup differences in body weight gain and/or food consumption.

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

no effects observed

Description (incidence and severity):

- Visual inspection of water bottles did not indicate any differences for the animals given the test item in relation to controls.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level. - At all dose levels, animals of either sex treated with the test item (active ingredient) showed slightly higher prothrombin times in relation to the respective controls. With the exception of males treated with the low dose, statistical significance was achieved in all cases (p<0.01 for females receiving 300 or 1000 mg/kg bw/day and p<0.05 for the remaining dose groups) but dose-dependence was only apparent in females. Males from all treated dose groups and females from the high dose group also showed slightly higher activated partial thrompboplastin times in relation to the respective controls in a dose-dependent manner but without achieving statistical significance. The majority of individual values for both parameters were within the historical control data ranges and in the absence of any histopathology correlates from the high dose animals, these finding were considered unlikely to be of any toxicological significance. - At 1000 mg/kg bw/day, females showed slightly but statistically significantly higher values of red blood cell count and hematocrit in relation to controls (p<0.05). Dose-relationship was only evident for the latter but all individual values from the high dose females were within the background data ranges and these observations were considered unlikely to be of any toxicological relevance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level. - At all dose levels, males receiving the test item (active ingredient) showed statistically significantly lower aspartate aminotransferase activity in relation to controls (p<0.05 at 100 mg/kg bw/day and p<0.01 at 300 or 1000 mg/kg bw/day) but without any dose-relationship. All individual values were within the historical control data ranges and the corresponding values in females were similar to controls. There were no histopathological correlates in animals from the high dose group and this finding was considered to be of no toxicological relevance. - At all dose levels, females showed statistically significantly higher plasma levels of cholesterol when compared with controls (p<0.05). Plasma concentrations of bile acids in these females were also statistically significantly lower than controls (p<0.5). There was no dose-relationship for either parameter and all individual values from the test item-treated females were within the historical data ranges. In the corresponding males, these values were comparable with controls and, in the absence of any associated histopathological observations in females from the high dose group, these findings were considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- There were no intergroup differences related to treatment with the test item. - Motor activity evaluation during the final week of the treatment period revealed slightly but statistically significantly lower activity (last 20%) for males given the test item at 300 or 1000 mg/kg bw/day (active ingredient) in relation to controls (p<0.05). The remaining motor activity parameters for these animals were similar to controls and in the absence of any other signs of neurotoxicity, this finding was considered to be incidental. - Sensory reactivity scores across all test item-treated groups were similar to controls.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- Histopathological examination of the selected tissues from the high dose animals of either sex did not reveal any abnormalities, which could be unequivocally related to treatment with the test item. - There was an increase in hyaline droplets in the kidneys of 3/5 males in the high dose group. This was not associated with any damage to the cells or any sign of compromised function. The hyaline droplets within the tubules are consistent with the accumulation of alpha-2u-globulin, a common finding in untreated male rats (Khan KNM et al., 2002). Hyaline droplet accumulation is specific to the male rat and is considered not to be significant in man and in the absence of any other change in the kidney is considered not to be of toxicological significance in this study. There was also an increase in pigment deposition above the background level in the tubular cells in 2/5 females in the high dose group. This was minor, unrelated to any other changes and of doubtful significance (Greaves 2007) as it is considered to occur frequently in rats.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating: there was no effect of treatment on mating performance with all animals mating within four days after pairing. - Fertility: in total, five females (animal no. 18 from the control group, animal no. 62 from the intermediate dose group, and animal no. 86, 88 and 96 from the high dose groups) did not achieve pregnancy following evidence of positive mating. All non-pregnant females appeared to be cycling normally and there was no obvious reason for the lack of pregnancy with the exception of females 18 and 96; these females had successfully mated with male 6 (control group) and male 84 (high dose group), respectively, which showed marked or moderate tubular atrophy in the testes and this was considered likely to have caused impaired fertility or indeed infertility. There was no reason identified at histopathology examination for the lack of pregnancy in the remaining two high dose females and taken together with the high incidence of non-pregnancies in this dose group, this observation was considered to be equivocal. Female 62 was paired with male 50 and the latter showed reduced secretion in the seminal vesicles which may have affected fertility although this remains unclear. Female 63 from the intermediate dose group showed evidence of implantation but there was no litter. As there were no such findings in the high dose group, these observations was deemed unlikely to be treatment related.- Gestation length: with the exception of one female from the high dose group, gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was similar to controls; female 92 from the high dose group had a gestation length of 24½ days which may be due to its small litter size that included two pups, only one of which was born viable.

Details on results (P0)

HISTORICAL CONTROL DATA:- Hematology and clinical chemistry data as well as organ weights were compared to historical control data.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: unusual high number of non-pregnant females in the high dose group

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food efficiency:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

- Clinical signs detected in pups from all test item-treated groups included small size, pale, weak, cold, no milk in stomach, physical injury, scab formation found dead or missing and, were considered to be low incidence findings observed in offspring in studies of this type and as such unlikely to be related to the toxicity of the test item.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Offspring body weights and litter weights on days 1 and 4 post partum were generally comparable with controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Macroscopic necropsy findings for control offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment with the test item (active ingredient) on offspring development at 100, 300 or 1000 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

not specified

Description (incidence and severity):

- Surface righting reflex data did not reveal any detrimental effect of treatment with the substance at any dose level in relation to controls. At 300 mg/kg bw/day (active ingredient), the percentage of pups passing the test on Day 1 was statistically significantly higher than controls (p<0.05); however, as there were no statistically significant intergroup differences between the high dose and controls pups, this observation was considered to be incidental.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

VIABILITY, SEX RATIO AND SIZE (OFFSPRING)- There was no effect of treatment with the test item on the mean number of corpora lutea, implantation counts or pre- and post-implantation losses. - Of the litters born, litter size at birth and subsequently on days 1 and 4 post partum from females treated with the test item at all dose levels were comparable with controls. - There were no intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated dose groups when compared with controls.- Statistical analysis of these data did not identify any statistically significant differences and any minor intergroup differences were considered to be due to normal biological variation. OTHER FINDINGS (OFFSPRING)- Female 92 from the high dose group showed a total litter loss on Day 2 of lactation; the litter included two pups only one of which was born viable. As it is not uncommon for a litter of such small size not to reach term and due to the isolated nature of this incident, this finding was considered unlikely to be related to treatment with the test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analytical verification of dose formulations: the results indicate that the prepared formulations were within 91% to 108% of the nominal concentration and thus, the required content limit of +/-10% with reference to the nominal content was met. In addition, the test item was found to be stable in the formulations when kept for 20 days in the refrigerator (4°C) due to results which met the variation limit of 10% from the time-zero mean. In conclusion, the results indicate the accurate use of the test item and RO Water as vehicle during this study. The formulations were found to be homogeneously prepared (visual inspection) and sufficient formulation stability under storage conditios was proven.

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of the substance (active ingredient) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 1000 mg/kg bw/day was well tolerated. Microscopic examination of the selected tissues from the high dose animals did not identify any treatment-related abnormalities and based on the available data the NOAEL for systemic toxicity was considered to be 1000 mg/kg bw/day. The NOEL for reproductive toxicity, within the confines of this screening study, was considered to be 300 mg/kg bw/day. This was due to the unusually high number of non-pregnant females in the high dose group and, although there was no effect of treatment with the test item at any dose level on any other reproduction or offspring developmental parameters this observation remains equivocal because an effect of treatment cannot be completely discounted.