Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 10 August 2011 and November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: -
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:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) as at July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EC 440/2008 (laying down test methods pursuant to Regulation EC 1907/2006) as at 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Animals: Rat, RccHanTM: WIST(SPF)- Rationale: Recognized by international guidelines as a recommended test system.- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands- Number of Animals: 40 males (10 per group), 40 females (10 per group)- Age (at Start of Treatment): 11 weeks- Body Weight Range (at Start of Treatment): Male (289 to 333 g), Females (186 to 216 g)- Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.ENVIRONMENTAL CONDITIONS- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK and ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105110601, 72 and 100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 28/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
DOSE FORMULATIONSThe dose formulations were prepared weekly using the test item as supplied by the Sponsor.Polyphosphoric acids, esters with triethanolamine, sodium salts was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Each formulation was divided in daily aliquots.Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.STORAGE OF DOSE FORMULATIONSDose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.TREATMENT- Method: Oral, by gavage- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.- Frequency of Administration: Once daily- Target Dose Levels: Group 1: 0 mg/kg/day (control group), Group 2: 100 mg/kg/day, Group 3: 500 mg/kg/day, Group 4: 1000 mg/kg/day- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, using dose levels of 0, 100, 300 and 1000 mg/kg/day.- Dose Volume: 10 mL/kg body weight (daily adjusted to the actual body weight of individual animal)- Duration of Acclimatization Period: 6 days- Duration of Treatment Period: Males (28 days), females (approximately 6 weeks)
Details on mating procedure:
MATING, GESTATION AND LACTATIONDuring the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:- the daily vaginal smear was sperm positive, or- a copulation plug was observed.The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONSOn the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on the first treatment day. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytical Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.The samples were analyzed by their free content of phosphate using ion chromatography coupled to a conductivity detector following an analytical procedure developed by Harlan Laboratories. The test item was used as the analytical standard. The phosphate peak in polyphosphoric acids, esters with triethanolamine, sodium salts was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of phosphate in polyphosphoric acids, esters with triethanolamine, sodium salts and, therefore, the absence of the test item in the vehicle control samples (purified water) was confirmed.The application formulations investigated during the study were found to comprise polyphosphoric acids, esters with triethanolamine, sodium salts in the range of 90.1% to 100.3% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of polyphosphoric acids, esters with triethanolamine, sodium salts in the preparations was approved because single results found did not deviate more than 1.7% (<15%) from the corresponding mean.In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.In conclusion, the results indicate the accurate use of the test item polyphosphoric acids, esters with triethanolamine, sodium salts and purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
- Males: 28 days- Females: Approximately 6 weeks
Frequency of treatment:
- Once daily
Details on study schedule:
MALES- Acclimatization: 5 days minimum- First Test Item Administration: Day 1 of pre-pairing- Pre-Pairing: 14 days- Pairing: 14 days maximum- Treatment Ends: On day before sacrifice- Necropsy: After treatment for 28 days, when no longer needed for assessment of reproductive effectsFEMALES- Acclimatization: 5 days minimum- First Test Item Administration: Day 1 of pre-pairing- Pre-Pairing: 14 days- Pairing: 14 days maximum- Gestation: Approximately 21 days- Treatment Ends: On day 4 post partum- Necropsy: On day 5 post partum (pups on day 4 post partum)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
VIABILITY/MORTALITY- Twice dailyCLINICAL SIGNS AND OBSERVATIONSDaily cage-side clinical observation was performed once daily during acclimatization period. Thereafter, up to the necropsy, daily clinical observation was performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. An additional observation was made five hours after dosing during the normal working week. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.FOOD CONSUMPTION- Males (weekly during pre-pairing period and days 1 - 5 and 5 - 9 during after pairing period).- Females (pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum). No food consumption was recorded during the pairing period.WATER CONSUMPTION- Monitored daily by visual inspection of water bottles. BODY WEIGHTS- Recorded daily from treatment start to day of necropsy.DETAILED CLINICAL OBSERVATIONSDetailed clinical observations were performed outside the home cage in all P generation animals. In all males of P generation it was performed once prior to the first administration of the test item and weekly thereafter. In females of P generation, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior. FUNCTIONAL OBSERVATION BATTERYAt one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:- Cage-side observations: Faeces-balls, urine and posture as well as resistance to removal.- Hand-held observations: Muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.- Open field observations: Level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.- Reflexes: Blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).- Measurements / Counts: Hind limb / fore limb grip strength, landing foot splay, rectal temperature.Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.CLINICAL LABORATORY INVESTIGATIONSBlood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 17.5 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.HEMATOLOGYThe following hematology parameters were setermined:Complete Blood Cell Count:- Erythrocyte count- Hemoglobin- Hematocrit- Mean corpuscular volume- Red cell volume distribution width- Mean corpuscular hemoglobin- Mean corpuscular hemoglobin concentration- Hemoglobin concentration distribution width- Leukocyte count, total- Differential leukocyte count:- Platelet countCoagulation:- Prothrombin time (= Thromboplastin time)- Activated partial Thromboplastin timeCLINICAL BIOCHEMISTRYThe following clinical biochemistry parameters were determined:- Glucose - Urea - Creatinine - Bilirubin, total - Cholesterol, total- Triglycerides - Aspartate aminotransferase - Alanine aminotransferase - Alkaline phosphatase- Gamma-glutamyl-transferase - Bile acids- Sodium - Potassium - Chloride - Calcium - Phosphorus - Protein, total- Albumin- Globulin- Albumin/Globulin ratio
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
During histopathology examination, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Litter observations:
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible) and (with identification) on days 1 and 4 post partum. In addition a surface righting assessment was performed on day 1 post partum.
Postmortem examinations (parental animals):
TERMINATION AND NECROPSYMales were sacrificed after treatment for 28 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum. All parent animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes.For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.ORGAN WEIGHTSAt the scheduled sacrifice following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken of all parental males:- Testes (as pairs)- Epididymides (as pairs)- Seminal vesicles (as pairs)- ProstateOf all parental females:- Ovaries (as pairs)- Uterus and CervixOf all parental males and females: - Adrenal glands (weighed as pairs)- Brain - Heart- Kidneys (weighed as pairs)- Liver- Pituitary - Thymus- Spleen- ThyroidTISSUE PRESERVATIONTissues from the following reproductive organs from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution (or in Bouin’s fixative, if indicated):- Prostate- Seminal vesicles with coagulating gland - Testes (in Bouin’s fixative)- Epididymides (in Bouin’s fixative) Tissues from the following reproductive organs from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:- Ovaries - Uterus- Vagina- CervixIn addition, from all males and females at scheduled necropsy and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:- Gross lesions- Brain (including cerebrum, cerebellum and pons)- Spinal cord (cervical, mid-thoracic and lumbar)- Small and large intestines (incl. Peyer’s patches)- Stomach- Liver - Kidneys- Adrenals- Spleen- Heart- Thymus- Thyroids, and parathyroids if possible- Trachea and lungs (preserved by inflation with fixative and then immersion)- Urinary bladder- Lymph nodes (mesenterial, mandibular)- Peripheral nerve (sciatic)- Aorta (thoratic)- Bone and bone marrow (femur including stifle joint)- Bone and bone marrow (sternum)- Muscle (skeletal)- Oesophagus - Pancreas- Pituitary- Salivary glands (submaxillary / mandibular)- Skin (abdominal region)- Eyes ( fixed in Davidson's fluid)- Mammary tissueHISTOTECHNIQUEAll organ and tissue samples to be examined by the principal investigator for histopathology phase were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS-hematoxylin. Special stains were used at the discretion of the principal investigator.HISTOPATHOLOGYSlides of all organs and tissues collected at terminal sacrifice from the first five males and first five females which gave birth and slides of all reproductive organs from all males and females of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions, to all animals, which died spontaneously or had to be terminated in extremis and to all unfertile animals. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made. A histopathology peer review was performed a principal investigator (AnaPath GmbH).
Postmortem examinations (offspring):
TERMINATION AND NECROPSYPups were sacrificed on day 4 post partum.All pups sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all pups were sacrificed by an injection of sodium pentobarbital.Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
Statistics:
The following statistical methods were used to analyze functional observational battery, locomotor activity, food consumption, body weights, reproduction data, clinical laboratory investigations, organ weight and ratios and incidence and severity of histopathological findings:- Means and standard deviations of various data were calculated and included in the report.- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, pre- and post-implantation losses, mean litter size.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex rations and postnatal losses (up to day 4 post partum).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidentally, statistically significantly lower value of hemoglobin concentration distribution width (HDW) was noted in males at the dose level of 1000 mg/kg bw/day; mean value at the high dose level was 1.56 compared to 1.72 in the control group. The value at the high dose level was within the 95% of a tolerance limit for the historical controls (a range from 1.55 to 2.14) and therefore the difference was considered not to be test item-related.
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Males:No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.At the dose level of 100 mg/kg bw/day, statistically significantly higher testes weights to brain weights ratio was noted; mean ratio at the low dose level was 193.18 compared to 176.78 in the control group. No significant differences of this ratio was noted at the mid and high dose level and the value at the high dose level was very close to the background of the historical controls (containing mean group values from 155.90 to 193. 13). For these reasons higher testes weights to brain weights ratio at the low dose level was considered not to be related to the treatment with the test item.Females:No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.At the dose level of 100 mg/kg bw/day, statistically significantly higher adrenals weights to body weights ratio was noted; mean ratio at the mid dose level was 0.037 compared to 0.033 in the control group. No significant differences of this ratio was noted at the high dose level and therefore higher adrenals weights to body weights ratio at the mid dose level was considered not to be related to the treatment with the test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:No test item-related findings were noted during necropsy of males at any dose level.Following findings were considered to be within the range of normal background alterations:- enlarged liver found in two males in the control group and two males at each: mid and high dose levels (nos. 6, 9, nos. 29, 30 and nos. 36, 38, respectively),- pelvic dilation found in two males in the control group and one male at the high dose level (nos. 1, 3 and no. 38, respectively),- foci on thymus found in four males in the control group and two males at each: low, mid and high dose levels (nos. 1, 2, 5, 7, nos. 14, 17, nos. 26, 29 and nos. 33, 34, respectively),- foci or discoloration on mandibular lymph node found in one male in the control group, one male at the low dose level and two males at the high dose level (no. 3, no. 11, nos. 31 and 33, respectively).No further findings were noted in males at any dose level.Females:No test item-related findings were noted during necropsy of females at any dose level.Following findings were considered to be within the range of normal background alterations:- discoloration of stomach found in one female in the control group, one female at each: low and high dose levels and two females at the mid dose level (no. 42, no. 59, nos. 62, 69 and no. 76, respectively),- pelvic dilation found in one female in the control group and one female at the high dose level (no. 49 and no.76 , respectively),- watery cyst on ovary of one female at mid dose level (no. 69),- discoloration of ovaries found in one female in the control group, one female at low dose level and four females at high dose level (no. 43, no. 57 and nos. 72, 77, 79, respectively),- mucous content of uterus found in one female at the high dose level (no. 77),- foci or discoloration of thymus found in one female at each: low and mid dose levels (no. 59 and nos. 61, respectively),- thymus reduced in size found in one female in the control group and two females at each: mid and high dose levels (no. 45, nos. 62, 64 and nos. 71, 78 respectively).- In addition eschar in one female at the low dose level (no. 59) and alopecia in one female at the high dose level (no. 78) which were observed already during the in live phase, were also recorded during the necropsy.No further findings were noted in females at any dose level.
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

IN-LIVE DATA - PARENTAL ANIMALSVIABILITY / MORTALITYAll animals survived the scheduled study period.CLINICAL OBSERVATIONS (DAILY)No test item-related clinical signs were found in males or females at any dose level.Incidentally, breathing noises were noted from day 8 of the after pairing period onwards in one male at the dose level of 1000 mg/ kg bw/day (no. 38), scabs on the neck and shoulder were noted from day 13 of the gestation period onwards in one female at the dose level of 100 mg/kg bw/day (no. 59) and hair loss was noted from day 23 of the gestation period onwards in one female at the dose level of 1000 mg/kg bw/day (no. 78).No further observations were noted in males or females at any dose level.DETAILED CLINICAL OBSERVATIONS (WEEKLY)No test item-related effects were noted during detailed weekly clinical observations in males or females at any dose level.The only one finding recorded was a crust on the neck and shoulder in one female at the dose level of 100 mg/kg bw/day (no. 59).FUNCTIONAL OBSERVATIONAL BATTERYNo test item-related effects were noted during functional observational battery in males or females at any dose level.At the dose level of 1000 mg/kg bw/day in males, statistically significantly higher grip strength of forelimb was noted; mean forelimb grip strength was 0.35 kg compared to 0.23 kg in the control group. No further statistically significant differences were noted during examination of grip strength of males or females at any dose level. Value at the high dose level was in the range of the historical controls which contained values from 0.314 kg to 0.801 kg. For this reason the difference in grip strength was considered not to be test item-related.Incidentally, decreased rearing was noted in one male in the control group (no. 3) and one male at the dose level of 1000 mg/kg bw/day (no. 34) as well as increased rearings were noted in two females at the dose level of 100 mg/kg bw/day (nos. 54 and 56).No further findings were noted during functional observational battery in males or females at any dose level.LOCOMOTOR ACTIVITYNo test item-related effects were noted during measurement of locomotor activity in males or females at any dose level.At the dose level of 1000 mg/kg bw/day in males, a statistically significantly higher locomotor activity was noted; mean total beam crossing counts within 30 minutes of measurement was 1501 at the high dose level compared to 910 in the control group. No statistically significant differences in locomotor activity were noted in males at low and mid dose levels or in females at any dose level. Locomotor activity of males the high dose level was only slightly above the range of the historical controls which contained values from 571 to 1476 mean total beam crossing counts. For this reason the difference in locomotor activity was considered not to be test item-related.No effects on locomotor activity were noted in males up to the dose level of 500 mg/kg bw/day or in females at any dose level.In general, mean total beam crossing counts within 30 minutes of measurement were 910, 1220, 1333 and 1501 in males and 932, 823, 822 and 943 in females, given in the order of ascending dose levels.FOOD CONSUMPTION OF MALESPre-pairing and After Pairing Periods:No effects on food consumption of males were observed at any dose level.At the dose levels of 100, 500 and 1000 mg/kg bw/day, differences in mean food consumption were respectively: ±0%, -4% and ±0% over the pre-pairing period and -4%, -4% and ±0% over the after pairing period (percentages refer to the respective values in the control group). FOOD CONSUMPTION OF FEMALESPre-pairing, Gestation and Lactation Periods:No effects on food consumption of females were observed at any dose level.At the dose levels of 100, 500 and 1000 mg/kg bw/day, differences in mean food consumption were respectively: ±0%, +6% and +6% over the pre-pairing period, ±0%, -5% and 5% over the gestation period and +16%, +8% and +12% over the lactation period (percentages refer to the respective values in the control group). BODY WEIGHTS OF MALESPre-pairing, Pairing and After Pairing Periods:No effects on body weights or body weight gain of males were observed at any dose level.Overall differences in mean body weight gain at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day were respectively: +14.5%, +12.6%, +12.7% and +13.6% during the pre-pairing period, +2.9%, +1.9%, +2.6% and +2.6% during the pairing period and +4.6%, +3.7%, +4.2% and +3.7% during the after pairing period (percentages refer to the body weight gain within the period).BODY WEIGHTS OF FEMALESPre-pairing, Pairing, Gestation and Lactation Periods:No effects on body weights or body weight gain of females were observed at any dose level.Overall differences in mean body weight gain at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day were respectively: +7.4%, +6.9%, +6.6% and +8.6% during the pre-pairing period, +58.8%, +60.3%, +53.3% and +57.8% during the gestation period and +4.2%, +5.4%, +4.3% and +6.0% during the lactation period (percentages refer to the body weight gain within the period).CLINICAL LABORATORY INVESTIGATIONSHEMATOLOGYMales:No effects on hematology parameters, which were considered to be test item-related, were noted at any dose level.Incidentally, statistically significantly lower value of hemoglobin concentration distribution width (HDW) was noted at the dose level of 1000 mg/kg bw/day; mean value at the high dose level was 1.56 compared to 1.72 in the control group. The value at the high dose level was within the 95% of a tolerance limit for the historical controls (a range from 1.55 to 2.14) and therefore the difference was considered not to be test item-related.No significant differences were noted among remaining values.Females: No effects on hematology parameters were noted at any dose level.CLINICAL BIOCHEMISTRYMales: No effects on clinical biochemistry parameters were noted at any dose level.Females: No effects on clinical biochemistry parameters were noted at any dose level.REPRODUCTION AND BREEDING DATAMATING PERFORMANCE AND FERTILITYNo test item-related effect on mating performance or fertility was observed at any dose level.With exception of one female, all females were mated within four days after initiation of pairing. One female in the mid dose group (no. 61) mated after six days of pairing. Mean (median) precoital times were 2.3 (2), 2.7 (4), 2.8 (4) and 2.6 (3) days at the dose level of 0, 100, 500 and 1000 mg/kg bw/day, respectively.Six females were not pregnant; one in the control group (female no. 43, mated with male no. 3), three at the low dose level (female nos. 51, 57 and 60 mated with male nos. 11, 17 and 20, respectively) and two at the high dose level (female nos. 72 and 77 mated with male nos. 32 and 37, respectively). Consequently, fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 90%, 70%, 100% and 80% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively.The incidence of infertility did not follow a dose dependency, and the number of non pregnant females per group was within the historical background control range, for these reasons this was not considered to be treatment-related.All pregnant females gave birth to living pups. Consequently, the gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all groups.CORPORA LUTEA COUNTNo effects on corpora lutea count were observed at any dose level.Mean number of corpora lutea per dam was 14.1, 13.9, 13.8 and 14.8 in order of ascending dose levels.PRE-IMPLANTATION LOSSNo effects on pre-implantation loss were observed at any dose level.Mean pre-implantation loss per dam was 2.8, 1.0, 1.5 and 1.0 days, in order of ascending dose level.DURATION OF GESTATIONNo effects on duration of gestation were observed at any dose level.Mean duration of gestation was 21.3, 21.3, 21.9 and 21.5 days, in order of ascending dose level.IMPLANTATION RATE AND POST-IMPLANTATION LOSSNo effects on implantation rate or post implantation loss were observed at any dose level.Mean number of implantations per dam was 11.3, 12.9, 12.3 and 14.0 whereas mean number of post-implantation loss per dam was 0.4, 0.3, 1.2 and 0.6; both cited in order of ascending dose levels.LITTER SIZE AT FIRST LITTER CHECKNo effects on litter size were observed at any dose level.Mean numbers of living pups per dam at first litter check were 10.9, 12.6, 11.1 and 13.4, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 96.1%, 97.8%, 90.2% and 95.5% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively. POSTNATAL LOSS DAYS 0 - 4 POST PARTUMNo effects on postnatal loss were observed at any dose level.No dead pups were found at first litter check at any dose level. At the dose level of 500 mg/kg bw/day, 4 pups (from three litters, nos. 62, 66 and 67) were lost during the four days of the lactation period; one pup was found dead on day 2 post partum, two pups were missing on day 2 post partum and one pup was missing on day 3 post partum. No further mortality of pups was observed at any dose level. Because the incidence of pups mortality did not correlate with the dose levels, the postnatal loss was considered not to be test item-related.TERMINAL FINDINGS - PARENT ANIMALSORGAN WEIGHTSMales:No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.At the dose level of 100 mg/kg bw/day, statistically significantly higher testes weights to brain weights ratio was noted; mean ratio at the low dose level was 193.18 compared to 176.78 in the control group. No significant differences of this ratio was noted at the mid and high dose level and the value at the high dose level was very close to the background of the historical controls (containing mean group values from 155.90 to 193. 13). For these reasons higher testes weights to brain weights ratio at the low dose level was considered not to be related to the treatment with the test item.Females:No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.At the dose level of 100 mg/kg bw/day, statistically significantly higher adrenals weights to body weights ratio was noted; mean ratio at the mid dose level was 0.037 compared to 0.033 in the control group. No significant differences of this ratio was noted at the high dose level and therefore higher adrenals weights to body weights ratio at the mid dose level was considered not to be related to the treatment with the test item.MACROSCOPICAL FINDINGSMales:No test item-related findings were noted during necropsy of males at any dose level. Following findings were considered to be within the range of normal background alterations:- enlarged liver found in two males in the control group and two males at each: mid and high dose levels (nos. 6, 9, nos. 29, 30 and nos. 36, 38, respectively),- pelvic dilation found in two males in the control group and one male at the high dose level (nos. 1, 3 and no. 38, respectively),- foci on thymus found in four males in the control group and two males at each: low, mid and high dose levels (nos. 1, 2, 5, 7, nos. 14, 17, nos. 26, 29 and nos. 33, 34, respectively),- foci or discoloration on mandibular lymph node found in one male in the control group, one male at the low dose level and two males at the high dose level (no. 3, no. 11, nos. 31 and 33, respectively).No further findings were noted in males at any dose level.Females:No test item-related findings were noted during necropsy of females at any dose level. Following findings were considered to be within the range of normal background alterations:- discoloration of stomach found in one female in the control group, one female at each: low and high dose levels and two females at the mid dose level (no. 42, no. 59, nos. 62, 69 and no. 76, respectively),- pelvic dilation found in one female in the control group and one female at the high dose level (no. 49 and no.76 , respectively),- watery cyst on ovary of one female at mid dose level (no. 69),- discoloration of ovaries found in one female in the control group, one female at low dose level and four females at high dose level (no. 43, no. 57 and nos. 72, 77, 79, respectively),- mucous content of uterus found in one female at the high dose level (no. 77),- foci or discoloration of thymus found in one female at each: low and mid dose levels (no. 59 and nos. 61, respectively),- thymus reduced in size found in one female in the control group and two females at each: mid and high dose levels (no. 45, nos. 62, 64 and nos. 71, 78 respectively).- In addition eschar in one female at the low dose level (no. 59) and alopecia in one female at the high dose level (no. 78) which were observed already during the in live phase, were also recorded during the necropsy.No further findings were noted in females at any dose level.HISTOPATHOLOGY FINDINGSThe test item polyphosphoric acids, esters with triethanolamine, sodium salts produced no histological evidence of toxicological properties in the organs and tissues examined.No test item-related histological findings were recorded in ovary on females that did not give birth and the reproductive organs of infertile males.Sperm staging: no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.In particular thorough histopathological examination of all reproductive organs did not reveal any evidence of treatment related toxicity up tp and including the highest permissible dose of 1000 mg/kg

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
for reproduction and development
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 1000 mg/kg/day, the highest dose level used.
Key result
Dose descriptor:
NOEL
Remarks:
for general toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No signs of general toxicity in males or females and was observed up to 1000 mg/kg bw day, the highest dose level used.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
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 DATA - F1 PUPSEXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATIONNo test item-related findings were noted in pups at any dose level.Incidentally, a pale body in one pup (from litter no. 53) and a bluish head in another pup (from litter no. 58) at the dose level of 100 mg/kg bw/day and a bite on a back in one pup (from litter no. 62) at the dose level of 500 mg/kg bw/day were noted at first litter check.No further findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.SEX RATIOSPups sex ratio was not affected by exposure to the test item at any dose level. At first litter check, percentages of male pups were 51%, 50%, 46% and 48%, in order of ascending dose level.RIGHTING REFLEXNo effects on righting reflex of pups were observed at any dose level.Percentages of pups for which righting reflex was observed within the first one minute of the test were 80%, 81%, 95% and 94% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively. BODY WEIGHTS TO DAY 4 POST PARTUMNo effects on pups body weights or body weight gain were noted at any dose level.Overall mean body weights of pups on day 1 post partum were: 6.2 g, 5.8 g, 6.2 g and 5.9 g, at the dose levels of 0, 100, 500 and 1000 mg/kg/day respectively. Overall, body weight gain of pups during the first four days of lactation period was +46.8%, +48.3%, +50.0% and +44.1%, respectively.MACROSCOPICAL FINDINGSNo findings were noted during the necropsy of pups at any dose level.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Remarks:
for development to day 4 post partum
Generation:
F1
Effect level:
ca. 1 000 other: mg/kg bw/day received by parental animals. Offspring were not treated.
Based on:
other: act. ingr received by parent 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 findings were noted in pups to day 4 post partum

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

SUMMARY OF PERFORMANCE
P Animals Breeding for F1 Litters

Group (mg/kg/day)

1 (0)

2 (100)

3 (500)

4 (1000)

Female numbers

41-50

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Numbers of females, which were not pregnant (A)

1

3

0

2

Number of females which reared their pups until day 4 post partum

9

7

10

8

(A)Female nos. 43, 51, 57, 60, 77 and 72.

Applicant's summary and conclusion

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item polyphosphoric acids, esters with triethanolamine, sodium salts to rats over approximately 28 days. Polyphosphoric acids, esters with triethanolamine, sodium salts was administered in highly purified water as vehicle at dosages of 100, 500, and 1000 mg/kg body weight/day, and controls received the vehicle only. Polyphosphoric acids, esters with triethanolamine, sodium salts was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected at any dose level.Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.
Executive summary:

The purpose of this study was to generate information concerning the effects of polyphosphoric acids, esters with triethanolamine, sodium salts on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.  

Polyphosphoric acids, esters with triethanolamine, sodium salts was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum, for a period of approximately of 6 weeks.

 

The following dose levels were applied:

 

Group 1:                0 mg/kg body weight/day (control group)

Group 2:            100 mg/kg body weight/day

Group 3:            500 mg/kg body weight/day

Group 4:          1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

 

PARENTAL ANIMALS

Mortality and General Tolerability

 

All animals survived the scheduled study period.

 

No test item-related effects were noted during daily clinical or detailed weekly clinical observations in males or females at any dose level.

 

Functional Observational Battery

 

No test item-related effects were noted during functional observational battery in males or females at any dose level.

 

Food Consumption

 

No effects on food consumption of males or females were observed at any dose level.

 

Body Weights

 

No effects on body weights or body weight gain of males or females were observed at any dose level.

 

Clinical Laboratory Investigations

 

No test item-related effects on hematology or clinical biochemistry parameters were noted in males or females at any dose level.

 

Reproduction and Breeding Data

 

No test item-related effect on mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were observed at any dose level.

 

Organ Weights

 

No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted in males or females at any dose level.

 

Macroscopical Findings and Histopathological Examinations

 

No test item-related findings were noted during necropsy of males or females at any dose level.

 

During histological examination, no evidence of toxicological properties in the organs and tissues examined was found. No test item-related histological findings were recorded in ovaries of females that did not give birth or in the reproductive organs of infertile males. During the sperm staging no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals were found.

 

 

LITTERDATA- F1 PUPS

Findings at First Litter Check and during Lactation

 

No test item-related findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

 

No effects on righting reflex were observed in pups at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

 

No effects on pups body weights or body weight gain were noted at any dose level.

 

Macroscopical Findings

 

No findings were noted during the necropsy of pups at any dose level.

Conclusion

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw day, the highest dose level used.