Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined repeated dose toxicity study with the Reproduction/Developmental Toxicity Screening Test in the Rat:
Test substance 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.
The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg bw day, the highest dose level used.

Link to relevant study records
Reference
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.
Reason / purpose:
reference to same study
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
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.
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)
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
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).
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
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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
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.
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
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.
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
Key result
Reproductive effects observed:
no
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.
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database consists of one reliable (Klimisch 1) study which is suitable for completing this endpoint. Whilst it is acknowledged that the available OECD 422 “Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test” does not address all aspects of reproduction that can be derived from an EOGRTS study, the available study is sufficient in order to conclude that no reproductive toxicity was observed at any dose level tested. Given the low degree of toxicity observed, lack of any reprotoxic response at the highest permitted dose level and the low levels of exposure a waiving argument has been proposed for further reproductive toxicity endpoints.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Combined repeated dose toxicity study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD Guideline 422).

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. The test item 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. The test item 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.

Six females were not pregnant; one in the control group, three at the low dose level and two at the high dose level. The increased number of non-pregnant females in the low dose group was considered to be incidental with no one single factor that could be isolated to provide a cause for this infertility. A background of spontaneous non-pregnant females is an expected event for reproduction toxicology studies. Variable rates of infertility can also be expected in studies of relatively small group size. There were no indications to suggest any issues to show that the health or reproductive capacity of this strain of animal has been compromised. The conclusion is therefore that reduced reproductive performance on this study was a spontaneous event.

The low dose level had only seven pregnancies, which is lower than that prescribed for the study. Of the seven pregnancies there were no findings to suggest an effect on reproduction and development. This finding does not contradict the conclusions of a lack of treatment effect at the higher treatment levels where sufficient pregnancies were seen thereby allowing the appropriate

evaluation to take place and therefore the reduced number of pregnancies is not considered to compromise the validity of the study.

Based on these results, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.

Effects on developmental toxicity

Description of key information

OECD 414 Prenatal Developmental study in the female rats treated by gavage during gestation (from day 5 to day 19 of gestation on a daily basis) at dose levels of 100, 500 and 1000 mg/kg bw/day did not result in any toxicologically significant developmental effects in the foetuses. The ‘No Observed Effect Level’ (NOEL, developmental, rat) was considered to be 1000 mg/kg bw/day the highest dose level used.

OECD 422 Combined Repeat Dose Toxicity Study With Reproduction / Developmental Screening at dose levels of 100. 500 and 1000 mg/kg bw/day (gavage) in male rats treated for 28 days and female rats treated for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. The No Observed Effect Level (NOEL, developmental rat) was considered to be 1000 mg/kg bw day, the highest dose level used.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 16 January 2014 and 10 April 2014. The in-life phase of the study was conducted between 19 January 2014 (first day of treatment) and 06 February 2014 (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A positive result was seen in the CHO HPRT forward mutation assay and a toxicokinetic assessment based on available data showed limited evidence of absorption of the test substance in view of this a pre-natal developmental toxicity study was conducted to investigate the substances effect on developmental toxicity and address concerns for worker safety. The study also fills an Annex IX REACH data gap.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 194 to 272g.The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The animals were housed in a single air-conditioned room. 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 computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets. The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on exposure:
Test Item Formulation and Experimental Preparation:For the purpose of the 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 was previously determined and results showed the formulations to be stable for at least seven days. Formulations were prepared on four occasions and stored at approximately +4 °C in the dark.Procedure:Four dose groups were allocated:Control: 0 mg/kg bw/day (active ingredient)Low: 100 mg/kg bw/day (active ingredient)Intermediate: 500 mg/kg bw/day (active ingredient)High: 1000 mg/kg bw/day (active ingredient)See any other information on materials and methods section for furtehr details on treatment groups. The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage using a Stainless Steel dosing cannula attached to a graduated Syringe. The volume of test and control item administered to each animal was based on the most recent scheduled body weight. Control animals were treated in an identical manner with the vehicle alone (Distilled water).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of Polyphosphoric acids,esters, with triethanolamine, sodium salts.The test item concentration in the test samples was determined by inductively coupled plasma with mass spectrometric detection (ICP-MS) using an external standard technique. The element analysed was phosphorous. The formulations investigated during the study were found to comprise test item in the range of 100% to 106% and the required content limit of ±10% with reference to the nominal content was met.The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method was validated and proven to be suitable for use.
Details on mating procedure:
The female animals were mated and pregnant prior to dose administration.
Duration of treatment / exposure:
From Day 5 to Day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
All females were terminated on Day 20 of gestation.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
No. of animals per sex per dose:
24 females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data, including a rat OECD 422 toxicity Study.
Maternal examinations:
CLINICAL OBSERVATIONS:Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.BODY WEIGHT:Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).FOOD CONSUMPTION:Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.WATER CONSUMPTION:Water intake was observed daily by visual inspection of the water bottles for any overt changes.POST MORTEM:All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes The ovaries and uteri of pregnant females were removed, examined and the following data recorded:i) Number of corpora luteaii) Number, position and type of intrauterine implantationiii) Fetal sexiv) External fetal appearancev) Fetal weightvi) Placental weightvii) Gravid uterus weightThe uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.Implantation types were divided into:Early Death/Resorption: No visible distinction between placental/decidual tissue and embryonic tissueLate Death/ Resorption: Separate embryonic/fetal and placental tissue visibleDead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposesAll implantations and viable fetuses were numbered according to their intrauterine position.
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and visceral alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
Data was assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test.Probability values (p) are presented as follows:p<0.01 **p<0.05 *p≥0.05 (not significant)
Indices:
Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.Pre and Post Implantation Loss:Percentage pre-implantation loss was calculated as: (number of corpora lutea - number of implantations / number of corpora lutea) x 100Percentage post-implantation loss was calculated as:(number of implantations - number of live fetuses / number of implantations) x 100Sex ratio:Sex ratio was calculated as:% male fetuses (sex ratio) = Number of male fetuses / Total number of fetuses x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.Females treated with 1000 and 500 mg/kg bw/day showed a statistically significant increase in body weight gain between Days 5 and 6 and Days 5 and 11 (respectively). An increase in body weight gain is not considered to represent an adverse effect of treatment. Females treated with 1000 and 500 mg/kg bw/day also showed a statistically significant reduction in body weight gain between Days 11 and 14. A true dose related response was not evident and no effect was apparent on overall body weight development therefore the intergroup differences were considered of no toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Dead fetuses:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Please see attached background material for data tables.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.One fetus from a female treated with 1000 mg/kg bw/day was small and had a cleft lip, shortened upper jaw, malpositioned pinnae, bulging eyes and a cleft palate. Whilst these observations are considered to represent malformation of the fetus, in isolation and without any other treatment-related findings the intergroup difference was considered to be a spontaneous event and not related to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.Females treated with 1000 and 500 mg/kg bw/day showed a statistically significant increase in the percent of fetuses showing ossified odontoid. Females treated with 500 and 100 mg/kg bw/day also showed a statistically significant increase in the percent of fetuses showing ossification of the ventral arch of vertebra 1. The observations of isolated variants at a higher incidence compared with controls is not significant when evaluated in isolation. Both observations are also consistent with precocious ossification rather than delayed ossification. The distribution of the changes seen indicated a lack of specificity of affected ossification which suggests the differences seen are of a random pattern. The number of parameters routinely evaluated makes it highly likely that one or more finding will be seen at a higher level in dosed groups compared with controls. A true developmental effect is only seen when a number of variants or a syndrome of variants is observed, therefore the intergroup difference is considered of no biological significance.
Visceral malformations:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Key result
Developmental effects observed:
no

Please see attached background material for results:

Body weight, body weight change and gravid uterine weight,

Body weight change corrected for gravid uterine weight

Number of pregnant and non-pregnant dams

Number of dams with abortions, early deliveries, stillbirths, resorptions, and/or dead foetuses

Pre-and postimplantation loss, number and percent

Mean number and percent of live offspring

Mean foetal/pup body weight by sex and sexes combined

Number and percent of foetuses and litters with malformation and/or variation, description and incidences of malformations and main variations (and/or retardation).

Historical control data

Conclusions:
The oral administration of Polyphosphoric acids, esters with triethanolamine, sodium salts to pregnant rats by gavage during gestation at dose levels of 100, 500 and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%), did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction:

The study was designed to investigate the effects of the test item on adult pregnant females and embryonic and fetal development following repeated administration by gavage to the pregnant female rat during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 500, and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%)). A further group of twenty-four time mated females was exposed to the vehicle only to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality:

There were no unscheduled deaths.

Clinical Observations:

No clinically observable signs of toxicity were evident in treated females.

Body Weight:

No toxicological significant effects in body weight development were detected.

Food Consumption:

No treatment-related effects were detected in food consumption.

Water Consumption:

No adverse effects on water consumption were detected.

Post Mortem Studies:

No macroscopic abnormalities were detected.

Litter Data and Litter Placental and Fetal Weights:

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Fetal Examination:

No treatment-related effects were detected on fetal external findings. No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings in fetuses from females treated with 100, 500 or 1000 mg/kg bw/day.

Conclusion:

The oral administration ofPolyphosphoricacids, esters with triethanolamine, sodium saltsto pregnant rats by gavage during gestation at dose levels of 100, 500 and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%)), did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Endpoint:
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: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the studywas conducted under GLP conditions.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) as at 22nd March 1996
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
Species:
rat
Strain:
Wistar
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.
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.Based upon the results of stability analyses performed within the Harlan Laboratories study nos. D34115 and D39381 (Development and Implementation of an analytical method for dose formulation analysis), dose formulations were stable for at least one week.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, Harlan Laboratories Study D35115, 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)
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.
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.
Duration of treatment / exposure:
- Males: 28 weeks- Females: Approximately 6 weeks
Frequency of treatment:
- Once daily
Duration of test:
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)
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
Maternal 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- 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. 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 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 on day 4 post partum, relevant parameters were performed with five P 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 from 5 lactating females from each group 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.Hematology:The following hematology parameters were determined: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 time Clinical Biochemistry:The 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 TERMINATION AND NECROPSYDams 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. At the scheduled sacrifice, all parent animals were sacrificed by an injection of sodium pentobarbital and exsanguinated.. All parent animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. 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 females:- Ovaries (as pairs) - Uterus and Cervix - 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 females were preserved in neutral phosphate buffered 4% formaldehyde solution:- Ovaries - Uterus- Vagina- Cervix In addition, from all females at scheduled necropsy 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 tissue HISTOTECHNIQUEAll 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.HISTOPATHOLOGYSlides of all organs and tissues collected at terminal sacrifice from the first five females which gave birth and slides of all reproductive organs from all females of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all unfertile animals. Histological examination of ovaries was carried out on any females that did not give birth.A histopathology peer review was performed a principal investigator (AnaPath GmbH).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination on day 5 post partum.Examinations included:- Gravid uterus weight: No- Number of corpora lutea: Yes - Number of implantations: Yes- Number of early resorptions: No- Number of late resorptions: No
Fetal examinations:
Not performed
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.
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.
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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):
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
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Six females were not pregnant; one in the control group, three at the low dose level and two at the high dose level. 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 mat ed) 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.
Details on maternal toxic effects:
Maternal toxic effects:no effectsDetails on maternal toxic effects:IN-LIVE DATA - PARENTAL ANIMALSVIABILITY / MORTALITYAll animals survived the scheduled study period.CLINICAL OBSERVATIONS (DAILY)No test item-related clinical signs were found in females at any dose level.Incidentally, 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 females at any dose level.DETAILED CLINICAL OBSERVATIONS (WEEKLY)No test item-related effects were noted during detailed weekly clinical observations in 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 females at any dose level.Incidentally, 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 females at any dose level.LOCOMOTOR ACTIVITYNo effects were noted during measurement of locomotor activity in females at any dose level.In general, mean total beam crossing counts within 30 minutes of measurement were 932, 823, 822 and 943 in females, given in the order of ascending dose levels.FOOD CONSUMPTIONPre-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 WEIGHTSPre-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 INVESTIGATIONSHematology:No effects on hematology parameters were noted at any dose level.Clinical Biochemistry:No effects on clinical biochemistry parameters were noted at any dose level.ORGAN WEIGHTSNo 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 FINDINGSNo 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.
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive and developmental toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
other: No signs of reproductive or developmental toxicity in males or females was observed up to 1000 mg/kg bw day, the highest dose level used.
Key result
Dose descriptor:
NOEL
Remarks:
General toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: other:
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Key result
Dose descriptor:
NOEL
Remarks:
for development to day 4 post partum
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:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
Remarks on result:
other: No test item-related findings were noted in pups to day 4 post partum
Key result
Developmental effects observed:
no
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.
VIABILITY / MORTALITY OF MALES

All animals survived the scheduled study period.

 

CLINICAL OBSERVATIONS (DAILY) OF MALES

No test item-related clinical signs were found in males 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).

 

DETAILED CLINICAL OBSERVATIONS (WEEKLY) OF MALES

No effects were noted during detailed weekly clinical observations in males at any dose level.

 

FUNCTIONAL OBSERVATIONALBATTERYOF MALES

No test item-related effects were noted during functional observational battery in males 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).

No further findings were noted during functional observational battery in males at any dose level.

 

LOCOMOTOR ACTIVITY OF MALES

No test item-related effects were noted during measurement of locomotor activity in males 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.

 

In general, mean total beam crossing counts within 30 minutes of measurement were 910, 1220, 1333 and 1501 in the order of ascending dose levels.

 

FOOD CONSUMPTION OF MALES

Pre-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).

 

BODY WEIGHTS OF MALES

Pre-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).

 

CLINICAL LABORATORY INVESTIGATIONS OF MALES

Hematology:

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.

 

Clinical Biochemistry:

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.

 

ORGAN WEIGHTS OF 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.

 

MACROSCOPICAL FINDINGS OF 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.

 

HISTOPATHOLOGY FINDINGS OF MALES

The 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 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. 

 

MATING PERFORMANCE AND FERTILITY

No 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 COUNT

No 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 LOSS

No 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 GESTATION

No 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 LOSS

No 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 CHECK

No 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 PARTUM

No 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.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

No 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 RATIOS

Pups 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 REFLEX

No 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 OF PUPS TO DAY 4 POST PARTUM

No 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 FINDINGS OF PUPS

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

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database consists of two reliable (Klimisch 1) studies, the prenatal developmental toxicity in rats (OECD 414) and one for developmental screening in rats (OECD 422). The data presented meet the requirements of REACH Annexes VII to X for this endpoint.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal developmental toxicity (OECD 414)

This study investigated the effects of the test item (Polyphosphoric acids, esters with triethanolamine, sodium salts)

on adult pregnant females and embryonic and fetal development following repeated administration by gavage to the pregnant female rat during gestation including the period of organogenesis. The test item was administered between Days 5 and 19 of gestation inclusive at dose levels 100, 500, and 1000 mg/kg bw/day and controls received the vehicle only.

Treatment with the test item did not result in any toxicological significant effects in parental females or toxicologically significant changes in offspring parameters.

The maternal ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 1000 mg/kg bw/day.

The developmental ‘No Observed Effect Level’ (NOEL) was considered to be 1000 mg/kg bw/day.

Combined repeated dose toxicity study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD Guideline 422).

This study is a valid investigtion 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. The test item 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. The test item 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.

Six females were not pregnant; one in the control group, three at the low dose level and two at the high dose level. The increased number of non-pregnant females in the low dose group was considered to be incidental with no one single factor that could be isolated to provide a cause for this infertility. A background of spontaneous non-pregnant females is an expected event for reproduction toxicology studies. Variable rates of infertility can also be expected in studies of relatively small group size. There were no indications to suggest any issues to show that the health or reproductive capacity of this strain of animal has been compromised. The conclusion is therefore that reduced reproductive performance on this study was a spontaneous event.

The low dose level had only seven pregnancies, which is lower than that prescribed for the study. Of the seven pregnancies there were no findings to suggest an affect on reproduction and development. This finding does not contradict the conclusions of a lack of treatment effect at the higher treatment levels where sufficient pregnancies were seen thereby allowing the appropriate evaluation to take place.

Justification for classification or non-classification

Both the prenatal developmental toxicity (OECD 414) and the Combined Repeat Dose Toxicity with Reproduction/Developmental Toxicity Screening Test (OECD 422) in rats showed no treatment related effects in the reproductive parameters examined or on foetal development at any dose level including the highest permitted dose level of 1000 mg/kg. Therefore, there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development. Therefore the substance is not classified for reproductive or developmental toxicity.