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EC number: 223-989-2 | CAS number: 4156-21-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on generations indicated in Effect levels (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21-Jun-2012 to [include experimental completion date]
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Well performed GLP and OECD guideline study
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate
- EC Number:
- 223-989-2
- EC Name:
- Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate
- Cas Number:
- 4156-21-2
- Molecular formula:
- C9H6Cl2N4O3S.Na
- IUPAC Name:
- sodium 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate
- Test material form:
- other: White powder (referred to pure substance)
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system 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: 44 males (11 per group) and 44 females (11 per group)- Age (at Start of Treatment): 11 weeks- Body Weight Range (at Start of Treatment): 298 to 340 g (males) and 183 to 212 g (females)- Identification: Parent animals by cage card and individual animal number (ear tattoo). On day 1 post partum, pups were individually tattooed with Indian ink.- Randomization: Performed after four days of acclimatization using a computer-generated random algorithm. 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%). Values outside of these ranges occasionally occurred, usually following room cleaning, which is considered not to have any influence on the study. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards 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 (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105120601, 02105120301 and 6960C.CS-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. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Analyses for contaminants were performed. - Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- other: highly purified water
- Details on exposure:
- DOSE FORMULATIONSThe dose formulations were prepared weekly using the test item as supplied by the Sponsor. Dose levels were calculated based on the active ingredient.Granofin Easy F-90 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. 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 in the refrigerator (5 ± 3 °C) in glass beakers.Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study D51272 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations were stable for at least 7 days if stored in the refrigerator.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: 0 mg/kg/day (Group 1, control group), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)- 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 D51272, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.- Dose Volume: 10 mL/kg body weight- Dose Concentrations: 0 mg/mL/day (Group 1, control group), 10 mg/mL/day (Group 2), 30 mg/mL/day (Group 3) and 100 mg/mL/day (Group 4)
- Details on mating procedure:
- During 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:
- METHODOn the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 0.5 g of each concentration were taken from the middle only of each aliquot used on day 7 of the treatment. 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 the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.The samples were analyzed following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were discarded.Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were discarded.RESULTSThe Granofin Easy F-90 peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of Granofin Easy F-90 and, therefore, the absence of the test item in the vehicle control samples (highly purified water) was confirmed. The application formulations investigated during the study were found to comprise Granofin Easy F-90 in the range of 88% to 99.4% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Granofin Easy F-90 in the preparations was approved because single results found did not deviate more than 1.0% (acceptance criterion: <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 Granofin Easy F-90 and highly 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 daysFemales: Approximately 7 weeks
- Frequency of treatment:
- Once daily
- Details on study schedule:
- MALES- Acclimatization : 7 days- First Test Item Administration : Day 1 of pre-pairing- Pre-Pairing : 14 days- Pairing : 14 days maximum- Treatment Ends : On day before sacrifice- Blood Sampling : Day 14 of pre-pairing- Necropsy : After treatment of at least 28 days, when no longer needed for assessment of reproductive effectsFEMALES- Acclimatization : 7 days- 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 3 post partum- Blood Sampling : Day 14 of pre-pairing- Necropsy : Females and pups on day 4 post partum
Doses / concentrations
- Remarks:
- Doses / Concentrations:0, 100, 300 and 1000 mg/kg bw/dayBasis:actual ingested
- No. of animals per sex per dose:
- 11
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies. Rationale for Choice of Species, Route of Administration and Dose LevelsThe rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (non-GLP Harlan Laboratories Study D51272).
- Positive control:
- Not required
Examinations
- Parental animals: Observations and examinations:
- VIABILITY/MORTALITYTwice dailyCLINICAL SIGNSDaily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.FOOD CONSUMPTION- Males: Pre-Pairing period days 1 - 8, 8 - 13; after pairing period weekly.- Females: Pre-Pairing period days 1 - 8, 8 - 13; gestation days 0 - 7, 7 14 and 14 - 21 and days 1 - 4 of the lactation.No food consumption was recorded during the pairing period.BODY WEIGHTSWas recorded once during acclimatization and daily from treatment start to day of necropsy.DETAILED CLINICAL OBSERVATIONSDetailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, 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 were also reported.FUNCTIONAL OBSERVATIONAL 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, constitution, skin, pupil 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, and 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 day 14 of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn sublingually under light isoflurane anesthesia. The animals were fasted for approximately 18 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.The assay was performed under the responsibility of R. Draheim at Harlan Laboratories Ltd. (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.Blood sampling after 14 days: 11-Jul-2012The 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 timeThe 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):
- Not examined
- Sperm parameters (parental animals):
- Not examined
- 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), 1 and 4 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. Females and pups were sacrificed on day 4 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 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. Special attention was directed at the organs of the reproductive system.Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. 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, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken. - Adrenal glands (weighed as pairs)- Brain (including cerebrum, cerebellum and pons)- Heart- Kidneys (weighed as pairs)- Liver- Thymus- SpleenTISSUE PRESERVATIONThe following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:- Prostate- Seminal vesicles with coagulating gland - Testes (in Bouin’s fixative)- Epididymides (in Bouin’s fixative)The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:In addition, from the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:- Gross lesions- Brain (including cerebrum, cerebellum, and pons)- Spinal chord- 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)- Uterus (with vagina)- Urinary bladder- Lymph nodes (mesenterial, mandibular)- Peripheral nerve (sciatic)- Bone marrowHISTOTECHNIQUEAll organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.HISTOPATHOLOGYSlides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.Test item-related morphologic changes were detected in liver, stomach, spleen and kidney in high-dose animal, therefore those same organs from the mid- and low-dose group were examined to establish a no-effect level.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, if necessary. A histopathology peer review was performed and the results were included in the histopathology phase report.
- Postmortem examinations (offspring):
- TERMINATION AND NECROPSYPups were sacrificed on day 4 post partum by an injection of sodium pentobarbital. All pups were examined macroscopically for any structural changes. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
- Statistics:
- The following statistical methods were used to analyze food consumption, body weights, locomotor activity, organ weights and ratios, macroscopical findings, clinical laboratory data and reproduction data:- Means and standard deviations of various data were calculated.- 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: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index
- Offspring viability indices:
- From the on-line reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day bedding in mouth noted in both genders was considered a sign of discomfort caused by the test item
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, reduction of food consumption and body weight gain noted in males at the beginning of the pre-pairing period and absolute body weights also during pairing. This effect was reversible.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, reduction of food consumption and body weight gain noted in males at the beginning of the pre-pairing period and absolute body weights also during pairing. This effect was reversible.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment with the test item led to morphological changes in liver, stomach, spleen and kidney in males down to 100 mg/kg bw/day and in females down to 300 mg/kg bw/day.
- Other effects:
- not examined
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
Details on results (P0)
Effect levels (P0)
open allclose all
- Dose descriptor:
- NOAEL
- Remarks:
- for general toxicity
- Sex:
- male
- Basis for effect level:
- other: Due to findings in the liver noted down to the low-dose level (100 mg/kg bw/day).
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified
- Dose descriptor:
- NOAEL
- Remarks:
- for general toxicity
- Effect level:
- 100 mg/kg bw/day
- Sex:
- female
- Basis for effect level:
- other: As a consequence of histopathology findings noted at 1000 and 300 mg/kg bw/day
- Dose descriptor:
- NOEL
- Remarks:
- for reproduction
- Effect level:
- 1 000 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: The relevant reproduction parameters investigated within this study were not affected by the treatment with the test item at any dose level
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
Details on results (F1)
Effect levels (F1)
- Dose descriptor:
- NOEL
- Remarks:
- for development
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: The relevant developmental parameters investigated within this study were not affected by the treatment with the test item at any dose level
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Any other information on results incl. tables
SUMMARY OF PERFORMANCE
P animals breeding for F1 Litters
Group | 1 | 2 | 3 | 4 |
Female numbers | 45-55 | 56-66 | 67-77 | 78-88 |
Number of females paired | 11 | 11 | 11 | 11 |
Number of females mated | 11 | 11 | 11 | 11 |
Number of non pregnant females (A) | 1 | 1 | 0 | 3 |
Numbers of pregnant females, which did not deliver any pups (B) | 0 | 0 | 1 | 0 |
Number of females which reared their pups until day 4 post partum | 10 | 10 | 10 | 8 |
(A) Female Nos. 54, 65, 78, 85 and 87.
(B) Female No. 72 had implantations only
Applicant's summary and conclusion
- Conclusions:
- This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Granofin Easy F-90 to rats. Granofin Easy F-90 was administered in vehicle (highly purified water) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. 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. GENERAL TOXICITYAll animals survived the scheduled study period. With exception for bedding in mouth noted in males and females at the dose level of 1000 mg/kg bw/day, which was considered to be sign of discomfort caused by the test item, no clinical signs related to the treatment were noted at any dose level.Food consumption, body weight gain and absolute body weights were temporarily reduced in males at the dose level of 1000 mg/kg bw/day during the first days of treatment but these values recovered and were similar to or higher than the control values during the remaining study period. Therefore these effects were considered not to be adverse.In females no effects on food consumption, body weight gain or absolute body weights were noted at any dose level.During clinical laboratory investigations multiple changes were noted in hematology parameters at the dose levels of 1000 mg/kg bw/day, most of them in both genders: reduced hematocrit, increased red cell volume distribution width, increased mean corpuscular hemoglobin concentration, increased hemoglobin concentration distribution width, increased number of total leucocytes, increased concentration of neutrophils and increased number of large unstained cells. Increased mean corpuscular hemoglobin concentration was noted also at the dose level of 300 mg/kg bw/day in both genders. No cause for these findings was identified within this study. However taking into consideration the extent of these changes at the high-dose level, a systemic adverse effect as a cause for these changes could not be excluded. Effect at the dose level of 300 mg/kg bw/day was minor and therefore considered possibly not related to any adverse systemic effect.Changes in clinical biochemistry parameters were noted at the dose level of 1000 mg/kg bw/day: increased activity of alanine aminotransferase in males and females and increased concentration of glucose in females. These findings were considered to be test item-related.Further findings were: increased weights of thymus, kidneys and spleen in males and increased weights of kidneys and spleen in females at the dose level of 1000 mg/kg bw/day. These changes were considered to be test item-related but not adverse in the absence of any related adverse histopathological change.An increased incidence and mean severity of extramedullary hemopoiesis in spleen were noted at the dose level of 1000 mg/kg bw/day in both genders and at the dose level of 300 mg/kg bw/day in females. Reason for these findings was not clear. The finding may represent an adaptation to an increased demand in erythrocytes, which is supported by the hematology findings in this study, and therefore was considered to be reversible.Renal tubular vacuolation of mainly cortical tubules was recorded in kidneys of females at the dose level of 1000 mg/kg bw/day. This finding was associated with increased organ weight ratios. In the absence of any degenerative or increased regenerative lesions this finding was considered to be an adaptive change and reversible.Findings in the stomach: increased incidence and/or severity of submucosal inflammatory infiltrate, mucosal inflammatory foci, mucosal erosions, and acanthosis/hyperkeratosis of the limiting ridge noted in males in all dose groups and in females at the dose levels of 300 and 1000 mg/kg bw/day in both genders and mucosal ulceration in males at this dose level were considered to reflect a local irritant action and to represent a portal of entry effect unrelated to systemic effects.During histopathological examination, inflammatory cell foci were found at elevated incidence and / or severity in the liver at the dose levels of 1000, 300 and 100 mg/kg bw/day in males and at the dose levels of 1000 and 300 mg/kg bw/day in females. The reason for this finding is not clear at this stage, the finding is deemed to be adverse.REPRODUCTION AND DEVELOPMENTAL TOXICOLOGYNo indication of any test item-related effect on reproduction or development was noted during the study at any dose level.CONCLUSIONBased on these results, no NOAEL (No Observed Adverse Effect Level) for general toxicity was established in males due to findings in the liver noted down to the low-dose level. NOAEL in females was established at the dose level of 100 mg/kg bw/day.The NOEL (No Observed Effect Level) for reproduction and 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 preliminary information concerning the effects of Granofin Easy F-90 on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.
Granofin Easy F-90 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 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: 300 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:
MORTALITY AND GENERAL TOLERABILITY IN PARENTAL ANIMALS
All animals survived the scheduled study period.
At the dose level of 1000 mg/kg bw/day, bedding in mouth was noted in males and females. This finding was considered to be sign of discomfort caused by the test item.
No further findings were noted at any dose level.
FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS
No test item-related findings were noted during functional observational battery in males or females at any dose level. No effects on locomotor activity were noted in males or females at any dose level.
FOOD CONSUMPTION OF PARENTAL ANIMALS
At the dose level of 1000 mg/kg bw/day, reduction of food consumption was noted in males at the beginning of the pre-pairing period. This effect was reversible; food consumption increased thereafter and was slightly higher than the control values during the remaining study period.
No effects on food consumption were noted in males at the dose levels of 100 and 300 mg/kg bw/day or in females at any dose level.
BODY WEIGHTS OF PARENTAL ANIMALS
At the dose level of 1000 mg/kg bw/day, reduced body weight gain in males was noted during the pre-pairing period. Afterwards body weight gain recovered and was similar or higher than the control values. Absolute body weights of males at the high dose level were slightly lower during the pre-pairing and pairing periods and similar to the control values during after pairing period.
No test item- related effects on absolute body weights or body weight gain were noted in males at the dose level of 100 and 300 mg/kg bw/day or in females at any dose level.
CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS
Test item related changes in hematology parameter were noted in males and females at the dose levels of 1000 and 300 mg/kg bw/day.
Following changes were noted at the dose level of 1000 mg/kg bw/day: reduced hematocrit (in males), increased red cell volume distribution width (in both genders), increased mean corpuscular hemoglobin concentration (in both genders), increased hemoglobin concentration distribution width (in both genders), increased number of total leucocytes (in both genders), increased concentration of neutrophils (in both genders) and increased number of large unstained cells (in females). All these changes were considered to be related to the treatment with the test item. These changes were considered to possibly indicate a systemic adverse effect.
At the dose level of 300 mg/kg bw/day, increased mean corpuscular hemoglobin concentration was noted in both genders. This effect was considered to be test item-related, but not adverse.
No further effects on hematology parameters were noted in males or females at any dose level.
At the dose level of 1000 mg/kg bw/day, increased activity of alanine aminotransferase was noted in males and females and increased concentration of glucose noted in females. These findings were considered to be test item-related.
No further effects on biochemistry parameters were noted in males or females at any dose level.
REPRODUCTION AND BREEDING DATA OF PARENTAL ANIMALS
Mating performance, fertility, number of corpora lutea, duration of gestation, implantation rate and post-implantation loss, litter size and postnatal loss were not affected by the treatment with the test item any dose level.
ORGAN WEIGHTS OF PARENTAL ANIMALS
At the dose level of 1000 mg/kg bw/day in males, higher weights of thymus, kidneys and spleen were noted. In females at the high dose level increased weights of liver, kidneys and spleen were noted. These changes were considered to be test item-related.
No further test item-related effects on organ weights were noted in males or females at any dose level.
MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS
Type and distribution of findings recorded during necropsy did not indicate any test item-related effect in males or females.
Under the conditions of this experiment, Granofin Easy F-90 led to morphological changes in liver, stomach, spleen and kidney. Findings in the liver (increased incidence of hepatic inflammatory cell foci) were recorded at the dose levels of 1000, 300 and 100 mg/kg bw/day in males and at the dose levels of 1000 and 300 mg/kg bw/day in females. These findings were considered to be adverse.
Findings in the stomach (increased incidence and/or severity of submucosal inflammatory infiltrate, mucosal inflammatory foci, mucosal erosions, acanthosis/hyperkeratosis of the limiting ridge and mucosal ulceration) were recorded at the dose levels of 1000, 300 and 100 mg/kg bw/day in males and at the dose levels of 1000 and 300 mg/kg bw/day in females. These findings were considered to reflect a local irritant action and to represent a portal of entry effect; an effect unrelated to systemic effects.
Increased incidence and/or mean severity of extramedullary hemopoiesiswere recorded at the dose level of 1000 mg/kg bw/day in males and at the dose levels of 1000 and 300 mg/kg bw/day in females. The cause for the finding in the spleen was unclear. It was considered to probably represent an adaptation to an increased demand in erythrocytes and was considered to be reversible.
Finding in the kidneys (renal tubular vacuolation of mainly cortical tubules) recorded in females at the dose level of 1000 mg/kg bw/day was considered to represent an adaptive change and to be reversible.
FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related findings were noted in pups during first litter check and during lactation in any dose group.
Pup sex ratio was not affected by the exposure to the test item at any dose level.
BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM
Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.
MACROSCOPICAL FINDINGS OF PUPS
No test item-related findings were noted at macroscopic examination of pups in any dose group.
CONCLUSION
Based on these results, no NOAEL (No Observed Adverse Effect Level) for general toxicity was established in males due to findings in the liver noted down to the low-dose level. NOAEL in females was established at the dose level of 100 mg/kg bw/day.
The NOEL (No Observed Effect Level) for reproduction and developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.
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