Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

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:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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)

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)

VIABILITY/MORTALITYAll animals survived scheduled study period.DAILY CLINICAL SIGNS OR OBSERVATIONSAt the dose level of 1000 mg/kg bw/day, bedding in mouth was noted in all males and females starting from day 12 of the pre-pairing period until the end of the treatment. This finding was considered to be sign of discomfort caused by the test item.No further findings were noted at any dose level.FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONSNo findings were noted during detailed weekly clinical observations in males or females at any dose level.FUNCTIONAL OBSERVATIONAL BATTERYNo test item-related findings were noted during functional observational battery in males or females at any dose level.Incidentally, vocalization was noted in one female at the dose level of 100 mg/kg bw/day.No further findings were noted during functional observational battery in males or females at any dose level.LOCOMOTOR ACTIVITYNo effects on locomotor activity were noted in males or females at any dose level.Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1369, 1307, 1342 and 1106 in males and 953, 808, 896 and 854 in females.FOOD CONSUMPTION OF MALESAt the dose level of 1000 mg/kg bw/day, a statistically significant reduction of food consumption was noted from day 1 to 8 of the pre-pairing period. Mean food consumption was 24.7 g/ animal/day at the high dose level compared to 28.1 g/animal/day in the control group. Afterwards, food consumption recovered and was slightly higher than the control values.No effects on food consumption were noted at the dose levels of 100 and 300 mg/kg bw/day.Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: -0.4%, +3.6% and -4.3% during the pre-pairing period and +1.1%, +4.2% and +8.3% during the after pairing period (percentages refer to the respective values in the control group).FOOD CONSUMPTION OF FEMALESNo effects on food consumption were noted in females at any dose level.Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: -13.4%, -12.4%, and -8.0% during the pre-pairing period, -6.7%, -1.3% and +3.6% during the gestation period and +1.2%, +7.5% and +0.4% during the lactation period (percentages refer to the respective values in the control group).BODY WEIGHTS OF MALESAt the dose level of 1000 mg/kg bw/day, reduced body weight gain was noted during the pre-pairing period. The reduction was statistically significant from day 5 to 13 of this period. Afterwards body weight gain recovered and was similar or higher than the control values. On days 4, 9 and 10 of the after pairing period increase in body weight gain was statistically significant. 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. The differences were not statistically significant at any time of the study.No effects on absolute body weights or body weight gain were noted at the dose level of 100 and 300 mg/kg bw/day.Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +14% and +10% during the pre-pairing period, +3%, +3%, +2% and +3% during the pairing period and +8%, +8%, +8% and +9% during the after pairing period (percentages refer to the body weight change within the respective period).BODY WEIGHTS OF FEMALESNo test item-related effects on absolute body weights or body weight gain were noted in females at any dose level.At the dose level of 1000 mg/kg bw/day, body weight loss by 2% was noted during lactation. Body weight gain in the control group was +4% during this period. Because the mean value of the body weight loss at the high-dose level was due to changes in the body weights of two females (no. 81 and 86) and body weight gain of the remaining females was similar to the control values, this change was considered to be incidental and not related to the treatment with the test item.Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +6%, +5%, +8% and +8% during the pre-pairing period, +60%, +56%, +59% and +59% during the gestation period and +4%, +5%, +2% and -2% during the lactation period (percentages refer to the body weight change within the respective period).2. CLINICAL LABORATORY INVESTIGATIONSHEMATOLOGY- In males, the following changes at the dose level of 1000 mg/kg bw/day were considered to be test item-related: reduced hematocrit (HCT; 0.44 versus 0.48 in the control group), increased red cell volume distribution width (RDW; 0.139 versus 0.113 in the control group), increased mean corpuscular hemoglobin concentration (MCHC; 22.45 mmol/l versus 21.47 mmol/l in the control group), increased hemoglobin concentration distribution width (HDW; 2.16 mmol/l versus 1.64 mmol/l in the control group), increased number of total leucocytes (WBC; 14.69 x 109/l versus 10.24 x 109/l in the control group) and increased concentration of neutrophils (NEUT; 3.98 x 109/l versus 1.06 x 109/l in the control group). All these changes were statistically significant and a similar pattern was noted in females. For these reasons they were considered to be related to the treatment with the test item.At the dose level of 300 mg/kg bw/day, increased MCHC was noted. Mean value at the mid-dose level was 22.02; the difference to the control was statistically significant. Because of dose-dependent increase of this value which was noted also in females, this change was considered to be test item-related.No further effects on hematology parameters were noted in males at any dose level.- In females, the following changes at the dose level of 1000 mg/kg bw/day were considered to be test item-related: increased RDW (0.139 versus 0.119 in the control group), increased MCHC (22.50 mmol/l versus 21.84 mmol/l in the control group), increased HDW (1.84 mmol/l versus 1.43 mmol/l in the control group), increased number of WBC (7.13 x 109/l versus 4.93 x 109/l in the control group), increased concentration of NEUT (1.76 x 109/l versus 0.66 x 109/l in the control group) and increased number of large unstained cells (LUC; 0.12 x 109/l versus 0.06 x 109/l in the control group). With exception for increased number of WBC, all these changes were statistically significant and with exception for LUC, they were noted also in males, with statistical significance. For these reasons they were considered to be related to the treatment with the test item.At the dose level of 300 mg/kg bw/day, increased MCHC was noted. Mean value at the mid-dose level was 22.34; the difference to the control was statistically significant. Because of dose-dependent increase of this value which was noted also in males, this change was considered to be test item-related.No further effects on hematology parameters were noted in females at any dose level.CLINICAL BIOCHEMISTRY- In males, at the dose level of 1000 mg/kg bw/day, increased activity of alanine aminotransferase (ALAT) was noted. Mean value of ALAT was 72.7 U/l at the high dose level compared to 42.6 U/l in the control group. Change of ALAT concentration was statistically significant. This finding was considered to be test item-related.No further test item-related changes of biochemistry parameters were noted in males at any dose level.At the dose level of 300 mg/kg bw/day, statistically significantly increased concentration of glucose was noted. Because value at the mid-dose level was within the 95% of tolerance limit and because of lack of similar change at the high-dose level, this effect was considered not to be related to the treatment.- In females, at the dose level of 1000 mg/kg bw/day, increased activity of ALAT was noted. Mean value of ALAT was 62.2 U/l at the high dose level compared to 30.8 U/l in the control group. Change of ALAT concentration was statistically significant. This finding was considered to be test item-related. Further, statistically significantly increased concentration of glucose was noted at the high-dose level. Mean concentration of glucose in this group was 8.84 mmol/l compared to 7.58 mmol/l; value at the high-dose level was beyond the 95% of tolerance limit (containing values from 4.41 to 7.42 mmol/l). For this reason increase in concentration of glucose was considered to possibly be test item-related.No further changes of biochemistry parameters were noted in females at any dose level.3. TERMINAL FINDINGSORGAN WEIGHTSAt the dose level of 1000 mg/kg bw/day in males, higher weights of thymus, kidneys and spleen were noted. Increase in absolute weights of these organs, increase in their weights relative to brain weights and increase in spleen weight to body weight were statistically significant. In females at the high dose level increased weights of kidneys and spleen were noted. Increase in absolute weight of spleen and increase in liver, kidneys and spleen weights relative to brain weights were statistically significant. 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.Further statistically significant changes: lower epididymides weights to body weight ratio (unilateral) and higher testis weights to brain weights ratios (bilateral), were noted in males at the dose level of 1000 mg/kg bw/day. Because absolute weights of these organs were similar to the controls and only one of the calculated ratios was changed, these effects were considered not to be related to the treatment with the test item. In females at the dose level of 300 mg/kg bw/day, statistically significantly lower heart weights to body weights ratio was noted. In the absence of any effect on heart weights at the dose level of 1000 mg/kg bw/day, this change was considered not to be related to the treatment with the test item.MACROSCOPICAL FINDINGS- Males: During necropsy, findings were noted in individual animals. In the control group one male (no. 2) had red foci on the lung and on the thymus, one male (no. 3) had red foci on the thymus and one male (no. 10) had reddish discoloration of seminal vesicles. At the low-dose level, one male (no. 12) had enlarged liver, pelvic dilation, and red foci on the thymus. At the mid-dose level, one male (no. 24) had crateriform retraction in the stomach and one male (no. 25) had enlarged liver. At the high-dose level, one male (no. 34) had red foci in the stomach, enlarged liver, pelvic dilation and red foci on thymus, one male (no. 36) had firm, gray white nodule in the stomach, one male (no. 37) had enlarged liver and one male (no. 38) had red foci on the stomach and pelvic dilation. No further findings were noted in males at any dose level. Type and distribution of these findings did not indicate any test item-related effect.- Females: During necropsy, findings were noted in individual animals. In the control group one female (no. 50) had thymus reduced in size. At the low-dose level, one female (no. 56) had watery cyst on left ovary, one female (no. 57) had thymus reduced in size, one female (no. 58) had reddish foci on clitoral glands, one female (no. 62) had red discoloration of ovaries, one female (no. 64) had ectopic splenic tissue and one female (no. 65) had red discoloration of thymus. At the mid-dose level, two females (nos. 69 and 71) had red discoloration of ovaries, one female (no. 70) had reddish discoloration of pancreas and one female (no. 72) had dilation of uterus (unilateral). At the high-dose level three females (nos. 78, 85 and 87) had red/reddish discoloration of ovaries and one female (no. 87) had pus-like fluid in uterus. No further findings were noted in females at any dose level. Type and distribution of these findings did not indicate any test item-related effect.HISTOPATHOLOGY FINDINGSUnder the conditions of this experiment, Granofin Easy F-90 led to morphological changes in liver, stomach, spleen and kidney.- Liver: Inflammatory cell foci were found at elevated incidence and / or severity in males of groups 2-4 and females of groups 3-4. - Stomach: The full spectrum of inflammatory findings (submucosal inflammation, inflammatory mucosal foci), acanthosis/hyperkeratosis of limiting ridge and impaired integrity of glandular mucosa (erosion and / or ulceration) was found in group 4 animals of both genders. Additionally, increased incidence and or severity of submucosal inflammatory infiltrate was found in males of groups 2-3 and, mucosal erosions were recorded in animals of both genders in group 3- Spleen: Substantially increased incidence and/or mean severity of extramedullary hemopoiesis was recorded in group 3 females and group 4 animals of both genders. - Kidneys: Renal tubular vacuolation of mainly cortical tubules was found in kidneys of dose group 4 females. The finding was paralleled by increased organ weight ratios. - Other Findings: The remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.- Qualitative Sperm Staging: There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. - Findings in Non-Pregnant Animals: Animals 54, 65, 72, 78, 85 and 87 were found to be not pregnant. In animal 72 (group 3) cornual dilation of the uterus was found to be causative for the failure to deliver. However, animal 72 showed pregnancy related changes in the uterus, including decidual reaction and hemorrhagic implantation sites. In animal 87 (group 4) the finding of a uterine/cervical retention cyst was deemed to be responsible for missed conception.In all further animals no reason for the missed pregnancy could be recorded. 4. REPRODUCTION AND BREEDINGMATING PERFORMANCE AND FERTILITYMating performance and fertility were not affected by the treatment at any dose level. All females mated within the first pairing period.Mean (median) precoital times were 3.2 (3), 3.1 (3), 2.8 (3) and 2.5 (2) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.Five females were not pregnant: one each in control and low-dose group and three in the high-dose group. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 90.9%, 90.9%, 100.0% and 72.7% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. One female at the mid-dose level had two implantation sites but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and high-dose levels and 90.9% at the mid-dose level.CORPORA LUTEA COUNTNo effects on corpora lutea count were observed at any dose level.Mean number of corpora lutea per dam was 13.6, 12.3, 13.3 and 12.9 in order of ascending dose levels.DURATION OF GESTATIONNo effects on duration of gestation were observed at any dose level.Mean duration of gestation was 21.5, 21.4, 21.5 and 21.4 days, in order of ascending dose level.IMPLANTATION RATE AND POST-IMPLANTATION LOSSNo effects on implantation rate and post-implantation loss were observed at any dose level.In order of ascending dose levels, mean number of implantations per dam was 12.6, 11.6, 12.3 and 12.3 whereas mean incidence of post-implantation loss per dam was 1.3, 0.7, 1.1 and 1.9 per dam.LITTER SIZE AT FIRST LITTER CHECKNo effects on litter size were noted at any dose level.At the dose level of 1000 mg/kg bw/day, two pups (from two litters) were found dead during the first litter check. This finding was considered to be in the normal biological range.Mean number of living pups per dam at first litter check was 11.3, 10.9, 11.2 and 10.8 in order of ascending dose levels.Birth index (number of pups born alive as a percentage of implantations) was 89.7%, 94.0%, 91.1% and 87.8% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.SUMMARY OF PERFORMANCE P animals breeding for F1 Litters Group (mg/kg/day) 1(0)2(100)3(300)4(1000)Female numbers 45-5556-6667-7778-88Number of females paired 11111111Number of females mated 111111 11Number of non pregnant females (A)1103Numbers of pregnant females,which did not deliver any pups (B)0010 (A) Female Nos. 54, 65, 78, 85 and 87.(B) Female No. 72 had implantations only.  POSTNATAL LOSS DAYS 0 – 4 POST PARTUM No test item-related effects on postnatal loss were noted at any dose level. In the control group one pup was missing on day 3, at the low-dose level one pup was missing on day 2, at the high-dose level two pups (from one litter) were missing on days 2 and 3. Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.0% and 0.3% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 99.1%, 99.1%, 100.0% and 97.7% in order of ascending dose levels. EXTERNAL EXAMINATION OF 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 RATIOS Pup sex ratio was not affected by exposure to the test item at any dose level. At first litter check, percentages of male pups were 50%, 54%, 53% and 44% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day. 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. In order of ascending dose levels, mean body weights of pups were: 5.9 g, 6.0 g, 6.2 g and 6.0 g on day 1 post partum and 8.9 g, 9.1 g, 9.2 g and 8.7 g on day 4 post partum. Mean differences in body weights during lactation were +50.9%, +50.9%, +47.8% and +44.5%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively. MACROSCOPICAL FINDINGS OF PUPS No test item-related findings were noted at macroscopic examination of pups in any dose group.

Effect levels (P0)

1

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

2

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

3

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)

POSTNATAL LOSS DAYS 0 – 4 POST PARTUMNo test item-related effects on postnatal loss were noted at any dose level.In the control group one pup was missing on day 3, at the low-dose level one pup was missing on day 2, at the high-dose level two pups (from one litter) were missing on days 2 and 3.Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.0% and 0.3% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 99.1%, 99.1%, 100.0% and 97.7% in order of ascending dose levels. EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATIONNo test item-related findings were noted in pups during first litter check and during lactation in any dose group.PUP SEX RATIOSPup sex ratio was not affected by exposure to the test item at any dose level. At first litter check, percentages of male pups were 50%, 54%, 53% and 44% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUMBody weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.In order of ascending dose levels, mean body weights of pups were: 5.9 g, 6.0 g, 6.2 g and 6.0 g on day 1 post partum and 8.9 g, 9.1 g, 9.2 g and 8.7 g on day 4 post partum. Mean differences in body weights during lactation were +50.9%, +50.9%, +47.8% and +44.5%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.MACROSCOPICAL FINDINGS OF PUPSNo test item-related findings were noted at macroscopic examination of pups in any dose group.

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 (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (100)
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.