Heptanal, oligomeric reaction products with aniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Hepteen Base® in rats by oral gavage including a preliminary dose range finding study.

Parental NOAEL: Females: 50 mg/kg/day / Males: 150 mg/kg/day

Reproduction NOAEL: 150 mg/kg/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2016 to 06 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see "Any other infomration on materials and methods incl. tables" for details.

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

yes

Remarks:

see "Any other infomration on materials and methods incl. tables" for details.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Deviations:

not applicable

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

Internal identification number: 207368/A
Test item: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Laboratories Den Bosch BV)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)
Purity/composition correction factor: No correction factor required
Test item handling No specific handling conditions required
pH (1% in water, indicative range): 8.0 – 7.8 (determined by Charles River Laboratories Den Bosch BV)
Specific gravity/density: 0.9130
Stability in vehicle (1% aqueous carboxymethyl cellulose with 0.1% Tween-80): Stability for at least 24 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL, Project 512979.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.

Source F0 Charles River Deutschland, Sulzfeld, Germany.
Age at start pretest Females: approximately 11 weeks.
Age at start F0-treatment Males: approximately 11 weeks.
Females: approximately 13 weeks.
Number of F0-animals: 48 females and 40 males.
At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and continued in the study. The remaining females were removed from the study.
Acclimatization: F0 At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection: F0 At least upon receipt of the animals.
Randomization: F0 Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: F0 During pretest (females) and treatment (males and females): by earmark and tattoo.
Mating procedures Following a minimum of 14 days of dosing for the males and females, the animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug.
This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. All females were mated within 5 days.
Parturition: The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 328 pups.
Identification of pups: On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by indelible ink on the feet.
Culling: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups . Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Animal Husbandry
Room number: Room A0.09 (A0.23 for motor activity measurements).
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food.
Water: Free access to tap-water. During motor activity measurements, animals did not have access to water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures.

Route of administration:

oral: gavage

Vehicle:

other: 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, IJsselstein, The Netherlands; water: Elix, Millipore S.A.S., Molsheim, France) + 0.1% Tween-80 (Merck-Schuchardt, Hohenbrunn, Germany).

Details on exposure:

Vehicle: 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, IJsselstein, The Netherlands; water: Elix, Millipore S.A.S., Molsheim, France) + 0.1% Tween-80 (Merck-Schuchardt, Hohenbrunn, Germany).
Rationale: for vehicle Based on trial formulations performed at Charles River Laboratories Den Bosch BV.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Storage conditions: At room temperature.

Details on mating procedure:

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of
copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.

During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (12 August 2016) according to a validated method (Test Facility Study No. 512979).
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 512979).

Duration of treatment / exposure:

Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and during 13-15 days after delivery (up to and including the day before scheduled necropsy). Females which failed to deliver healthy offspring were dosed for 40 days.

Female nos. 42, 43, 44, 49 and 50 (Group 1), 60 (Group 2), and 63 (Group 3) were not dosed on one occasion, and female no. 66 (Group 3) was not dosed on two following days. The reason for this was that these females were littering at the time of dosing. The omission of 1-2 days of dosing over a period of several weeks was not considered to affect the toxicological
evaluation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a 10-day dose range finding study (Test Facility Study No. 512993;), the selected dose levels for the combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were 50, 150 and 500 mg/kg/day.

Positive control:

Not required

Parental animals: Observations and examinations:

Mortality / Viability
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

Clinical signs
At least once daily from start of treatment onwards up to the day prior to necropsy detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena (collected under Test Facility Study No. 512995 for logistic reasons and reported under Test Facility Study No. 512992). These clinical observations were conducted at least 1 hour (± 30 min) after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 =normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).

Total movements and ambulations are reported.
Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) at least 1 hour (± 30 min) after dosing.

Body weights
Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. All Group 4 animals were additionally weighed on Day 6 in order to monitor the health status of the animals.

Food consumption
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.. Cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Clinical Laboratory Investigations (F0-Generation only)
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 F0 animals/sex/group (see Allocation) under inhalation anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

Blood Sampling for Thyroid Hormone Analysis
F0-generation, males and females:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire).

Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30
a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was kept for possible measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.

In general:
Total T4 was measured in serum using the Immulite 1000® (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands). TSH was measured in serum, if required.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Not specified

Litter observations:

Each litter was examined to determine the following, if practically possible:
Mortality / Viability
The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.

Clinical signs
At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check were presented in the respective tables.

Body weights
Live pups were weighed on PND 1, 4, 7 and 13.

Sex
Sex was determined for all pups on PND 1 and 4.

Anogenital distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/nipple retention
On PND 13, all males in each litter were examined for the number of areola/nipples.

Blood Sampling for Thyroid Hormone Analysis
F1-generation, PND 4 pups:
From 2 surplus pups per litter at culling (if possible). Blood samples from the 2 pups per litter were collected into one serum tube. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. Blood samples were collected by decapitation, between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.

F1-generation, PND 13-15 pups:
From 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 μL serum for measurement of thyroxine (T4) and the remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).

Postmortem examinations (parental animals):

F0-generation - Termination
The animals were deprived of food overnight (with a maximum of 24 hours) before blood
sampling, but water was available.
All animals surviving to the end of the observation period and all moribund animals were
deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and
subsequently exsanguinated.

Necropsy was conducted on the following days:
Condition Day of necropsy
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Females: which delivered PND 14-16.
Females: which failed to deliver (nos. 62 and 67)
Post-coitum: Day 25 (females with evidence of mating).
Euthanized in extremis (all Group 4
animals and Group 3 female no. 70) : When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites were recorded for all paired females. As for female no. 67 (Group 3) no macroscopically visible implantation sites were present, the nongravid uterus was stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)). Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Selected 5 animals/sex/group (see Allocation) and all animals that were euthanized in extremis:
Identification marks: not processed; (Nasopharynx) Adrenal glands (Esophagus); (Aorta) Ovaries; Brain - cerebellum, mid-brain, cortex (7-levels); (Pancreas);
Caecum Peyer's patches [jejunum, ileum] if detectable; Cervix; Pituitary gland; Clitoral gland; Preputial gland; Colon; Prostate gland;Coagulation gland; Rectum; (Cowper’s gland); (Salivary glands - mandibular, sublingual); Duodenum Sciatic nerve; Epididymides; Seminal vesicles; Eyes (with optic nerve (if detectable) and Harderian gland); Skeletal muscle; (Skin); Mammary gland area (males and females); Spinal cord -cervical, midthoracic, lumbar; Femur including joint; Spleen; (Glans penis) Sternum with bone marrow; (Levator ani plus bulbocavernosus muscle complex (LABC)); Stomach; Testes; Heart; Thymus; Ileum Thyroid including parathyroid if detectable; Jejunum; (Tongue); Kidneys; Trachea; (Lacrimal gland, exorbital); Urinary; bladder; (Larynx); Uterus; Liver; Vagina; Lung, infused with formalin; All gross lesions; Lymph nodes - mandibular, mesenteric.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

All remaining animals, males that failed to sire and females which failed to deliver:
Cervix Preputial gland; Clitoral gland Prostate gland; Coagulation gland Seminal vesicles; Cowper’s glands Testes; Epididymides; Thyroid including parathyroid if detectable; Glans penis Uterus; Levator ani plus bulbocavernosus muscle complex; (LABC); Vagina; All gross lesions; Mammary gland area (males and females) Identification marks: not processed; Ovaries

F0-generation – Organ Weights
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group :
Adrenal glands; Brain; Cowper’s glands; Epididymides; Glans penis; Heart; Kidneys; Levator ani plus bulbocavernosus muscle complex (LABC); Liver; Ovaries; Prostate; Seminal vesicles including coagulating glands; Spleen; Testes; Thymus; Thyroid (including parathyroid if detectable); Uterus (including cervix)
All remaining animals:
Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Testes; Thyroid;
Absolute organ weights and organ to body weight ratios are reported.

Postmortem examinations (offspring):

F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

F0-generation – Histopathology
The following slides were examined by a pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 3.
• Additional slides of the testes of the selected 5 males of Groups 1 and 3 and all males that failed to sire to examine staging of spermatogenesis.
• The preserved organs and tissues of all animals that were euthanized in extremis.
• Thyroid glands and spleen of the selected 5 males of Group 2, based on (possible) treatment-related changes in these organs in males of Group 3.
• All gross lesions of all animals (all dose groups).
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

F1-generation Pups - Pathology
F1-generation Pups - Termination
Pups, younger than 7 days were killed by decapitation.
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.

On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.

Necropsy was conducted on the following days:
Condition Day of necropsy
Culling PND 4.
Terminal sacrifice PND 13-15.

Spontaneous deaths As soon as possible after death and always within 24 hours.

F1-generation pups - Macroscopic Examination
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
At terminal sacrifice (PND 13-15), the thyroid from 1 male and 1 female pup per litter were preserved in 10% buffered formalin. If possible, these were the same pups as selected for blood sampling. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test (Ref. 4) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%)
(Number of females mated/ Number of females paired) x 100

Fertility index (%)
(Number of pregnant females/ Number of females paired) x 100

Conception index (%)
(Number of pregnant females/ Number of females mated) x 100

Gestation index (%)
(Number of females bearing live pups/ Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%)
(Total number of offspring born/ Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% when the number of offspring exceeds the number of implantation sites recorded.

Live birth index (%)
(Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%)
(Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%)
(Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100

Viability index (%)
(Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100

Lactation index (%)
(Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clear signs of toxicity were observed in 4 out of 10 females at 150 mg/kg/day (nos. 61, 65, 69, 70), including the female that was euthanized preterm. Slight to moderate lethargy, piloerection, hunched posture, flat gait, pale appearance and/or ptosis were noted on one or more days towards the end of the post-coitum phase (around expected delivery date) and/or during lactation.

Also one female at 50 mg/kg/day and one male at 150 mg/kg/day were noted with piloerection on 1-2 days towards the end of the treatment period. However, as piloerection was observed for one control female as well, and since for the treated animals this was an isolated finding that occurred in the absence of any other sign of toxicity, no toxicological significance was attached to this observation.

Salivation seen after dosing among animals of the 50 mg/kg/day (one male only) and 150 mg/kg dose group (6 males and 7 females) from the third week of treatment onwards was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test item.

Alopecia was noted for one female at 150 mg/kg/day. It occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, it was not considered to be a test-item related.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals of the high dose group (500 mg/kg/day) had to be euthanized after 6-7 days of
treatment due to unacceptable high toxicity, and one female at the mid dose level (150 mg/kg/day) was euthanized on Day 21 post-coitum.

500 mg/kg/day
Treatment was poorly tolerated by both males and females. After 4-5 days, animals started to show severe signs of toxicity. These consisted of hunched posture, piloerection, diarrhoea, ptosis, chromodacryorrhoea (eye or snout) and/or a lean appearance. At the end of the first week of treatment, significant body weight loss was recorded, reaching up to 21% at the individual level. In addition, food consumption was approximately 50% lower for males and females during Days 1-8 as compared to their concurrent controls. Due to this severe toxicity, one high dose male was euthanized for humane reasons on Day 7 of the premating period, followed by the remaining animals on the day thereafter. At necropsy in the stomach an irregular surface, nodules and/or focus/foci (microscopic correlate: ulcer) and thickening of the limiting ridge (microscopic correlate: squamous hyperplasia) were recorded. In the intestinal tract (yellowish)14 gelatinous or watery cloudy contents and/or distension with gas were recorded (no microscopic correlate). The microscopic findings in the forestomach
consisting of ulceration was considered to be the main cause of moribundity for the majority of these animals. Related to their poor condition 3/10 males and 3/10 females were noted with a thymus reduced in size (microscopic correlate lymphoid depletion and/or increased lymphocytolysis), and a microscopic finding considered secondary to the inflammatory process in the forestomach was present in the bone marrow-sternum (increased granulopoiesis).

150 mg/kg/day
One female (no. 70) was euthanized on Day 21 post-coitum. Clinical signs indicative of a poor condition were noted from Day 20 post-coitum onwards and included lethargy (at a slight to moderate degree), hunched posture, an abnormal gait, ptosis, piloerection and/or a pale appearance. She lost 5% of her weight from Days 14-17 post-coitum, followed by almost no recovery, and her food intake decreased markedly from Days 14-20 post-coitum. Macroscopic findings at necropsy revealed gelatinous content in the ileum and caecum (with no microscopic correlate), and the spleen, thymus and mesenteric lymph nodes were reduced in size (microscopic correlates: lymphoid atrophy or depletion). Ulceration of the forestomach was considered to be the main cause of moribundity for this female. In each uterine horn there were seven viable foetuses; all without any external abnormalities. There were no unscheduled deaths in the 50 mg/kg/day and control group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly lower body weight gains and consequently lower body weights were noted in females at 150 mg/kg/day on Days 17 and 20 post-coitum. When compared to the concurrent control group mean body weight gain was decreased by approximately 25%. During lactation, body weight gain returned to normal values again, but body weights remained significantly lower.

At the individual level, up to 3-5% body weight loss was recorded for three females (nos. 65, 69 and 70) that also showed clinical signs of toxicity. Recovery was only seen for one of these females (no. 69). The relatively low body weight/body weight gain recorded for female no. 62 throughout the study was not related to treatment, but due to the fact that she had implantation sites only.

No treatment-related effects were noted for males at 50 and 150 mg/kg/day and females at 50 mg/kg/day. Body weights and body weight gains remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For females at 150 mg/kg/day, there was a trend towards slightly lower values for food consumption (before and after correction for body weight) during the entire treatment period. Changes as compared to the concurrent control group were relatively small, and did not always reach statistical significance.

At the individual level, two females showed clear treatment-related effects on food intake. The female that was sacrificed preterm (no. 70) had a markedly reduced food consumption during the last week of the post-coitum phase. In addition, another female (no. 69) had a relatively low food intake during Days 1-4 of lactation, which was followed by complete recovery thereafter. The same female was shown to be affected by treatment based on clinical signs and body weight loss early in lactation (see previous sections). The relatively low absolute food intake recorded for female no. 62 from the second week post-coitum onwards
was not related to treatment, but due to the fact that she had no live fetuses (implantation sites only).

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted for males at 50 and 150 mg/kg/day and females at 50 mg/kg/day. The slightly lower absolute and relative food intake recorded for females at 150 mg/kg/day during the first four days of lactation was not considered adverse as the difference with the concurrent control group was slight only (reaching no statistical significance) and values recovered to normal thereafter.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 150 mg/kg/day, statistically significantly changes in red blood cell parameters were observed as compared to the concurrent control group. This included a lower value for red blood cell (RBC; 8.32 10E12/L) count with correlating higher values for relative reticulocyte count (4.0 % RBC) and red blood cell distribution width (RDW; 14.0%). Mean values for red blood cell and reticulocyte count were at the higher end or above the historical control range.

Also males at 50 mg/kg/day had a higher value for reticulocyte count (3.2 % RBC). No changes in haematology parameters were noted in female rats following treatment up to 150 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were not considered not to be affected by treatment.

The statistically significantly lower calcium concentration recorded in males at 150 mg/kg/day when compared to controls, was considered to have arisen as a result of a slightly high control value (2.69 mmol/L), and therefore was not regarded as treatment related.

Thyroid hormone analyses:
Serum levels of T4 in F0-males were not affected by treatment up to 150 mg/kg/day.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Note: No data are available from the 500 mg/kg/day group which was euthanized preterm.

Hearing ability, pupillary reflex, static righting reflex and grip strength were considered to be normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval
that decreased over the duration of the test period.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A test item-related microscopic finding after treatment with Hepteen Base® was noted in the spleen of the 50 and 150 mg/kg/day group males.

Spleen, a slightly increased incidence and severity of extramedullary hematopoiesis was recorded in males at 50 and 150 mg/kg/day. The remainder of the recorded microscopic findings were within the range of background
pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.
All females had regular cycles of 4 days.
Note: For females of the 500 mg/kg/day that were sacrificed preterm only data from pretest and the first 8 days of the treatment period were available.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No reproduction data are available from the 500 mg/kg/day group, as these animals were sacrificed before start of mating.

There were 2/10 couples treated at 150 mg/kg/day, that failed to deliver healthy pups.
Histopathologic examination did not reveal any changes in the reproductive organs that could explain this reproductive failure.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.

Mating index
Mating was not affected by treatment at 50 and 150 mg/kg/day. All females showed evidence of mating.

Fertility and Conception index
Fertility and conception index were not considered to be affected by treatment up to 150 mg/kg/day.
One female in Group 3 (no. 67) was not pregnant. This was not considered to be test-item related as it was an isolated case and there were no morphological findings in the reproductive organs of this female or the corresponding male (no. 27) she was mated with that could explain this reproductive failure.

Precoital time
Precoital time was not considered to be affected by treatment.

Number of implantation sites
Number of implantation sites was not considered to be affected by treatment.
One female in Group 3 (no. 62) had 2 implantation sites only. Such low numbers are occasionally seen for rats. As all remaining pregnant females in this group had completely normal litter sizes and in the absence of any morphological findings in the reproductive organs of either this female or the male she was mated with (no. 32), a relation to treatment could be excluded.
All females showed evidence of mating.
For female nos. 41 (Group 1), 53, 57, 58 (Group 2) and 69 (Group 3), the number of pups were slightly higher than the number of implantations. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Days 14 or 16 of lactation.

Gestation index and duration
Gestation index and duration of gestation were not considered to be affected by treatment.
In the 150 mg/kg/day group, two out of the nine pregnant females failed to deliver healthy offspring. One female had implantation sites only (see section 7.4.1.4) and one female had to be euthanized due to complications during parturition (see section 7.2.1). As a consequence, the gestation index was markedly lower at 150 mg/kg/day as compared to the control and 50 mg/kg/day groups (78% versus 100%). This finding, however, was not considered to be
treatment related. Females with implantation sites only or parturition problems are occasionally seen in this type of studies. At the isolated incidence and in the absence of any morphological findings in the reproductive organs, it was regarded an incidental finding.

Two females in the 150 mg/kg/day group (nos. 63 and 66) had 23 days of gestation. This is slightly longer than seen for the remaining females in this study, but still within the normal range of biological variation. Therefore, it was not considered to be related to treatment.

Parturition/maternal care
One female at 150 mg/kg/day (no. 70) had to be euthanized before parturition on Day 21 post-coitum due to unacceptable high toxicity.

Another female in the 150 mg/kg/day group (no.69) delivered on Day 23 post-coitum, In her
litter with 11 pups, three pups were found cold at first litter check, one pup had little milk and one dead pup had no milk in its stomach (note: another dead pup could not be examined due to advanced autolysis). These pup observations suggestive for poor maternal care were considered to be secondary to the poor condition of the dam noted just before parturition and during the first days of lactation based on clinical signs, body weight loss and markedly reduced food consumption. In the subsequent period she recovered completely and all remaining pups survived until scheduled necropsy.

Female no. 66 (Group 3) was noted with signs of littering on two following days. No toxicological relevance was attached to this finding. Based on the available in-life data there were no indications for any delivery problems. Furthermore, she delivered a healthy litter of 13 pups.

No difficulties in parturition or deficiencies in maternal care were observed in the control and 50 mg/kg/day groups.

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Post-implantation survival index
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Not applicable

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be a direct effect of treatment.
One control pup was noted with scabbing on the right front leg. As the control group was treated with the vehicle alone, a possible test item related effect could be excluded.
The findings of less milk in the stomach (pup 3) and cold appearance (pups 7, 9, 11) in litter 69 were considered to be secondary to maternal toxicity.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
The significantly lower mean number of living pups at first litter check recorded for the 150 mg/kg/day group as compared to the concurrent control group (11.0 versus 13.1) was caused by the relatively large litter sizes of the control animals. All values remained within the available range of historical control data17 (mean ± standard deviation: 11.0 ± 2.7; P5 value: 6.0).

One pup of the control group (pup 13/litter 41), one pup at 50 mg/kg (pup 9/litter 59) and three pups at 150 mg/kg/day (pup/litter 8/64, 6/69 and 10/69) were found dead at first litter check. It should be noted that two out of the three dead pups in the 150 mg/kg/day were from the litter of dam no. 69 who had a poor condition around parturition
.
Therefore, the death of these two pups was not considered to be a direct effect of the treatment, but secondary to maternal toxicity. When excluding this litter, the mortality incidence seen in this study was within the range considered normal for pups of this age.

Viability index
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
One pup in the 50 mg/kg/day group (pup 7/litter 51) was found missing on lactation Day 3. This pup was most likely cannibalised. There were no findings noted for this pup during lactation Days 1 and 2 which could explain its death. No toxicological relevance was attributed to this finding since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg/day, a trend towards slightly lower body weights of pups was noted from lactation Day 4 onwards, reaching statistical significance on lactation Day 13.
Body weights of pups in the 50 mg/kg/day group remained in the same range as for pups in the control group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings noted among pups that were considered to be a direct test-item related effect on pup development.
One pup that was found dead at first litter check had no milk in its stomach. It should be noted that the dam of this litter (no. 69) was in a poor condition around parturition. Other incidental macroscopic findings of pups that were found dead included partial cannibalism and autolysis. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant. There were no macroscopic findings among surviving pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio
Sex ratio was not considered to be affected by treatment.

Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Areola/nipple retention
Treatment up to 150 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No developmental effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start mating.
One female at 150 mg/kg/day had to be euthanized before parturition on Day 21 post-coitum due to unacceptable high toxicity. As in this group there were two females that failed to produce live offspring (both not considered to be treatment-related), seven litters were available for evaluation of possible test-item related effects on pup development at 150 mg/kg/day. Although relatively low, the available results were considered to be sufficient for a proper evaluation.
One female in the 150 mg/kg/day group had in total 11 pups of which three pups were found cold at first litter check, one pup had little milk and one dead pup had no milk in its stomach (note: another dead pup could not be examined due to advanced autolysis). These pup observations suggestive for poor maternal care were considered to be secondary to the poor condition of the dam noted just before parturition and during the first days of lactation based on clinical signs, body weight loss and markedly reduced food consumption. In the subsequent period she recovered completely and all remaining pups survived until scheduled
necropsy.

At 150 mg/kg/day, a trend towards slightly lower body weights of pups was noted from lactation Day 4 onwards, reaching statistical significance on lactation Day 13. No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration of gestation, postimplantation survival index, live birth index, viability index, lactation index, clinical signs, sex ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels or macroscopy) by treatment at 150 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

no

F0-GENERATION - POSTCOITUM

 

DAY OF THE PAIRING PERIOD	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG
NUMBER OF FEMALES MATED
1	1	3	3
2	1	-	-
3	4	3	4
4	3	4	3
5	1	-	-
MEDIAN PRECOITAL TIME	3	3	3
MEAN PRECOITAL TIME	3.2	2.8	2.7
N	10	10	10

 

Developmental data F0-GENERATION –LACTATION

 

	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG
LITTERS TOTAL	10	10	7
DURATION OF GESTATION
MEAN (+)	21.4	21.4	21.9
ST.DEV.	0.5	0.5	0.9
N	10	10	7
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	1	1	2
TOTAL	1	1	3
MEAN (+)	0.1	0.1	0.4
ST.DEV.	0.3	0.3	0.8
N	10	10	7
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	44 / 56	45 / 55	52 / 48
TOTAL	131	120	77
MEAN (+)	13.1	12.0	11.0 ++
ST.DEV.	1.7	2.1	1.4
N	10	10	7
POSTNATAL LOSS
% OF LIVING PUPS	0.0	0.8	0.0
LITTERS AFFECTED (#)	0	1	0
TOTAL (#)	0	1	0
MEAN (+)	0.0	0.1	0.0
ST.DEV.	0.0	0.3	0.0
N	10	10	7
CULLED PUPS
TOTAL	51	39	21
LIVING PUPS DAY 4 P.P.
TOTAL	80	80	56
MEAN (+)	8.0	8.0	8.0
ST.DEV.	0.0	0.0	0.0
N	10	10	7
BREEDING LOSS DAYS 5 - 13 P.P. % OF LIVING PUPS AT DAY 4 P.P.	0.0	0.0	1.8
LITTERS AFFECTED (#)	0	0	1
TOTAL (#)	0	0	1
MEAN (+)	0.0	0.0	0.1
ST.DEV.	0.0	0.0	0.4
N	10	10	7
LIVING PUPS DAY 13 P.P.
% OF MALES / FEMALES (#)	48 / 52	49 / 51	51 / 49
TOTAL	79	80	55
MEAN (+)	7.9	8.0	7.9
ST.DEV.	0.3	0.0	0.4
N	10	10	7
VIABILITY INDEX (#)	100.0	99.2	100.0
WEANING INDEX (#)	98.8	100.0	98.2

Viability index = (Number of alive pups on day 4 p.p. / Number of pups born alive) *100

Weaning index =(Number of alive pups on day 13 p.p. / Number of alive pups on day 4 p.p.) *100

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Body weights of pups F0-GENERATION -LACTATION

 

DAY	SEX		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG
1	M	MEAN	6.5	6.6	6.4
		ST.DEV.	0.4	0.5	0.8
		N	10	10	7
	F	MEAN	6.2	6.1	6.0
		ST.DEV.	0.5	0.3	0.6
		N	10	10	7
	M+F	MEAN	6.3	6.3	6.2
		ST.DEV.	0.5	0.4	0.7
		N	10	10	7
4	M	MEAN	9.5	9.6	9.1
		ST.DEV.	0.8	0.8	1.6
		N	10	10	7
	F	MEAN	9.1	9.0	8.5
		ST.DEV.	0.9	0.6	1.3
		N	10	10	7
	M+F	MEAN	9.3	9.3	8.7
		ST.DEV.	0.8	0.7	1.4
		N	10	10	7
7	M	MEAN	16.1	15.8	14.9
		ST.DEV.	1.1	1.1	2.2
		N	10	10	7
	F	MEAN	15.7	15.1	14.1
		ST.DEV.	1.6	1.0	1.6
		N	10	10	7
	M+F	MEAN	15.9	15.4	14.5
		ST.DEV.	1.3	1.1	1.9
		N	10	10	7
13	M	MEAN	30.7	30.4	27.4 *
		ST.DEV.	1.9	1.7	3.0
		N	9	9	5
	F	MEAN	29.6	29.0	26.7 *
		ST.DEV.	2.3	1.7	2.3
		N	9	9	5
	M+F	MEAN	30.1	29.6	27.1 *
		ST.DEV.	2.0	1.6	2.6
		N	9	9	5

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

DEVELOPMENTAL DATA PER LITTER FEMALES

F0-GENERATION –LACTATION

 

		First litter check					P.natal loss			Living pups
LITTER	DURATION OF GESTATION	DEAD M	PUPS F	LIVING M	PUPS F	TOT.	DAYS M	0 - 4 F	CULLED PUPS TOT.	DAY 4 M	P.P. F	TOT.
GROUP 1 (CONTROL)
41	21	0	1	5	7	12	0	0	4	4	4	8
42	22	0	0	4	6	10	0	0	2	4	4	8
43	21	0	0	5	9	14	0	0	6	4	4	8
44	22	0	0	3	11	14	0	0	6	3	5	8
45	21	0	0	4	9	13	0	0	5	4	4	8
46	21	0	0	8	7	15	0	0	7	4	4	8
47	21	0	0	7	9	16	0	0	8	4	4	8
48	22	0	0	7	6	13	0	0	5	4	4	8
49	21	0	0	8	4	12	0	0	4	4	4	8
50	22	0	0	7	5	12	0	0	4	4	4	8
TOTAL		0	1	58	73	131	0	0	51	39	41	80
N	10	10	10	10	10	10	10	10	10	10	10	10
MEAN	21.4	0.0	0.1	5.8	7.3	13.1	0.0	0.0	5.1	3.9	4.1	8.0
ST.DEV.	0.5	0.0	0.3	1.8	2.2	1.7	0.0	0.0	1.7	0.3	0.3	0.0
GROUP 2 (50 MG/KG)
51	21	0	0	4	8	12	1	0	3	3	5	8
52	21	0	0	6	6	12	0	0	4	4	4	8
53	22	0	0	4	11	15	0	0	7	4	4	8
54	21	0	0	4	4	8	0	0	0	4	4	8
55	22	0	0	4	8	12	0	0	4	4	4	8
56	21	0	0	6	8	14	0	0	6	4	4	8
57	22	0	0	5	5	10	0	0	2	4	4	8
58	21	0	0	9	5	14	0	0	6	4	4	8
59	21	0	1	4	7	11	0	0	3	4	4	8
60	22	0	0	8	4	12	0	0	4	4	4	8
TOTAL		0	1	54	66	120	1	0	39	39	41	80
N	10	10	10	10	10	10	10	10	10	10	10	10
MEAN	21.4	0.0	0.1	5.4	6.6	12.0	0.1	0.0	3.9	3.9	4.1	8.0
ST.DEV.	0.5	0.0	0.3	1.8	2.2	2.1	0.3	0.0	2.1	0.3	0.3	0.0
GROUP 3 (150 MG/KG)
61	21	0	0	5	7	12	0	0	4	4	4	8
63	23	0	0	4	7	11	0	0	3	4	4	8
64	21	0	1	6	4	10	0	0	2	4	4	8
65	22	0	0	4	6	10	0	0	2	4	4	8
66	23	0	0	8	5	13	0	0	5	4	4	8
68	21	0	0	8	4	12	0	0	4	4	4	8
69	22	1	1	5	4	9	0	0	1	4	4	8
TOTAL		1	2	40	37	77	0	0	21	28	28	56
N	7	7	7	7	7	7	7	7	7	7	7	7
MEAN	21.9	0.1	0.3	5.7	5.3	11.0	0.0	0.0	3.0	4.0	4.0	8.0
ST.DEV.	0.9	0.4	0.5	1.7	1.4	1.4	0.0	0.0	1.4	0.0	0.0	0.0

 

 

DEVELOPMENTAL DATA PER LITTER FEMALES F0-GENERATION –LACTATION

 

GROUP 1 (CONTROL)
	Breeding loss		Living pups
Litter	Days 5-13		Day 13 - PP
	M	F	M	F	Total
41	0	0	4	4	8
42	0	0	4	4	8
43	0	0	4	4	8
44	0	0	3	5	8
45	0	0	4	4	8
46	0	0	4	4	8
47	0	0	4	4	8
48	0	0	4	4	8
49	0	0	3	4	7
50	0	0	4	4	8
TOTAL	0	0	38	41	79
N	10	10	10	10	10
MEAN	0.0	0.0	3.8	4.1	7.9
ST.DEV.	0.0	0.0	0.4	0.3	0.3
GROUP 2 (50 MG/KG)
	Breeding loss		Living pups
Litter	Days 5-13		Day 13 - PP
	M	F	M	F	Total
51	0	0	3	5	8
52	0	0	4	4	8
53	0	0	4	4	8
54	0	0	4	4	8
55	0	0	4	4	8
56	0	0	4	4	8
57	0	0	4	4	8
58	0	0	4	4	8
59	0	0	4	4	8
60	0	0	4	4	8
TOTAL	0	0	39	41	80
N	10	10	10	10	10
MEAN	0.0	0.0	3.9	4.1	8.0
ST.DEV.	0.0	0.0	0.3	0.3	0.0
GROUP 3 (150 MG/KG)
	Breeding loss		Living pups
Litter	Days 5-13		Day 13 - PP
	M	F	M	F	Total
61	0	0	4	4	8
63	0	0	4	4	8
64	0	0	4	4	8
65	0	1	4	3	7
66	0	0	4	4	8
68	0	0	4	4	8
69	0	0	4	4	8
TOTAL	0	1	28	27	55
N	7	7	7	7	7
MEAN	0.0	0.1	4.0	3.9	7.9
ST.DEV.	0.0	0.4	0.0	0.4	0.4

 

Conclusions:

Hepteen Base® was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day. Due to unacceptable high toxicity, animals in the high dose group had to be euthanized after the first week of treatment. In the remaining groups, males were dosed for 2 weeks prior to mating, during mating, and up to the day prior to termination (for 29 days). The females of the remaining groups that delivered were dosed for 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation (for 50-56 days; up to the day prior to necropsy). Females that failed to deliver healthy offspring were exposed for 40 days.

Formulation analysis demonstrated that the formulations were prepared accurately and homogenously.

Parental results
All animals in the 500 mg/kg/day group and one female in the 150 mg/kg/day group had to be euthanized preterm.

Treatment at 500 mg/kg/day was poorly tolerated by the animals (both sexes). After 4-5 days, animals started to show severe signs of toxicity. These consisted of hunched posture, piloerection, diarrhoea, ptosis, chromodacryorrhoea (eye or snout) and/or a lean appearance. At the end of the first week of treatment, significant body weight loss was recorded, reaching up to 21% at the individual level. In addition, food consumption was approximately 50% lower for males and females during Days 1-8 as compared to their concurrent controls. Due to this severe toxicity, one high dose male was euthanized for humane reasons on Day 7 of the premating period, followed by the remaining animals on the day thereafter. At necropsy in the stomach an irregular surface, nodules and/or focus/foci (microscopic correlate: ulcer) and thickening of the limiting ridge (microscopic correlate: squamous hyperplasia) were recorded. In the intestinal tract (yellowish)18 gelatinous or watery cloudy contents and/or distension with gas were recorded (no microscopic correlate). The microscopic findings in the forestomach consisting of ulceration was considered to be the main cause of moribundity
for the majority of these animals. Related to their poor condition 3/10 males and 3/10 females were noted with a thymus reduced in size (microscopic correlate lymphoid depletion and/or increased lymphocytolysis), and a microscopic finding considered secondary to the inflammatory process in the forestomach was present in the bone marrow-sternum (increased granulopoiesis).

In addition, one female at 150 mg/kg/day was euthanized on Day 21 post-coitum after 37 days of treatment. Clinical signs indicative of a poor condition were noted from Day 20 post-coitum onwards and included lethargy (at a slight to moderate degree), hunched posture, a flat gait, ptosis, piloerection and/or a pale appearance. She lost 5% of her weight from Days 14-17 post-coitum, followed by almost no recovery, and her food intake decreased markedly during Days 14-20 post-coitum. Macroscopic findings at necropsy revealed gelatinous content in the ileum and caecum (no microscopic correlate), and the spleen, thymus and mesenteric lymph nodes were reduced in size (microscopic correlates: lymphoid atrophy or depletion). Ulceration of the forestomach was considered to be the main cause of moribundity for this female. In each uterine horn there were seven viable fetuses; all without any external abnormalities.

Clear signs of toxicity were observed in 4 out of 10 females at 150 mg/kg/day, including the female that was euthanized preterm (see above). Slight lethargy (at a slight to moderate degree), piloerection, hunched posture, flat gait, pale appearance and/or ptosis were noted on one or more days towards the end of the post-coitum phase (around expected delivery date) and/or during lactation.

Significantly lower body weight gains and consequently lower body weights were noted in females at 150 mg/kg/day on Days 17 and 20 post-coitum. When compared to the concurrent control group mean body weight gain was decreased by approximately 25%. During lactation, body weight gain returned to normal values again, but body weights remained significantly lower.

In addition, for females at the mid dose there was a trend towards slightly lower values for food consumption (before and after correction for body weight) during the entire treatment period. Changes in the 150 mg/kg/day group as compared to the concurrent control group were relatively small, and did not always reach statistical significance.

In males at 150 mg/kg/day, statistically significantly changes in red blood cell parameters were observed as compared to the concurrent control group. This included a lower value for red blood cell count with correlating higher values for relative reticulocyte count and red blood cell distribution width. Mean values for red blood cell and reticulocyte count were at the higher end or above the historical control range. Also males at 50 mg/kg/day had a higher value for relative reticulocyte count. At the microscopic level, a slightly increased incidence and severity of extramedullary hematopoiesis (up to slight degree) was recorded in male rats at 50 and 150 mg/kg/day. Extramedullary hematopoiesis in the spleen can be seen as a spontaneous finding and the slight increase in incidence and severity is considered to be nonadverse.

No comparable treatment-related effects were noted in females at 50 and 150 mg/kg/day.

At 150 mg/kg/day, no treatment-related effects were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, clinical biochemistry, thyroid hormone analysis, macroscopic examination, and organ weights).

No parental toxicity was observed at 50 mg/kg/day.

Reproductive results
No reproductive effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start of mating.

No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day.
All reproductive parameters investigated in this study (i.e. mating, fertility and conception index, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of the reproductive organs) were considered to be unaffected by treatment.

Developmental results
No developmental effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start mating.

One female at 150 mg/kg/day had to be euthanized before parturition on Day 21 post-coitum due to unacceptable high toxicity. As in this group there were two females that failed to produce live offspring (both not considered to be treatment-related), seven litters were available for evaluation of possible test-item related effects on pup development at 150 mg/kg/day. Although relatively low, the available results were considered to be sufficient for a proper evaluation.

One female in the 150 mg/kg/day group had in total 11 pups of which three pups were found cold at first litter check, one pup had little milk and one dead pup had no milk in its stomach (note: another dead pup could not be examined due to advanced autolysis). These pup observations suggestive for poor maternal care were considered to be secondary to the poor condition of the dam noted just before parturition and during the first days of lactation based on clinical signs, body weight loss and markedly reduced food consumption. In the subsequent period she recovered completely and all remaining pups survived until scheduled necropsy.

At 150 mg/kg/day, a trend towards slightly lower body weights of pups was noted from lactation Day 4 onwards, reaching statistical significance on lactation Day 13.

No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration of gestation, postimplantation survival index, live birth index, viability index, lactation index, clinical signs, sex ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels or
macroscopy) by treatment at 150 mg/kg/day.

No developmental toxicity was observed in the 50 mg/kg/day group.

In conclusion, treatment with Hepteen Base® by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day revealed severe parental toxicity at 500 mg/kg/day. All high dose animals had to be euthanized after 6-7 days of treatment. No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day. Developmental toxicity was indicated by lower fetal pup weights and a single litter with a slightly higher incidence of pup mortality and clinical signs at 150 mg/kg/day. This was considered to be secondary to maternal toxicity.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: Females: 50 mg/kg/day
Males: 150 mg/kg/day
Reproduction NOAEL: 150 mg/kg/day
Developmental NOAEL: 50 mg/kg/day

Note: The entire 500 mg/kg/day group (males and females) had to be euthanized during the premating period due to unacceptable high toxicity. Therefore no reproductive and developmental effects could be evaluated at this dose.

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicityscreening test of Hepteen Base® in rats by oral gavage including a preliminary dose rangefinding study.

Guidelines

The study was based on the following guidelines:

 

•OECD 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016

•OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

•OECD 421: Reproduction/Developmental Toxicity Screening Test, July 2016

•OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000.

•EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

•OECD 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

•OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents, July 2000.

 

Rationale for dose levels

Based on the results of a 10-day dose range finding study (Test Facility Study No. 512993; the selected dose levels for the combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were 50, 150 and 500 mg/kg/day.

 

Study outline

The test item, Hepteen Base®, formulated in 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, was administered by daily oral gavage to SPF-bred Wistar Han rats. One control group (treated with the vehicle alone) and three treated groups (50, 150 and 500 mg/kg/day) were tested, each consisting of 10 males and 10 females.

 

Due to unexpected high toxicity, animals of the highest dose group were euthanized for humane reasons after only 6-7 days of treatment on Days 7 and 8 of the premating period. Males of the remaining groups were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to termination. Females of the remaining groups were dosed for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation (up to the day prior to necropsy). Females which failed to deliver healthy offspring were treated for 40 days.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conceptionindices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

 

Formulations were analyzed once during the study to assess accuracy and homogeneity.

 

Results/discussion

Accuracy and homogeneity of formulations were demonstrated by analyses.

 

Parental results

All animals in the 500 mg/kg/day group and one female in the 150 mg/kg/day group had to be euthanized preterm.

 

Treatment at 500 mg/kg/day was poorly tolerated by the animals (both sexes). After 4-5 days, animals started to show severe signs of toxicity. These consisted of hunched posture, piloerection, diarrhoea, ptosis, chromodacryorrhoea (eye or snout) and/or a lean appearance. At the end of the first week of treatment, significant body weight loss was recorded, reaching up to 21% at the individual level. In addition, food consumption was approximately 50% lower for males and females during Days 1-8 as compared to their concurrent controls. Due to this severe toxicity, one high dose male was euthanized for humane reasons on Day 7 of the premating period, followed by the remaining animals on the day thereafter. At necropsy in the stomach an irregular surface, nodules and/or focus/foci (microscopic correlate: ulcer) and thickening of the limiting ridge (microscopic correlate: squamous hyperplasia) were

recorded. In the intestinal tract (yellowish) gelatinous or watery cloudy contents and/or distension with gas were recorded (no microscopic correlate). The microscopic findings in the forestomach consisting of ulceration was considered to be the main cause of moribundity for the majority of these animals. Related to their poor condition 3/10 males and 3/10 females were noted with a thymus reduced in size (microscopic correlate lymphoid depletion and/or increased lymphocytolysis), and a microscopic finding considered secondary to the inflammatory process in the forestomach was present in the bone marrow-sternum (increased granulopoiesis).

 

In addition, one female at 150 mg/kg/day was euthanized on Day 21 post-coitum after 37 days of treatment. Clinical signs indicative of a poor condition were noted from Day 20 post-coitum onwards and included lethargy (at a slight to moderate degree), hunched posture, a flat gait, ptosis, piloerection and/or a pale appearance. She lost 5% of her weight from Days 14-17 post-coitum, followed by almost no recovery, and her food intake decreased markedly during Days 14-20 post-coitum. Macroscopic findings at necropsy revealed gelatinous content in the ileum and caecum (no microscopic correlate), and the spleen, thymus and mesenteric lymph nodes were reduced in size (microscopic correlates: lymphoid atrophy or depletion). Ulceration of the forestomach was considered to be the main cause of moribundity for this female. In each uterine horn there were seven viable fetuses; all without any external abnormalities.

 

Clear signs of toxicity were observed in 4 out of 10 females at 150 mg/kg/day, including the female that was euthanized preterm (see above). Slight lethargy (at a slight to moderate degree), piloerection, hunched posture, flat gait, pale appearance and/or ptosis were noted on one or more days towards the end of the post-coitum phase (around expected delivery date) and/or during lactation.

Significantly lower body weight gains and consequently lower body weights were noted in females at 150 mg/kg/day on Days 17 and 20 post-coitum. When compared to the concurrent control group mean body weight gain was decreased by approximately 25%. During lactation, body weight gain returned to normal values again, but body weights remained significantly lower.

 

In addition, for females at the mid dose there was a trend towards slightly lower values for food consumption (before and after correction for body weight) during the entire treatment period. Changes in the 150 mg/kg/day group as compared to the concurrent control group were relatively small, and did not always reach statistical significance. In males at 150 mg/kg/day, statistically significantly changes in red blood cell parameters were observed as compared to the concurrent control group. This included a lower value for

red blood cell count with correlating higher values for relative reticulocyte count and red blood cell distribution width. Mean values for red blood cell and reticulocyte count were at the higher end or above the historical control range. Also males at 50 mg/kg/day had a higher value for relative reticulocyte count. At the microscopic level, a slightly increased incidence and severity of extramedullary hematopoiesis (up to slight degree) was recorded in male rats at 50 and 150 mg/kg/day. Extramedullary hematopoiesis in the spleen can be seen as a spontaneous finding and the slight increase in incidence and severity is considered to be nonadverse.

No comparable treatment-related effects were noted in females at 50 and150 mg/kg/day.

 

At 150 mg/kg/day, no treatment-related effects were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, clinical biochemistry, thyroid hormone analysis, macroscopic examination, and organ weights).

 

No parental toxicity was observed at 50 mg/kg/day.

 

Reproductive results

No reproductive effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start of mating.

 

No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day.

 

All reproductive parameters investigated in this study (i.e. mating, fertility and conception index, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of the reproductive organs) were considered to be

unaffected by treatment.

 

Developmental results

No developmental effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start mating.

 

One female at 150 mg/kg/day had to be euthanized before parturition on Day 21 post-coitum due to unacceptable high toxicity. As in this group there were two females that failed to produce live offspring (both not considered to be treatment-related), seven litters were available for evaluation of possible test-item related effects on pup development at 150 mg/kg/day. Although relatively low, the available results were considered to be sufficient for a proper evaluation.

 

One female in the 150 mg/kg/day group had in total 11 pups of which three pups were found cold at first litter check, one pup had little milk and one dead pup had no milk in its stomach (note: another dead pup could not be examined due to advanced autolysis). These pup observations suggestive for poor maternal care were considered to be secondary to the poor condition of the dam noted just before parturition and during the first days of lactation based on clinical signs, body weight loss and markedly reduced food consumption. In the subsequent period she recovered completely and all remaining pups survived until scheduled necropsy.

 

At 150 mg/kg/day, a trend towards slightly lower body weights of pups was noted from lactation Day 4 onwards, reaching statistical significance on lactation Day 13.

 

No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration of gestation, postimplantation survival index, live birth index, viability index, lactation index, clinical signs, sex ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels or macroscopy) by treatment at 150 mg/kg/day.

 

No developmental toxicity was observed in the 50 mg/kg/day group.

 

In conclusion, treatment with Hepteen Base® by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day revealed severe parental toxicity at 500 mg/kg/day. All high dose animals had to be euthanized after 6-7 days of treatment. No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day. Developmental toxicity was indicated by lower fetal pup weights and a single litter with a slightly higher incidence of pup mortality and clinical signs at 150 mg/kg/day. This was considered to be secondary to maternal toxicity.

 

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

 

Parental NOAEL: Females: 50 mg/kg/day

Males: 150 mg/kg/day

Reproduction NOAEL: 150 mg/kg/day

Developmental NOAEL: 50 mg/kg/day

 

Note: The entire 500 mg/kg/day group (males and females) had to be euthanized during the premating period due to unacceptable high toxicity. Therefore no reproductive and developmental effects could be evaluated at this dose.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1 - The study was conducted in a GLP accredited laboratory based on the following guidelines: OECD test guidelines 421, 422, 407 OPPTS test guidelines 870.3650, 870.3550, 870.3050

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Hepteen Base® in rats by oral gavage including a preliminary dose range finding study.

Rationale for dose levels

Based on the results of a 10-day dose range finding study (Test Facility Study No. 512993); the selected dose levels for the combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were 50, 150 and 500 mg/kg/day.

Study outline

The test item, Hepteen Base®, formulated in 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, was administered by daily oral gavage to SPF-bred Wistar Han rats. One control group (treated with the vehicle alone) and three treated groups (50, 150 and 500 mg/kg/day) were tested, each consisting of 10 males and 10 females.

Due to unexpected high toxicity, animals of the highest dose group were euthanized for humane reasons after only 6-7 days of treatment on Days 7 and 8 of the premating period. Males of the remaining groups were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to termination. Females of the remaining groups were dosed for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation (up to the day prior to necropsy). Females which failed to deliver healthy offspring were treated for 40 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity.

Accuracy and homogeneity of formulations were demonstrated by analyses.

Parental results

All animals in the 500 mg/kg/day group and one female in the 150 mg/kg/day group had to be euthanized preterm.

Treatment at 500 mg/kg/day was poorly tolerated by the animals (both sexes). After 4-5 days, animals started to show severe signs of toxicity. These consisted of hunched posture, piloerection, diarrhoea, ptosis, chromodacryorrhoea (eye or snout) and/or a lean appearance. At the end of the first week of treatment, significant body weight loss was recorded, reaching up to 21% at the individual level. In addition, food consumption was approximately 50% lower for males and females during Days 1-8 as compared to their concurrent controls. Due to this severe toxicity, one high dose male was euthanized for humane reasons on Day 7 of the premating period, followed by the remaining animals on the day thereafter. At necropsy in the stomach an irregular surface, nodules and/or focus/foci (microscopic correlate: ulcer) and thickening of the limiting ridge (microscopic correlate: squamous hyperplasia) were recorded. In the intestinal tract (yellowish)1 gelatinous or watery cloudy contents and/or distension with gas were recorded (no microscopic correlate). The microscopic findings in the forestomach consisting of ulceration was considered to be the main cause of moribundity for the majority of these animals. Related to their poor condition 3/10 males and 3/10 females were noted with a thymus reduced in size (microscopic correlate lymphoid depletion and/or increased lymphocytolysis), and a microscopic finding considered secondary to the inflammatory process in the forestomach was present in the bone marrow-sternum (increased granulopoiesis).

In addition, one female at 150 mg/kg/day was euthanized on Day 21 post-coitum after 37 days of treatment. Clinical signs indicative of a poor condition were noted from Day 20 post-coitum onwards and included lethargy (at a slight to moderate degree), hunched posture, a flat gait, ptosis, piloerection and/or a pale appearance. She lost 5% of her weight from Days 14-17 post-coitum, followed by almost no recovery, and her food intake decreased markedly during Days 14-20 post-coitum. Macroscopic findings at necropsy revealed gelatinous content in the ileum and caecum (no microscopic correlate), and the spleen, thymus and mesenteric lymph nodes were reduced in size (microscopic correlates: lymphoid atrophy or depletion). Ulceration of the forestomach was considered to be the main cause of moribundity for this female. In each uterine horn there were seven viable fetuses; all without any external abnormalities.

Clear signs of toxicity were observed in 4 out of 10 females at 150 mg/kg/day, including the female that was euthanized preterm (see above). Slight lethargy (at a slight to moderate degree), piloerection, hunched posture, flat gait, pale appearance and/or ptosis were noted on one or more days towards the end of the post-coitum phase (around expected delivery date) and/or during lactation. Significantly lower body weight gains and consequently lower body weights were noted in females at 150 mg/kg/day on Days 17 and 20 post-coitum. When compared to the concurrent control group mean body weight gain was decreased by approximately 25%. During lactation, body weight gain returned to normal values again, but body weights remained significantly lower.

In addition, for females at the mid dose there was a trend towards slightly lower values for food consumption (before and after correction for body weight) during the entire treatment period. Changes in the 150 mg/kg/day group as compared to the concurrent control group were relatively small, and did not always reach statistical significance. In males at 150 mg/kg/day, statistically significantly changes in red blood cell parameters were observed as compared to the concurrent control group. This included a lower value for red blood cell count with correlating higher values for relative reticulocyte count and red blood cell distribution width. Mean values for red blood cell and reticulocyte count were at the higher end or above the historical control range. Also males at 50 mg/kg/day had a higher value for relative reticulocyte count. At the microscopic level, a slightly increased incidence and severity of extramedullary hematopoiesis (up to slight degree) was recorded in male rats at 50 and 150 mg/kg/day. Extramedullary hematopoiesis in the spleen can be seen as a spontaneous finding and the slight increase in incidence and severity is considered to be nonadverse.

No comparable treatment-related effects were noted in females at 50 and150 mg/kg/day.

At 150 mg/kg/day, no treatment-related effects were noted in any of the remaining parental parameters investigated in this study (i.e. functional observations, clinical biochemistry, thyroid hormone analysis, macroscopic examination, and organ weights).

No parental toxicity was observed at 50 mg/kg/day.

Reproductive results

No reproductive effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start of mating.

No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day.

All reproductive parameters investigated in this study (i.e. mating, fertility and conception index, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of the reproductive organs) were considered to be unaffected by treatment.

In conclusion, treatment with Hepteen Base® by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day revealed severe parental toxicity at 500 mg/kg/day. All high dose animals had to be euthanized after 6-7 days of treatment. No reproduction toxicity was observed following treatment at 50 and 150 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: Females: 50 mg/kg/day

Males: 150 mg/kg/day

Reproduction NOAEL: 150 mg/kg/day

Note: The entire 500 mg/kg/day group (males and females) had to be euthanized during the premating period due to unacceptable high toxicity. Therefore no reproductive  effects could be evaluated at this dose.

Effects on developmental toxicity

Description of key information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Hepteen Base® in rats by oral gavage including a preliminary dose range finding study.

Developmental NOAEL: 50mg/kg/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1 - The study was conducted in a GLP accredited laboratory based on the following guidelines: OECD test guidelines 421, 422, 407 OPPTS test guidelines 870.3650, 870.3550, 870.3050

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Hepteen Base® in rats by oral gavage including a preliminary dose range finding study.

Rationale for dose levels

Based on the results of a 10-day dose range finding study (Test Facility Study No. 512993); the selected dose levels for the combined 28-day oral gavage study with the reproduction/developmental toxicity screening test were 50, 150 and 500 mg/kg/day.

Study outline

The test item, Hepteen Base®, formulated in 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, was administered by daily oral gavage to SPF-bred Wistar Han rats. One control group (treated with the vehicle alone) and three treated groups (50, 150 and 500 mg/kg/day) were tested, each consisting of 10 males and 10 females.

Due to unexpected high toxicity, animals of the highest dose group were euthanized for humane reasons after only 6-7 days of treatment on Days 7 and 8 of the premating period. Males of the remaining groups were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to termination. Females of the remaining groups were dosed for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation (up to the day prior to necropsy). Females which failed to deliver healthy offspring were treated for 40 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity.

Developmental results

No developmental effects at 500 mg/kg/day could be evaluated as all animals of this high dose group were terminated before start mating.

One female at 150 mg/kg/day had to be euthanized before parturition on Day 21 post-coitum due to unacceptable high toxicity. As in this group there were two females that failed to produce live offspring (both not considered to be treatment-related), seven litters were available for evaluation of possible test-item related effects on pup development at 150 mg/kg/day. Although relatively low, the available results were considered to be sufficient for a proper evaluation.

One female in the 150 mg/kg/day group had in total 11 pups of which three pups were found cold at first litter check, one pup had little milk and one dead pup had no milk in its stomach (note: another dead pup could not be examined due to advanced autolysis). These pup observations suggestive for poor maternal care were considered to be secondary to the poor condition of the dam noted just before parturition and during the first days of lactation based on clinical signs, body weight loss and markedly reduced food consumption. In the subsequent period she recovered completely and all remaining pups survived until scheduled necropsy.

At 150 mg/kg/day, a trend towards slightly lower body weights of pups was noted from lactation Day 4 onwards, reaching statistical significance on lactation Day 13.

No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration of gestation, postimplantation survival index, live birth index, viability index, lactation index, clinical signs, sex ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels or macroscopy) by treatment at 150 mg/kg/day.

No developmental toxicity was observed in the 50 mg/kg/day group.

Developmental toxicity was indicated by lower fetal pup weights and a single litter with a slightly higher incidence of pup mortality and clinical signs at 150 mg/kg/day. This was considered to be secondary to maternal toxicity.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Developmental NOAEL: 50 mg/kg/day

Note: The entire 500 mg/kg/day group (males and females) had to be euthanized during the premating period due to unacceptable high toxicity. Therefore no reproductive and developmental effects could be evaluated at this dose.