Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-20 - 2016-07-11 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-20 - 2016-07-11 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
Range-finding study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to other study
Remarks:
supporting study
Related information:
Composition 1
Reason / purpose:
reference to other study
Remarks:
supporting study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.422: "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST strain
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: At the start of treatment the males weighed 310 to 357g, the females weighed 190 to 224g.
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Rodent Facility.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.), ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage, ad libitum.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES:
From: 28 January 2016 (first day of treatment)
To: 14 March 2016 (final day of necropsy)
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 25, 75, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml / kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by laboratories' Analytical Services. Results show the formulations to be stable for up to 21 days. Formulations were initially prepared weekly and following further stability fortnightly and stored at approximately 4 °C in the dark.
Samples of test item formulation were taken and analyzed on three occasions for concentration of the test item at Analytical Services. The results indicate that the prepared formulations were within 10% of the nominal concentration.
Duration of treatment / exposure:
43 or 44 days (males)
until day 5 post partum (females)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary range-finding study, in accordance with OECD TG 422
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week and at weekends (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on five males selected from each test and control group prior to termination (Day 42 for males) and five selected females from the control, low or intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. (Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: No
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb) Erythrocyte count (RBC) Hematocrit (Hct)
Erythrocyte indices:
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic):
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by 'Innovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See above
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males from each dose level and five selected females treated with 100 and 300 mg/kg bw/day, prior to termination, together with an assessment of sensory reactivity to various stimuli. Functional performance tests and sensory reactivity assessment was also performed on four pregnant females retaining offspring to Day 4 post partum and females showing a total litter loss at 1000 mg/kg bw/day.
- Battery of functions tested: sensory activity / grip strength / motor activity
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalization, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

IMMUNOLOGY: No

OTHER: Reproductive Performance
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males from each dose group and five selected females from the control, low and intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. Some tissues were weighed from all remaining animals(A):
Adrenals, Brain, Epididymides(A), Heart, Kidneys, Liver, Ovaries(A), Pituitary (post-fixation)(A), Prostate and Seminal Vesicles(A), Spleen, Testes(A), Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)(A)

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group. For females treated with 1000 mg/kg bw/day four females with litters and four females showing total litter loss and all tissues preserved in buffered 10% formalin, except where stated. Some tissues were preserved from all remaining animals (A):
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland(A)
Colon
Duodenum
Epididymides(A) •
Esophagus Eyes *
Gross lesions(A)
Heart
Ileum (including peyer's patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland(A)
Muscle (skeletal)
Ovaries(A)
Pancreas
Pituitary(A)
Prostate(A)
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles(A)
Skin
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes(A) •
Thyroid/Parathyroid
Trachea
Thymus
Urinary bladder
Uterus & Cervix(A)
Vagina(A)

* Eyes fixed in Davidson's fluid
• preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of liver and thyroid glands (both sexes), pituitary (males only), spleen, thymus, lungs and the kidneys (females only) from animals in the low and intermediate groups
Statistics:
Due to limitations of this free-text field, see "any other information on materials and methods".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity for males or surviving females treated with 100, 300 or 1000 mg/kg bw/day.
One control male and one male treated with 1000 mg/kg bw/day were observed to have open wounds which later formed scabs between Days 20 to 33 and 17 to 33, respectively. An additional male treated with 1000 mg/kg bw/day showed signs of a swollen hind limb from Day 37 onwards. These observations were considered to be physical injuries and not related to treatment.
One male, each treated with 300 or 100 mg/kg bw/day had signs of noisy respiration on Days 1 to 5 and Day 8 (respectively). In the absence of a similar effect at 1000 mg/kg bw/day, these observations were considered to be incidental and of no toxicological importance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum. There were no clinical signs of toxicity prior to death and the macroscopic findings included an enlarged spleen and liver together with red colored fluid in the thoracic cavity. It was noted this female had a long gestation period of 24 days.
One female treated with 1000 mg/kg bw/day was killed in extremis due to showing symptoms of difficulty during parturition; clinical signs for this animal included labored respiration, decreased respiratory rate, pilo-erection, lethargy, hypothermia, hunched posture, pallor of the extremities and dehydration. The macroscopic findings included a pale brain, liver, mammary gland and pancreas and the small and large intestine (caecum, colon, duodenum, ileum and jejunum) had black colored contents. It was noted that this female would have had a gestation length of 24 days.
There were no further unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day throughout the treatment period.
Fluctuations in weekly group mean body weight gains were evident in treated males, in females treated with 1000 mg/kg bw/day during maturation and in females treated with 100 mg/kg bw/day during lactation, achieving statistical significance. There was generally no dose-dependance and, on a number of occasions group mean values for treated animals exceeded controls, therefore the intergroup differences were considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no adverse effects on food consumption or food conversion efficiency for males throughout the study or for females during pre-mating, gestation or lactation at dose levels of 100, 300 or 1000 mg/kg bw/day.
During gestation, treated females showed an increase in food intake throughout this phase, attaining statistical significance for females treated with 300 and 1000 mg/kg bw/day. An increase in food consumption is generally considered not to represent an adverse effect of treatment. Food intake for females during lactation was generally similar, however females treated with 100 mg/kg bw/day showed a statistically significant reduction between Days 1 and 4 post partum. In the absence of a dose related response the intergroup difference was considered of no toxicological significance.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period.
There was a significant increase in water consumption for animals of either sex from all treatment groups throughout the study for males and for females during pre-pairing, gestation and lactation phases. During pre-pairing, the overall increase in water intake for males was 15%, 15% and 34% (100, 300 and 1000 mg/kg bw/day, respectivity) and for females the overall increase was 25%, 59% and 73% (100, 300 and 1000 mg/kg bw/day, respectively). A statistically significant increase in water consumption was only evident in females treated with 1000 and 300 mg/kg bw/day throughout gestation and lactation.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treated males from all dose groups showed a statistically significant reduction in hemoglobin, hematocrit and erythrocyte count; a dose relationship was present in all cases. With the exception of one male from the high dose group, all individual values were within the background control ranges. However, these findings may be associated with the histopathological findings of hemopoiesis in the liver and spleen.
Females treated with 1000 mg/kg bw/day showed a reduction in neutrophil counts when compared to controls, attaining statistical significance. There was no dose relationship present and individual values were within the background control ranges.
There were no toxicologically significant effects detected in females treated with 300 or 100 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed a statistically significant increase in monocytes. Although the majority of the individual values were above the background control ranges no such effects were evident in 1000 mg/kg bw/day males or in corresponding females. Therefore in the absence of a true-dose related response the intergroup difference was considered of no toxicological significance.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males from all treatment groups showed statistically significant increases in cholesterol attaining statistical significance in all cases, without a dose related response. The majority of individual values were within the background control ranges however in view of the microscopic changes evident in the liver, an association to treatment cannot be excluded.
Females treated with 1000 or 300 mg/kg bw/day showed statistically significant increases in total protein. A dose relationship was evident but individual values were all within the background control ranges. However in view of the microscopic changes in the liver, an association to treatment cannot be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related effects detected.
Functional Performance Tests: There were no treatment-related changes in functional performance.
At 1000 mg/kg bw/day females showed a statistically significant increase in hind limb grip strength whilst females from all treatment groups showed a statistically significant increase in fore limb grip strength. Due to a lack of consistency and no dose relationship, these findings were considered to be incidental. At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.
At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver: Animals of either sex from all treatment groups showed a dose related increase in liver weight both absolute and relative to terminal body weight, attaining statistical significance, excluding females treated with 100 mg/kg bw/day. This can be associated with the centrilobular hepatocellular hypertrophy in these animals.
Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight with a dose related response. This may be associated with the histopathological findings identified.
Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weight both absolute and relative to terminal body weight with a dose related response.
Thyroid/Parathyroid: Animals of either sex showed a statistically significant increase in thyroid/parathyroid weights in both absolute and relative to terminal body weight. Females receiving 100 mg/kg bw/day did not attain statistical significance
Spleen: Females from all treatment groups showed an increase in spleen weights both absolute and relative to terminal body weight. Although a true dose response was not evident and statistical significance was only achieved at 300 mg/kg bw/day, in view of the histopathological changes identified, an association to treatment can not be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Early decedents
One female treated with 300 mg/kg bw/day that was found dead on Day 3 post partum showed an enlarged liver and spleen and a fluid filled thoracic cavity.
One female treated with 1000 mg/kg bw/day that was killed in extremis during parturition had black coloured contents in the caecum, colon, duodenum, ileum and jejunum. The brain, liver, mammary gland and pancreas were also pale.
Scheduled necropsy
At 1000 mg/kg bw/day seven males had enlarged livers, one of which was also mottled. A further one male (No.74) had a small prostate and seminal vesicles. Three females from this dose group and two females treated with 300 mg/kg bw/day had enlarged spleens.
One male (No.30) treated with 100 mg/kg bw/day was observed to have small epididymides and the testes were noted to be small and flaccid. This male did not produce a pregnancy in its female partner. One female at this level was noted to have fluid filled uterus and cervix. In the absence of a similar effect at 1000 mg/kg bw/day, these intergroup differences were considered to be incidental.
There were a number of animals of either sex observed to have reddened lungs including control animals. This was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Centrilobular hepatocellular hypertrophy, moderate or mild was present in all males treated with 1000 mg/kg bw/day and in eight females from this treatment group. In both sexes treated with 100 or 300 mg/kg bw/day centribular hepatocellular hypertrophy was evident from minimal to mild and showed a dose dependant trend.
Diffuse vacuolation (fat type), minimal or mild was present in 4/8 males and 5/9 females at 1000 mg/kg bw/day with periportal vacuolation being evident in another female. It was also present at a minimal level in both sexes at 100 or 300 mg/kg bw/day. Hematopoiesis was present in 3/9 1000 mg/kg bw/day females; 4/5 100 mg/kg bw/day females and 4/6 300 mg/kg bw/day females. The severity was increased at 300 mg/kg bw/day compared to 100 mg/kg bw/day.
Thyroid Gland: Mild follicular cell hypertrophy was present in all males and 8/9 females treated with 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day it was present at minimal or mild levels in males and females.
Pituitary: There was a minor increase in the amount of vacuolation in pars distalis (anterior lobe) of the pituitary gland in 1000 mg/kg bw/day males. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.
Spleen: Increased hematopoiesis was present in 7/9 1000 mg/kg bw/day females (mild to marked); 6/6 300 mg/kg bw/day females (mild to marked) and in 4/5 100 mg/kg bw/day females (mild or moderate).
Thymus: There was a reduction in the amount of involution noted in treated females from all dose groups when compared to control animals. Involution/atrophy is seen as a normal background change in pregnant and lactating animals therefore not generally recorded thus this may be linked to lower numbers of animals producing and feeding litters.
Lungs: There was a minor increase in the incidence of alveolar macrophage accumulation (focal) in the lungs of females at 1000 mg/kg bw/day only.
Kidneys: Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment.
Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Non-Pregnancy: A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.
Total Litter Loss: In total, 1 (No. 37), 5 (No. 61, 64, 68, 69 and 71) and 4 (No. 86, 88, 89 and 96) females showed total litter loss from 100, 300 and 1000 mg/kg bw/day dose groups, respectively. A further one female (No. 87) showed evidence of a pregnancy but no litter was observed. Histologically other than the general findings indicated above the only notable differences between control and treated females were fewer animals lactating (linked to lack of live pups) and 3 without implantation sites on the section considered to be linked to the reduction noted.
Premature Decedents: Female 64 had an enlarged liver and spleen, plus a fluid filled thorax at necropsy. There were numerous histopathological changes and septicemia was present. This is considered to be the cause of death. Although the initial cause is not clear it is likely to have originated in the uterus (degeneration was present) and this may have been a complication of pregnancy and parturition. Therefore, this is considered not to be related to treatment. Female 91 was killed due to issues with parturition and showed non-specific changes; most were considered to be agonal and its death could not be unequivocally related to treatment.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
other: based on the severity and adaptive response nature of the findings rather than an absence of findings.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
immune system
Organ:
spleen
thymus
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the repeated dose oral toxicity in rats (subacute; reproductive effects are discussed in the respective section).

Predominantly for the 1000 and 300 mg/kg bw/day dose groups microscopic examinations revealed treatment related changes in the liver, thyroid, pituitary, spleen, thymus, lungs and kidneys. The minimal centribular hepatocellular hypertrophy, minimal follicular cell hypertrophy in the thyroid gland and the mild increased hematopoiesis observed in the 100 mg/kg bw/day dose group were regarded as adaptive responses.
More specifically, the hypertrophy in the liver observed in animals from all dose groups was mainly centrilobular but due to the severity was spreading outwards towards the mid-zonal area. The histological changes noted in the thyroid gland (enlargement of follicles/ hypertrophy), pituitary (vacuolation males only) and the liver (hypertrophy) may be linked. Again, these findings are suggestive of an adaptive response to mixed function oxidase induction in the liver with concomitant increased incidence of follicular cell hypertrophy in the thyroid gland. In the pituitary, the enlarged vacuolated cells may reflect hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs); a common finding following administration of liver enzyme inducers where the underlying mechanism is considered to be increased hepatic clearance of thyroid hormones (causing hypertrophy of follicular cells) followed by a compensatory increase in the pituitary secretion of TSH. These histopathological changes can correlate with the increase in liver and thyroid gland weights observed at necropsy. The vacuolation in the liver of animals in all groups treated with the test item may be due to a perturbation of metabolic activity however a direct effect of the test item cannot be ruled out.
The increase in hematopoiesis in the spleen and liver of some females treated with the test item is of unknown etiology as there were no notable changes in the hematological picture but a direct effect of the test item cannot be ruled out. These finding can correlate to the increase in spleen weights observed at necropsy.
The reduction in thymic involution in treated females correlates with the increased weight noted but may be related to the reduced number of viable litters produced.
There were three animals with minimal or mild changes in the kidneys - basophilic (degenerating/regenerating) tubules one of which also had tubular dilation. These types of changes are seen at a low incidence as common background changes but none were apparent in control animals of either sex in this study. Whilst any significance within the study is equivocal, it was noted that there was an increase in kidney weight at necropsy.
At 1000 mg/kg bw/day there was an increase in the number of females with alveolar macrophage accumulation in the lungs and the significance of this finding is unclear.

Slight changes in hematological values (erythrocyte count, hemoglobin and hematocrit) and histopathological evidence of mild splenic/hepatic changes were considered to be non-adverse in nature at 100 mg/kg bw/day. It was considered that these changes are adaptive responses related to the administration of the test item at the lowest dose level as compared to the adverse effects noted for the two higher dose groups of 300 and 1000 mg/kg bw/day.
Collectively these findings, showing only mild differences compared to controls in the absence of a dose response relationship were not considered to be adverse. Therefore, the No Observed Adverse Effect Level was determined to be 100 mg/kg bw/day for systemic toxicity.

According to Regulation 1272/2008, Table 3.9.1, Categories for specific target organ toxicity-repeated exposure, Category 2 is required for substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance dose/concentration values are provided below (see 3.9.2.9) in order to help in classification.
Guidance Value Ranges (dose/concentration) to assist in Category 2 classification are i.a. 10 < C ≤ 100 mg/kg body weight/day for a Oral (rat) study over 90 days. For subacute studies, the limit value is 300 mg/kg bw. The Regulation states further, that the guidance values and ranges mentioned in paragraphs 3.9.2.9.6 and 3.9.2.9.7 are intended only for guidance purposes, i.e. to be used as part of the weight of evidence approach, and to assist with decisions about classification. They are not intended as strict demarcation values. Substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement; assessment shall take into consideration not only significant changes in a single organ or biological system but also generalised changes of a less severe nature involving several organs.
As stated above, there were effects in various organs noted, i.e. liver, thyroid, pituitary, spleen, thymus, lungs and kidneys. The increase in the number of females with alveolar macrophage accumulation in the lungs in the 1000 mg/kg is both of unclear toxicological significance and way above the guidance value for classification. Hence, Classification as STOT RE Cat. 2 (lungs) is not triggered.

Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight with a dose related response. This may be associated with the histopathological findings identified. Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment. Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change. Nevertheless those effects are only present at elevated dose levels which are not considered to justify a classification as STOT RE Cat. 2 (kidneys), and further the significance within the study is considered equivocal.

The reduction in thymic involution in treated females correlates with the increased weight noted but may be related to the reduced number of viable litters produced. In this case, this is considered as a secondary effect not directly related to the treatment, no classification is required.

As stated above, the effects noted in the other organs, i.e. centribular hepatocellular hypertrophy, follicular cell hypertrophy in the thyroid gland, vacuolation in pituitary (males), the increased hematopoiesis, and the increase in spleen weights are linked. They need to be regarded as adaptive response. Here, it cannot be absolutely distinguished to which dose the effect is not considered as toxic rather than adaptive, and at which dose exactly it has to be considered adverse or severe. Further, there are multiple organs involved which are connected, and it cannot be definitively distinguished whether there is a direct effect from the substance or secondary ones. The noted effects should be hence considered as a general systemic response rather than a single organ toxicity effect.

In consequence, clear evidence for classification as STOT RE Cat. 2 is not given, and classification can hence be omitted.
Executive summary:

Introduction

The present GLP study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development, presented in the respective section) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations, hematology and blood chemistry evaluations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from the control, low and intermediate dose groups on Day 4 post partum. Due to a high number of total litter losses evident in the high dosage group, four females with litters and four females with a total litter loss were investigated to allow comparison between the two sets of females which were in a different physiological state on Day 4 post partum.

Adult males were terminated on Days 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Mortality: One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum and one female treated with 1000 mg/kg bw/day was killed in extremis due to difficulty during parturition. There were no further unscheduled deaths.

Clinical Observations: There were no clinical signs of toxicity for males or the surviving females treated with 100, 300 or 1000 mg/kg bw/day.

Behavioral Assessment: There were no treatment-related effects detected.

Functional Performance Tests: There were no treatment-related changes in the functional performance.

Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

Body Weight: There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption: There were no adverse effects on food consumption or food conversion efficiency for males throughout the study or for females during pre-mating, gestation or lactation at dose levels of 100, 300 or 1000 mg/kg bw/day.

Water Consumption: Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period. An increase in water consumption was detected for animals of either sex from all treatment groups for the majority of the treatment period.

 

Laboratory Investigations

Hematology: Treated males from all groups showed a statistically significant reduction in hemoglobin, hematocrit and erythrocyte count; a dose relationship was present in all cases. With the exception of one male from the high dose group, all individual values were within the background control ranges. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in neutrophil count when compared to controls attaining statistical significance. No toxicologically significant effects were detected in females treated with 300 or 100 mg/kg bw/day.

Blood Chemistry: Males from all dose groups showed a statistically significant increase in cholesterol when compared to controls but without a dose relationship. At 1000 or 300 mg/kg bw/day females showed a statsistically significant increase in total protein when compared to controls with a dose related response.

 

Pathology

Necropsy:

Early decedents: One female treated with 300 mg/kg bw/day that was found dead on Day 3post partumshowed an enlarged liver and spleen and a fluid filled thoracic cavity. A further female treated with 1000 mg/kg bw/day that was killedin extremisduring parturition had black coloured contents in the caecum, colon, duodenum, ileum and jejunum. The brain, liver, mammary gland and pancreas were also pale.

Scheduled necropsy : At 1000 mg/kg bw/day seven males had enlarged livers of with one of these appearing mottled. Three females from this dose group and two females treated with 300 mg/kg bw/day were also observed with enlarged spleens. There were higher instances of small, weak and no milk in the stomach in offspring from litters of the high and intermediate dose groups.

 

Organ Weights

Liver: Animals of either sex from all treatment groups showed a dose related increase in liver weight both absolute and relative to terminal body weight. Statistical significance was achieved excluding females treated with 100 mg/kg bw/day.

Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increase in kidney weights both absolute and relative to terminal body weight with a dose related response.

Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weights both absolute and relative to terminal body weight with a dose related response.

Thyroid/Parathyroid: Animals of either sex from all treatment groups showed statistically significant increases in thyroid/parathyroid weight both absolute and relative to terminal body weight. Females receiving 100 mg/kg bw/day did not attain statistical significance.

Spleen: Females from all treatment groups showed an increase in spleen weights both absolute and relative to terminal body weight.

 

Histopathology

Liver: Centrilobular hepatocellular hypertrophy, moderate or mild was present in all males treated with 1000 mg/kg bw/day and in eight females from this treatment group. Both sexes treated with 100 or 300 mg/kg bw/day showed from minimal to mild centrilobular hepatocellular hypertrophy and showed a dose dependant trend. Diffuse vacuolation (fat type), minimal or mild was present in 4/8 males and 5/9 females at 1000 mg/kg bw/day with periportal vacuolation being evident in another female. It was also present at a minimal level in both sexes at 100 or 300 mg/kg bw/day. Hematopoiesis was present in 3/9 1000 mg/kg bw/day females; 4/5 100 mg/kg bw/day females and 4/6 300 mg/kg bw/day females. The severity of hematopoiesis was increased at 300 mg/kg bw/day compared to 100 mg/kg bw/day.

Thyroid Gland: Mild follicular cell hypertrophy was present in all males and 8/9 females treated with 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day it was present at minimal or mild severity in males and females.

Pituitary: There was a minor increase in the amount of vacuolation in pars distalis (anterior lobe) of the pituitary gland in males at 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.

Spleen: Increased hematopoiesis was present in 7/9 females at 1000 mg/kg bw/day (mild to marked); 6/6 females at 300 mg/kg bw/day females (mild to marked) and in 4/5 females at 100 mg/kg bw/day (mild or moderate).

Thymus : There was a reduction in the amount of involution noted in treated females from all dose groups when compared to control animals. Involution/atrophy is seen as a normal background change in pregnant and lactating animals therefore not generally recorded thus this may be linked to lower numbers of animals producing and feeding litters.

Lungs: There was a minor increase in the incidence of alveolar macrophage accumulation (focal) in the lungs of females at 1000 mg/kg bw/day only.

Kidneys: Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment. Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change.

Non-Pregnancy: A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.

Total Litter Loss: Several animals in the study showed total litter loss. Histologically other than the general findings indicated above the only notable differences between control and treated females were fewer animals lactating (linked to lack of live pups) and 3 without implantation sites on the section considered to be linked to the reduction noted. There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

 

Conclusion

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproductive toxicity was considered to be 100 mg/kg bw/day, based on the severity and adaptive response nature of the findings rather than an absence of findings.

Reason / purpose:
reference to other study
Remarks:
supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-05-25 - 2017-11-21 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see target record
Reason / purpose:
read-across source
Remarks:
target record
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.421: “Reproduction/Developmental Toxicity Screening Test” (adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008
Version / remarks:
This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark; used/formulated in light
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: males weighed 281 to 345g, the females weighed 196 to 245g
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records.
- Temperature (°C): The Study Plan target ranges for temperature were 22 ± 3 °C.
- Humidity (%): The Study Plan target ranges for relative humidity were 50 ± 20%.
Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
IN-LIFE DATES: The in-life phase of the study was conducted between 13 June 2016 (first day of treatment) and 06 August 2016 (final day of necropsy).
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Propylene Glycol. Test item formulations spanning the concentration ranges used in this study were previously shown to be homogenously prepared and stable for at least twenty-one days when stored refrigerated at approximately 4ºC, in the dark. Formulations were therefore prepared, aliquoted for use, and stored as above before use within the stability period.

VEHICLE
- Concentration in vehicle: 0, 12.5, 37.5, and 125 mg/ml
- Amount of vehicle (if gavage): 8 mL/kg
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were paired on a 1 male:1 female basis within each dose group, for a period of up to fourteen days. Following evidence of successful mating, the males were returned to their original cages.
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on three occasions and analyzed for concentration of the test item at the laboratories Analytical Services. The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within 101 to 111% of the nominal concentration.
Duration of treatment / exposure:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.
Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
Frequency of treatment:
daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
iii. On Day 15, animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. The male dose groups were killed and examined macroscopically on Day 44 or 45.
vii. At Day 13 post partum, all surviving offspring were killed and examined macroscopically. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for females in the morning of the day of necropsy.
viii. Where possible, blood samples were taken from two offspring on Day 4 post partum and one male and one female offspring on Day 13 post partum. In addition, blood samples were taken from all adult surviving animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels and dose volume were selected in collaboration with the Sponsor Representative and were based on available toxicity data including a 28 Day toxicity study in the rat (see chapter repeated dose toxicity). In the 28 Day study, administration of the test item to animals of either sex at dose levels of up to 1000 mg/kg bw/day was well tolerated. A high dose level of 1000 mg/kg bw/day was therefore considered to be suitable for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER: Thyroid Hormone Analysis
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.
All adult males at termination.
All adult females at termination.
All samples were dispatched to the Test Site where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator.
Estrous cyclicity (parental animals):
Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.
Vaginal smears were taken daily throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
After necropsy, detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data) (see deviations from study plan)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations)
One male and one female offspring from each litter at Day 13 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
- Maternal animals: All surviving animals. Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

GROSS NECROPSY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
The following organs were dissected free from fat and weighed before fixation from all animals (where applicable) from each dose group.
Cowpers glands
Epididymides
Glans penis
LABC (levator ani-bulbocavernous) muscle
Testes
Thyroid (weighed post-fixation with Parathyroid)

Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides ♦
Glans penis
LABC (levator ani-bulbocavernous) muscle
Mammary gland (females only)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulating gland)
Testes ♦
Thyroid/Parathyroid
Uterus/Cervix
Vagina
♦ Preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy, and any gross lesions were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
At the request of the Sponsor, thyroid/parathyroid from control and high dose adult females were routinely processed to paraffin wax, sectioned and stained with Hematoxylin and Eosin, and examined.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS NECROPSY
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.
Statistics:
Due to limitations of this free-text field, please see "Any other information on materials and methods"
Reproductive indices:
Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) * 100
Pregnancy Index (%) = (Number of pregnant females / number of animals mated) * 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of animals delivering live offspring / Number of pregnant females) * 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean post-implantation loss was calculated for each female/litter as follows:
Post-implantation loss (%) = ((Number of implantation sites – Total number of offspring born) / Number of implantation sites) * 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) * 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) * 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) * 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula: (Number of male offspring / Total number of offspring) * 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs considered to be related to the toxicity of the test item.
At 1000 mg/kg bw/day, animals of either sex showed sporadic instances of increased post-dose salivation from Days 2 (females) or 3 (males) which persisted towards the end of the dosing period. Such observations are common in this type of study and may reflect an irritant nature of the test item and/or formulation and as such are considered to be of no toxicological significance.
One female each from the 100 or 300 mg/kg bw/day dose groups showed clinical signs of noisy respiration on Days 22 and 4, respectively. These observations may be due to the dosing procedure and in the absence of similar findings in the remaining animals, they were considered unlikely to be related to treatment with the test item.
There were no clinical signs for any other animals throughout the treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 100 mg/kg bw/day, Female 47 was killed in extremis on Day 1 post partum (Day 41 relative to the start of dosing). Prior to death, clinical signs for this animal included decreased respiratory rate, ataxia, wet fur and increased post-dose salivation but there were no macroscopic observations at necropsy. In the absence of any premature decedents in the 300 or 1000 mg/kg bw/day dose groups, this death was considered unlikely to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment with the test item on body weight development in animals of either sex.
At all dose levels, males occasionally showed marginally higher body weight gains than controls which resulted in slightly higher overall body weight gains for these animals. There was generally no dose-dependence and the intergroup differences were not statistically significant. It is worth noting that a body weight increase of this nature in this type of study is generally considered to be of no toxicological significance.
During the pre-pairing phase of the study, females treated with the test item at all dose levels also showed slightly higher body weight gains than controls achieving statistical significance for the 1000 mg/kg bw/day females during the second week (p<0.05). During the gestation period, group mean body weight gains in the test item-treated females were slightly lower than controls resulting in slightly lower overall body weight gains across these dose groups. The intergroup differences were, however, neither strictly dose-related nor they achieved statistical significance and may be due to the slightly lower litter size, i.e. less fetuses, for these females and as such were considered not to be of any toxicological importance. During the first week of lactation, group mean body weight gains in test item-treated females remained lower than controls but there was no dose-relationship and statistical significance was only achieved for the 100 and 300 mg/kg bw/day females over Days 1 to 4 (p<0.05). This resulted in statistically significantly lower overall body weight gains for females treated with 300 or 1000 mg/kg bw/day over the first week of lactation (p<0.05), but without depicting any dose-dependence. Subsequent improvement was evident and group mean body weight gains for test item-treated females during the second week of lactation were comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, dietary intake for the males was comparable with controls throughout the study. The corresponding females also showed similar food consumption to controls during pre-pairing and early gestation. Over Days 14 to 20 of gestation, food intake for females receiving 100 or 300 mg/kg bw/day was slightly lower than controls (p<0.05) but a dose-relationship was not evident and the corresponding values in females from the 1000 mg/kg bw/day dose group was similar to controls. Fluctuations in dietary intake were also evident during the lactation phase of the study attaining occasional statistical significance for the 100 or 300 mg/kg bw/day females (p<0.01-0.05) but the corresponding values for the 1000 mg/kg bw/day dose group were comparable with controls.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item on food conversion efficiency (where calculated) for animals of either sex receiving the test item. Any fluctuations were deemed to be reflective of minor intergroup differences in body weight gain and/or food consumption.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any overt differences for animals of either sex receiving the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis: An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse effect ovserved.
At all dose levels, group mean absolute and body weight-related organ weights in males were comparable with controls.
Females treated with 1000 mg/kg bw/day showed slightly but statistically significantly higher absolute and body weight-relative thyroid weights in relation to controls. A small number (4/11) of absolute values exceeded the historical control data ranges. As there were no treatment-related occurrences in the relevant histopathological examination and thyroid hormone analysis, this finding was considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings at any dose level.
Incidental findings included small right seminal vesicle in one male each from the control and 100 mg/kg bw/day dose groups with another male from the latter dose group showing small left testis/epididymis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities at any dose level.
All of the histopathological findings encountered were considered to have occurred spontaneously or post mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.
Histopathological findings: neoplastic:
not examined
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study.
There were also no intergroup differences in the stage of estrous cycle on the day of necropsy.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating: There was no effect of treatment on mating performance. With the exception of one female from the 100 mg/kg bw/day dose group which mated on Day 14 after pairing, the remaining animals mated within four days after pairing.
Fertility: Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female each from the control, 100 and 300 mg/kg bw/day dose groups (Females 18, 39 and 68, respectively) and two females from the 1000 mg/kg bw/day dose group (Females 92 and 95) did not achieve pregnancy following positive evidence of mating. Male 27 (paired with Female 39) had severe unilateral tubular atrophy and a subsequent unilateral aspermia in the epididymis which may have prevented pregnancy. There were no histopathological changes in the tissues examined to account for non-pregnancy in the remaining animals and these non-pregnancies were deemed to be due to biological variation.
Gestation length: Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for test item-treated females appeared to be similar to controls.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (estrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs detected in pups from all treated groups included small size, cold, weak, no milk in the stomach, physical injury, found dead or missing. Females 23 (control), 41 and 45 (100 mg/kg bw/day), 70 (300 mg/kg bw/day) and 88 (1000 mg/kg bw/day) showed total litter losses. Such findings are often seen in this type of study and were considered unlikely to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Litter Responses: In total, eleven females each from the control, 100 and 300 mg/kg bw/day dose groups and ten females from the 1000 mg/kg bw/day dose group gave birth to a live litter. One litter each was lost in the control, 300 and 1000 mg/kg bw/day dose groups during the lactation phase of the study with the 100 mg/kg bw/day dose group showing two litter losses. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.
Offspring Litter Size, Sex Ratio and Viability: There was considered to be no adverse effect of treatment with the test item at any dose level on the mean number of implantations and post-implantation losses. Of the litters born, there was also no adverse effect of treatment with the maternal treatment on litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum indicating the lack of any adverse effect on offspring viability.
The mean number of implantation sites in females treated with the test item up to a dose level of 1000 mg/kg bw/day was slightly lower than controls. The majority of individual values were within the historical control data ranges and the intergroup differences were neither dose-related nor they attained statistical significance. Histopathological examination of the ovaries from the 1000 mg/kg bw/day did not identify any treatment-related findings and this observation was considered likely to be due to biological variation. Group mean post-implantation losses across all dose groups including controls were comparable, but due to the slightly lower number of implantation sites, litter size at birth and on subsequent days in the test item-treated dose groups remained lower than controls. These intergroup differences did not achieve statistical significance and were not dose-dependent, and as such were considered to be of no toxicological relevance.
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was considered to be no adverse effect of treatment with the test item on offspring body weights and body weight development.
At birth, group mean offspring body weights across all test item-treated dose groups were comparable with controls. Group mean offspring body weight gains from the 100 and 300 mg/kg bw/day dose groups remained slightly lower than controls over Days 1 to 7 of lactation which resulted in lower cumulative body weight gains for these pups, but improvement was noted over Days 7 to 13 of lactation. Consequently, the mean body weights on Day 4 and thereafter for these offspring were slightly lower than controls. It is worth noting that corresponding values for the 1000 mg/kg bw/day dose group were generally similar to controls and none of these intergroup differences achieved statistical significance. Due to the slightly lower litter size and lower body weight gains, litter weights for the 100 and 300 mg/kg bw/day dose groups remained lower than controls attaining statistical significance on Day 13 post partum for the latter. The corresponding values for the 1000 mg/kg bw/day dose group were, however, generally similar to controls, and these intergroup differences were considered not to be of any toxicological relevance.
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):
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The study was conducted under GLP according to OECD guideline 421 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the reproductive toxicity of the test item in rats (screening study). The oral (gavage) administration of the test item to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study also includes some endocrine disruptor relevant endpoints. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 28 July 2015).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Propylene Glycol).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male:one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance and visible nipple count (male offspring only).

Blood samples were taken at termination from all surviving adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and 1000 mg/kg bw/day dose groups was performed.

 

Results

Adult Responses

Mortality: There were no treatment-related deaths on the study.

Clinical Observations: There were no clinical signs considered to be related to the toxicity of the test item.

Body Weight: There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the dose administration period.

Food Consumption: There was no effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the treatment period.

Water Consumption: Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

Reproductive Performance

Mating: There was no effect of treatment with the test item on mating performance.

Fertility: There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Length: There were no treatment-related differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: There was no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development: There was no adverse effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1 post partum, visible nipple count in male offspring on Day 13 post partum or clinical signs up to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Pathology

Necropsy: Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any adverse effects up to a dose level of 1000 mg/kg bw/day.

Organ Weights: No adverse effects were detected in animals of either sex at any dose level.

Histopathology: Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

 

Conclusion

The oral (gavage) administration of4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12Hdibenzo[d,g][1,3,2]dioxaphosphocinto Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Reason / purpose:
reference to other study
Remarks:
supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2016-05-25 - 2017-11-21 (experimental phase)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is postulated that the results obtained from the available OECD 421 study on the target chemical 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin may be used to substantiate the conclusions derived in from the results of the available OECD 422 study on the registered substance 2,2'-methylenebis[6-cyclohexyl-p-cresol] itself. Analysis and stability determinations of the source chemical indicate that when the OECD 421 was conducted the test item contained more than 10% of 2,2'-methylenebis[6-cyclohexyl-p-cresol]. The NOAEL on reproductive toxicity was 1000 mg/kg in the OECD 421 study, allowing the conclusion that, with a content of min. 10% in the test item the NOAEL of 2,2'-methylenebis[6-cyclohexyl-p-cresol] would be 100 mg/kg when tested as such.
Further, both substances are structurally very related. The main component of the source chemical is basically the target chemical, only that the both hydroxyl groups are connected via phosphonic acid.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source Chemical: Technical product containing 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin (EC 277-633-6, CAS 73912-21-7, 438.5388g/mol, SMILES: Cc1cc2Cc3cc(C)cc(C4CCCCC4)c3OP(O)Oc2c(c1)C5CCCCC5) and its educt / degradation product 2,2'-methylenebis[6-cyclohexyl-p-cresol]. Directly after being produced, the product contains in general <10% 2,2'-methylenebis[6-cyclohexyl-p-cresol], most typically in the higher single digits.
Please see attached report, describing stability determinations of the source chemical 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin, showing its degradation into the target chemical 2,2'-methylenebis[6-cyclohexyl-p-cresol]
Target Chemical: 2,2'-methylenebis[6-cyclohexyl-p-cresol], EC 223-773-8, CAS 4066-02-8, 392.5735 g/mol, SMILES: Cc1cc(Cc2cc(C)cc(C3CCCCC3)c2O)c(O)c(c1)C4CCCCC4
The main component of the source chemical is 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin (EC 277-633-6, CAS 73912-21-7), which is the reaction product of Diethyl phosphonate (CAS 762-04-9) and the registered substance 2,2'-methylenebis[6-cyclohexyl-p-cresol] (CAS 4066-02-8).

3. ANALOGUE APPROACH JUSTIFICATION
The technical product, i.e. the source chemical, contains both 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin and its educt / degradation product 2,2'-methylenebis[6-cyclohexyl-p-cresol].
The main component of the source chemical is 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin, and the product contains up to 10% of the target chemical 2,2'-methylenebis[6-cyclohexyl-p-cresol] due to the production process. Further, the main component slowly decomposes into the target chemical, which was quantified in an aging experiment, report i.a. describing the analytical methods is attached.
To determine the content of the raw material 2,2'-methylenebis[6-cyclohexyl-p-cresol] in the product, it was analyzed using GC. Experiments were performed on a different batch as the one used in the present OECD 407 and 421 studies. However, this is not expected to have an impact on the conclusions, as only the initial amounts of 2,2'-methylenebis[6-cyclohexyl-p-cresol] differ, the degradation kinetics can be reasonable expected to be identical.
The content of the initial content of 2,2'-methylenebis[6-cyclohexyl-p-cresol] in the product was determined after production. After storage of that sample in the dark at 4 °C the measurement was repeated approx. 14 months later. The initial content was determined to 9.6%, the amount after storage for 14 months was 20.6%, the additionally formed 2,2'-methylenebis[6-cyclohexyl-p-cresol] amount to 11% of the total product. So it can be deducted that approx. 12.2% of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin were degraded over 14 months. Although it is not possible to determine the exact kinetic parameters with only two measurements, it was clearly shown that additional amounts of 2,2'-methylenebis[6-cyclohexyl-p-cresol] were formed. To enable estimations of the actual content of 2,2'-methylenebis[6-cyclohexyl-p-cresol] in the test item employed in the available OECD 407 and 421 studies, a linear course is assumed. This approach may be simplified but allows a rough estimation to show that the content of 2,2'-methylenebis[6-cyclohexyl-p-cresol] exceeds 10% in the test item.
So, assuming a linear course, approx. 0.87% of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin degrades, when stored at 4°C, into 2,2'-methylenebis[6-cyclohexyl-p-cresol] per month. The batch used in the OECD 407 and 421 studies had a content of 92.5%4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin, so it contained initially approx. 7.5% of 2,2'-methylenebis[6-cyclohexyl-p-cresol], neglecting a possible content of unknown impurities. The first day of treatment was 3 months (OECD 407) resp. 4.5 months (OECD 421), last day of treatment was 4 months (OECD 407) resp. 6 months (OECD 421) after the CoA was dated. So, assuming an approx. 0.87% degradation of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin per month, this would result in additional 2.4% (OECD 407) resp. 3.6% (OECD 421) at the start of treatment and 3.2% (OECD 407) resp. 4.8% (OECD 421) at the last day of treatment. In consequence, the actual total content of 2,2'-methylenebis[6-cyclohexyl-p-cresol] in the test item was 9.9% (OECD 407) resp. 11.1% (OECD 421) at the start of treatment and 10.7% (OECD 407) resp. 12.3% (OECD 421) at the last day of treatment.
So taking into account the observed NOAEL of 1000 mg/kg in both available OECD 407 and 421 studies, and neglecting possible matrix effects, the NOAEL resulting from only the content of 2,2'-methylenebis[6-cyclohexyl-p-cresol] in the test item would be 99-107 mg/kg (OECD 407) resp. 111-123 mg/kg (OECD 421).
As in the available OECD 422 study on only of 2,2'-methylenebis[6-cyclohexyl-p-cresol] a NOAEL of 100 mg/kg was found for both systemic and reproductive toxicity, the conclusions drawn OECD 407 and 421 studies can be used to support the conclusion that setting the NOAEL to 100 mg/kg is justified.
Further, both substances bear a rather high structural similarity: The main component of the source chemical is basically the target chemical, only that the both hydroxyl groups are connected via phosphonic acid. So the NOAEL of 1000 mg/kg derived from the structural analogue allows the conclusion that the derived NOAEL of 100 mg/kg from the registered substance itself can be considered as worst case.

4. DATA MATRIX
see attached justification
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.421: “Reproduction/Developmental Toxicity Screening Test” (adopted 28 July 2015)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008
Version / remarks:
This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark; used/formulated in light
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: males weighed 281 to 345g, the females weighed 196 to 245g
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records.
- Temperature (°C): The Study Plan target ranges for temperature were 22 ± 3 °C.
- Humidity (%): The Study Plan target ranges for relative humidity were 50 ± 20%.
Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
IN-LIFE DATES: The in-life phase of the study was conducted between 13 June 2016 (first day of treatment) and 06 August 2016 (final day of necropsy).
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Propylene Glycol. Test item formulations spanning the concentration ranges used in this study were previously shown to be homogenously prepared and stable for at least twenty-one days when stored refrigerated at approximately 4ºC, in the dark. Formulations were therefore prepared, aliquoted for use, and stored as above before use within the stability period.

VEHICLE
- Concentration in vehicle: 0, 12.5, 37.5, and 125 mg/ml
- Amount of vehicle (if gavage): 8 mL/kg
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were paired on a 1 male:1 female basis within each dose group, for a period of up to fourteen days. Following evidence of successful mating, the males were returned to their original cages.
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on three occasions and analyzed for concentration of the test item at the laboratories Analytical Services. The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within 101 to 111% of the nominal concentration.
Duration of treatment / exposure:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.
Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
Frequency of treatment:
daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
iii. On Day 15, animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. The male dose groups were killed and examined macroscopically on Day 44 or 45.
vii. At Day 13 post partum, all surviving offspring were killed and examined macroscopically. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for females in the morning of the day of necropsy.
viii. Where possible, blood samples were taken from two offspring on Day 4 post partum and one male and one female offspring on Day 13 post partum. In addition, blood samples were taken from all adult surviving animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels and dose volume were selected in collaboration with the Sponsor Representative and were based on available toxicity data including a 28 Day toxicity study in the rat (see chapter repeated dose toxicity). In the 28 Day study, administration of the test item to animals of either sex at dose levels of up to 1000 mg/kg bw/day was well tolerated. A high dose level of 1000 mg/kg bw/day was therefore considered to be suitable for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER: Thyroid Hormone Analysis
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.
All adult males at termination.
All adult females at termination.
All samples were dispatched to the Test Site where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator.
Estrous cyclicity (parental animals):
Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.
Vaginal smears were taken daily throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
After necropsy, detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data) (see deviations from study plan)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations)
One male and one female offspring from each litter at Day 13 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
- Maternal animals: All surviving animals. Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

GROSS NECROPSY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
The following organs were dissected free from fat and weighed before fixation from all animals (where applicable) from each dose group.
Cowpers glands
Epididymides
Glans penis
LABC (levator ani-bulbocavernous) muscle
Testes
Thyroid (weighed post-fixation with Parathyroid)

Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides ♦
Glans penis
LABC (levator ani-bulbocavernous) muscle
Mammary gland (females only)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulating gland)
Testes ♦
Thyroid/Parathyroid
Uterus/Cervix
Vagina
♦ Preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy, and any gross lesions were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
At the request of the Sponsor, thyroid/parathyroid from control and high dose adult females were routinely processed to paraffin wax, sectioned and stained with Hematoxylin and Eosin, and examined.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS NECROPSY
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.
Statistics:
Due to limitations of this free-text field, please see "Any other information on materials and methods"
Reproductive indices:
Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) * 100
Pregnancy Index (%) = (Number of pregnant females / number of animals mated) * 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of animals delivering live offspring / Number of pregnant females) * 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean post-implantation loss was calculated for each female/litter as follows:
Post-implantation loss (%) = ((Number of implantation sites – Total number of offspring born) / Number of implantation sites) * 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) * 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) * 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) * 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula: (Number of male offspring / Total number of offspring) * 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs considered to be related to the toxicity of the test item.
At 1000 mg/kg bw/day, animals of either sex showed sporadic instances of increased post-dose salivation from Days 2 (females) or 3 (males) which persisted towards the end of the dosing period. Such observations are common in this type of study and may reflect an irritant nature of the test item and/or formulation and as such are considered to be of no toxicological significance.
One female each from the 100 or 300 mg/kg bw/day dose groups showed clinical signs of noisy respiration on Days 22 and 4, respectively. These observations may be due to the dosing procedure and in the absence of similar findings in the remaining animals, they were considered unlikely to be related to treatment with the test item.
There were no clinical signs for any other animals throughout the treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 100 mg/kg bw/day, Female 47 was killed in extremis on Day 1 post partum (Day 41 relative to the start of dosing). Prior to death, clinical signs for this animal included decreased respiratory rate, ataxia, wet fur and increased post-dose salivation but there were no macroscopic observations at necropsy. In the absence of any premature decedents in the 300 or 1000 mg/kg bw/day dose groups, this death was considered unlikely to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment with the test item on body weight development in animals of either sex.
At all dose levels, males occasionally showed marginally higher body weight gains than controls which resulted in slightly higher overall body weight gains for these animals. There was generally no dose-dependence and the intergroup differences were not statistically significant. It is worth noting that a body weight increase of this nature in this type of study is generally considered to be of no toxicological significance.
During the pre-pairing phase of the study, females treated with the test item at all dose levels also showed slightly higher body weight gains than controls achieving statistical significance for the 1000 mg/kg bw/day females during the second week (p<0.05). During the gestation period, group mean body weight gains in the test item-treated females were slightly lower than controls resulting in slightly lower overall body weight gains across these dose groups. The intergroup differences were, however, neither strictly dose-related nor they achieved statistical significance and may be due to the slightly lower litter size, i.e. less fetuses, for these females and as such were considered not to be of any toxicological importance. During the first week of lactation, group mean body weight gains in test item-treated females remained lower than controls but there was no dose-relationship and statistical significance was only achieved for the 100 and 300 mg/kg bw/day females over Days 1 to 4 (p<0.05). This resulted in statistically significantly lower overall body weight gains for females treated with 300 or 1000 mg/kg bw/day over the first week of lactation (p<0.05), but without depicting any dose-dependence. Subsequent improvement was evident and group mean body weight gains for test item-treated females during the second week of lactation were comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At all dose levels, dietary intake for the males was comparable with controls throughout the study. The corresponding females also showed similar food consumption to controls during pre-pairing and early gestation. Over Days 14 to 20 of gestation, food intake for females receiving 100 or 300 mg/kg bw/day was slightly lower than controls (p<0.05) but a dose-relationship was not evident and the corresponding values in females from the 1000 mg/kg bw/day dose group was similar to controls. Fluctuations in dietary intake were also evident during the lactation phase of the study attaining occasional statistical significance for the 100 or 300 mg/kg bw/day females (p<0.01-0.05) but the corresponding values for the 1000 mg/kg bw/day dose group were comparable with controls.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item on food conversion efficiency (where calculated) for animals of either sex receiving the test item. Any fluctuations were deemed to be reflective of minor intergroup differences in body weight gain and/or food consumption.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any overt differences for animals of either sex receiving the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis: An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No adverse effect ovserved.
At all dose levels, group mean absolute and body weight-related organ weights in males were comparable with controls.
Females treated with 1000 mg/kg bw/day showed slightly but statistically significantly higher absolute and body weight-relative thyroid weights in relation to controls. A small number (4/11) of absolute values exceeded the historical control data ranges. As there were no treatment-related occurrences in the relevant histopathological examination and thyroid hormone analysis, this finding was considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings at any dose level.
Incidental findings included small right seminal vesicle in one male each from the control and 100 mg/kg bw/day dose groups with another male from the latter dose group showing small left testis/epididymis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities at any dose level.
All of the histopathological findings encountered were considered to have occurred spontaneously or post mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.
Histopathological findings: neoplastic:
not examined
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study.
There were also no intergroup differences in the stage of estrous cycle on the day of necropsy.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating: There was no effect of treatment on mating performance. With the exception of one female from the 100 mg/kg bw/day dose group which mated on Day 14 after pairing, the remaining animals mated within four days after pairing.
Fertility: Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female each from the control, 100 and 300 mg/kg bw/day dose groups (Females 18, 39 and 68, respectively) and two females from the 1000 mg/kg bw/day dose group (Females 92 and 95) did not achieve pregnancy following positive evidence of mating. Male 27 (paired with Female 39) had severe unilateral tubular atrophy and a subsequent unilateral aspermia in the epididymis which may have prevented pregnancy. There were no histopathological changes in the tissues examined to account for non-pregnancy in the remaining animals and these non-pregnancies were deemed to be due to biological variation.
Gestation length: Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for test item-treated females appeared to be similar to controls.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (estrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs detected in pups from all treated groups included small size, cold, weak, no milk in the stomach, physical injury, found dead or missing. Females 23 (control), 41 and 45 (100 mg/kg bw/day), 70 (300 mg/kg bw/day) and 88 (1000 mg/kg bw/day) showed total litter losses. Such findings are often seen in this type of study and were considered unlikely to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Litter Responses: In total, eleven females each from the control, 100 and 300 mg/kg bw/day dose groups and ten females from the 1000 mg/kg bw/day dose group gave birth to a live litter. One litter each was lost in the control, 300 and 1000 mg/kg bw/day dose groups during the lactation phase of the study with the 100 mg/kg bw/day dose group showing two litter losses. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.
Offspring Litter Size, Sex Ratio and Viability: There was considered to be no adverse effect of treatment with the test item at any dose level on the mean number of implantations and post-implantation losses. Of the litters born, there was also no adverse effect of treatment with the maternal treatment on litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum indicating the lack of any adverse effect on offspring viability.
The mean number of implantation sites in females treated with the test item up to a dose level of 1000 mg/kg bw/day was slightly lower than controls. The majority of individual values were within the historical control data ranges and the intergroup differences were neither dose-related nor they attained statistical significance. Histopathological examination of the ovaries from the 1000 mg/kg bw/day did not identify any treatment-related findings and this observation was considered likely to be due to biological variation. Group mean post-implantation losses across all dose groups including controls were comparable, but due to the slightly lower number of implantation sites, litter size at birth and on subsequent days in the test item-treated dose groups remained lower than controls. These intergroup differences did not achieve statistical significance and were not dose-dependent, and as such were considered to be of no toxicological relevance.
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was considered to be no adverse effect of treatment with the test item on offspring body weights and body weight development.
At birth, group mean offspring body weights across all test item-treated dose groups were comparable with controls. Group mean offspring body weight gains from the 100 and 300 mg/kg bw/day dose groups remained slightly lower than controls over Days 1 to 7 of lactation which resulted in lower cumulative body weight gains for these pups, but improvement was noted over Days 7 to 13 of lactation. Consequently, the mean body weights on Day 4 and thereafter for these offspring were slightly lower than controls. It is worth noting that corresponding values for the 1000 mg/kg bw/day dose group were generally similar to controls and none of these intergroup differences achieved statistical significance. Due to the slightly lower litter size and lower body weight gains, litter weights for the 100 and 300 mg/kg bw/day dose groups remained lower than controls attaining statistical significance on Day 13 post partum for the latter. The corresponding values for the 1000 mg/kg bw/day dose group were, however, generally similar to controls, and these intergroup differences were considered not to be of any toxicological relevance.
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):
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The study was conducted under GLP according to OECD guideline 421 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the reproductive toxicity of the test item in rats (screening study). The oral (gavage) administration of the test item to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study also includes some endocrine disruptor relevant endpoints. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 28 July 2015).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Propylene Glycol).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male:one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance and visible nipple count (male offspring only).

Blood samples were taken at termination from all surviving adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and 1000 mg/kg bw/day dose groups was performed.

 

Results

Adult Responses

Mortality: There were no treatment-related deaths on the study.

Clinical Observations: There were no clinical signs considered to be related to the toxicity of the test item.

Body Weight: There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the dose administration period.

Food Consumption: There was no effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the treatment period.

Water Consumption: Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

Reproductive Performance

Mating: There was no effect of treatment with the test item on mating performance.

Fertility: There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Length: There were no treatment-related differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: There was no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development: There was no adverse effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1 post partum, visible nipple count in male offspring on Day 13 post partum or clinical signs up to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Pathology

Necropsy: Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any adverse effects up to a dose level of 1000 mg/kg bw/day.

Organ Weights: No adverse effects were detected in animals of either sex at any dose level.

Histopathology: Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

 

Conclusion

The oral (gavage) administration of4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12Hdibenzo[d,g][1,3,2]dioxaphosphocinto Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.422: "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
n/a

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST strain
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks (only P generation)
- Weight at study initiation: At the start of treatment the males weighed 310 to 357g, the females weighed 190 to 224g. (only P generation)
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Rodent Facility.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.), ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage, ad libitum.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES:
From: 28 January 2016 (first day of treatment)
To: 14 March 2016 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 25, 75, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml / kg bw
Details on mating procedure:
- M/F ratio per cage: one male: one female
- Length of cohabitation: maximum of fourteen days.
- Proof of pregnancy: Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages.
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by laboratories' Analytical Services. Results show the formulations to be stable for up to 21 days. Formulations were initially prepared weekly and following further stability fortnightly and stored at approximately 4 °C in the dark.
Samples of test item formulation were taken and analyzed on three occasions for concentration of the test item at Analytical Services. The results indicate that the prepared formulations were within 10% of the nominal concentration.
Duration of treatment / exposure:
43 or 44 days (males)
until day 5 post partum (females)
Frequency of treatment:
daily
Details on study schedule:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females from the control, low and intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from the control, low and intermediate dose groups and from the high dose group, four females with litters and four females that showed a total litter loss for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary range-finding study, in accordance with OECD TG 422
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week and at weekends (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on five males selected from each test and control group prior to termination (Day 42 for males) and five selected females from the control, low or intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. (Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: No
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb) Erythrocyte count (RBC) Hematocrit (Hct)
Erythrocyte indices:
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic):
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by 'Innovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See above
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males from each dose level and five selected females treated with 100 and 300 mg/kg bw/day, prior to termination, together with an assessment of sensory reactivity to various stimuli. Functional performance tests and sensory reactivity assessment was also performed on four pregnant females retaining offspring to Day 4 post partum and females showing a total litter loss at 1000 mg/kg bw/day.
- Battery of functions tested: sensory activity / grip strength / motor activity
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalization, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

IMMUNOLOGY: No
Estrous cyclicity (parental animals):
Checked for normal cycling
Sperm parameters (parental animals):
Parameters examined in only P male parental generations: testis weight, epididymis weight, other: qualitative examination of the stages of spermatogenesis in the testes
Litter observations:
STANDARDISATION OF LITTERS
not required

PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
All live offspring were assessed for surface righting reflex on Day 1 post partum

GROSS EXAMINATION OF DEAD PUPS:
Yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 43 or 44
- Maternal animals: All surviving animals on day 5 post partum

GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males from each dose group and five selected females from the control, low and intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. Some tissues were weighed from all remaining animals(A):
Adrenals, Brain, Epididymides(A), Heart, Kidneys, Liver, Ovaries(A), Pituitary (post-fixation)(A), Prostate and Seminal Vesicles(A), Spleen, Testes(A), Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)(A)

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group. For females treated with 1000 mg/kg bw/day four females with litters and four females showing total litter loss and all tissues preserved in buffered 10% formalin, except where stated. Some tissues were preserved from all remaining animals (A):
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland(A)
Colon
Duodenum
Epididymides(A) •
Esophagus Eyes *
Gross lesions(A)
Heart
Ileum (including peyer's patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland(A)
Muscle (skeletal)
Ovaries(A)
Pancreas
Pituitary(A)
Prostate(A)
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles(A)
Skin
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes(A) •
Thyroid/Parathyroid
Trachea
Thymus
Urinary bladder
Uterus & Cervix(A)
Vagina(A)

* Eyes fixed in Davidson's fluid
• preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Postmortem examinations (offspring):
SACRIFICE
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Due to limitations of this free-text field, see "any other information on materials and methods".
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) * 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) * 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) * 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) * 100
Post-implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) * 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) * 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) * 100
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
(Number of male offspring / Total number of offspring) * 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity for males or surviving females treated with 100, 300 or 1000 mg/kg bw/day.
One control male and one male treated with 1000 mg/kg bw/day were observed to have open wounds which later formed scabs between Days 20 to 33 and 17 to 33, respectively. An additional male treated with 1000 mg/kg bw/day showed signs of a swollen hind limb from Day 37 onwards. These observations were considered to be physical injuries and not related to treatment.
One male, each treated with 300 or 100 mg/kg bw/day had signs of noisy respiration on Days 1 to 5 and Day 8 (respectively). In the absence of a similar effect at 1000 mg/kg bw/day, these observations were considered to be incidental and of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum. There were no clinical signs of toxicity prior to death and the macroscopic findings included an enlarged spleen and liver together with red colored fluid in the thoracic cavity. It was noted this female had a long gestation period of 24 days.
One female treated with 1000 mg/kg bw/day was killed in extremis due to showing symptoms of difficulty during parturition; clinical signs for this animal included labored respiration, decreased respiratory rate, pilo-erection, lethargy, hypothermia, hunched posture, pallor of the extremities and dehydration. The macroscopic findings included a pale brain, liver, mammary gland and pancreas and the small and large intestine (caecum, colon, duodenum, ileum and jejunum) had black colored contents. It was noted that this female would have had a gestation length of 24 days.
There were no further unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day throughout the treatment period.
Fluctuations in weekly group mean body weight gains were evident in treated males, in females treated with 1000 mg/kg bw/day during maturation and in females treated with 100 mg/kg bw/day during lactation, achieving statistical significance. There was generally no dose-dependance and, on a number of occasions group mean values for treated animals exceeded controls, therefore the intergroup differences were considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no adverse effects on food consumption or food conversion efficiency for males throughout the study or for females during pre-mating, gestation or lactation at dose levels of 100, 300 or 1000 mg/kg bw/day.
During gestation, treated females showed an increase in food intake throughout this phase, attaining statistical significance for females treated with 300 and 1000 mg/kg bw/day. An increase in food consumption is generally considered not to represent an adverse effect of treatment. Food intake for females during lactation was generally similar, however females treated with 100 mg/kg bw/day showed a statistically significant reduction between Days 1 and 4 post partum. In the absence of a dose related response the intergroup difference was considered of no toxicological significance.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period.
There was a significant increase in water consumption for animals of either sex from all treatment groups throughout the study for males and for females during pre-pairing, gestation and lactation phases. During pre-pairing, the overall increase in water intake for males was 15%, 15% and 34% (100, 300 and 1000 mg/kg bw/day, respectivity) and for females the overall increase was 25%, 59% and 73% (100, 300 and 1000 mg/kg bw/day, respectively). A statistically significant increase in water consumption was only evident in females treated with 1000 and 300 mg/kg bw/day throughout gestation and lactation.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treated males from all dose groups showed a statistically significant reduction in hemoglobin, hematocrit and erythrocyte count; a dose relationship was present in all cases. With the exception of one male from the high dose group, all individual values were within the background control ranges. However, these findings may be associated with the histopathological findings of hemopoiesis in the liver and spleen.
Females treated with 1000 mg/kg bw/day showed a reduction in neutrophil counts when compared to controls, attaining statistical significance. There was no dose relationship present and individual values were within the background control ranges.
There were no toxicologically significant effects detected in females treated with 300 or 100 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed a statistically significant increase in monocytes. Although the majority of the individual values were above the background control ranges no such effects were evident in 1000 mg/kg bw/day males or in corresponding females. Therefore in the absence of a true-dose related response the intergroup difference was considered of no toxicological significance.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males from all treatment groups showed statistically significant increases in cholesterol attaining statistical significance in all cases, without a dose related response. The majority of individual values were within the background control ranges however in view of the microscopic changes evident in the liver, an association to treatment cannot be excluded.
Females treated with 1000 or 300 mg/kg bw/day showed statistically significant increases in total protein. A dose relationship was evident but individual values were all within the background control ranges. However in view of the microscopic changes in the liver, an association to treatment cannot be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related effects detected.
Functional Performance Tests: There were no treatment-related changes in functional performance.
At 1000 mg/kg bw/day females showed a statistically significant increase in hind limb grip strength whilst females from all treatment groups showed a statistically significant increase in fore limb grip strength. Due to a lack of consistency and no dose relationship, these findings were considered to be incidental. At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.
At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver: Animals of either sex from all treatment groups showed a dose related increase in liver weight both absolute and relative to terminal body weight, attaining statistical significance, excluding females treated with 100 mg/kg bw/day. This can be associated with the centrilobular hepatocellular hypertrophy in these animals.
Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight with a dose related response. This may be associated with the histopathological findings identified.
Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weight both absolute and relative to terminal body weight with a dose related response.
Thyroid/Parathyroid: Animals of either sex showed a statistically significant increase in thyroid/parathyroid weights in both absolute and relative to terminal body weight. Females receiving 100 mg/kg bw/day did not attain statistical significance
Spleen: Females from all treatment groups showed an increase in spleen weights both absolute and relative to terminal body weight. Although a true dose response was not evident and statistical significance was only achieved at 300 mg/kg bw/day, in view of the histopathological changes identified, an association to treatment can not be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Early decedents
One female treated with 300 mg/kg bw/day that was found dead on Day 3 post partum showed an enlarged liver and spleen and a fluid filled thoracic cavity.
One female treated with 1000 mg/kg bw/day that was killed in extremis during parturition had black coloured contents in the caecum, colon, duodenum, ileum and jejunum. The brain, liver, mammary gland and pancreas were also pale.
Scheduled necropsy
At 1000 mg/kg bw/day seven males had enlarged livers, one of which was also mottled. A further one male (No.74) had a small prostate and seminal vesicles. Three females from this dose group and two females treated with 300 mg/kg bw/day had enlarged spleens.
One male (No.30) treated with 100 mg/kg bw/day was observed to have small epididymides and the testes were noted to be small and flaccid. This male did not produce a pregnancy in its female partner. One female at this level was noted to have fluid filled uterus and cervix. In the absence of a similar effect at 1000 mg/kg bw/day, these intergroup differences were considered to be incidental.
There were a number of animals of either sex observed to have reddened lungs including control animals. This was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Centrilobular hepatocellular hypertrophy, moderate or mild was present in all males treated with 1000 mg/kg bw/day and in eight females from this treatment group. In both sexes treated with 100 or 300 mg/kg bw/day centribular hepatocellular hypertrophy was evident from minimal to mild and showed a dose dependant trend.
Diffuse vacuolation (fat type), minimal or mild was present in 4/8 males and 5/9 females at 1000 mg/kg bw/day with periportal vacuolation being evident in another female. It was also present at a minimal level in both sexes at 100 or 300 mg/kg bw/day. Hematopoiesis was present in 3/9 1000 mg/kg bw/day females; 4/5 100 mg/kg bw/day females and 4/6 300 mg/kg bw/day females. The severity was increased at 300 mg/kg bw/day compared to 100 mg/kg bw/day.
Thyroid Gland: Mild follicular cell hypertrophy was present in all males and 8/9 females treated with 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day it was present at minimal or mild levels in males and females.
Pituitary: There was a minor increase in the amount of vacuolation in pars distalis (anterior lobe) of the pituitary gland in 1000 mg/kg bw/day males. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.
Spleen: Increased hematopoiesis was present in 7/9 1000 mg/kg bw/day females (mild to marked); 6/6 300 mg/kg bw/day females (mild to marked) and in 4/5 100 mg/kg bw/day females (mild or moderate).
Thymus: There was a reduction in the amount of involution noted in treated females from all dose groups when compared to control animals. Involution/atrophy is seen as a normal background change in pregnant and lactating animals therefore not generally recorded thus this may be linked to lower numbers of animals producing and feeding litters.
Lungs: There was a minor increase in the incidence of alveolar macrophage accumulation (focal) in the lungs of females at 1000 mg/kg bw/day only.
Kidneys: Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment.
Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change.

Non-Pregnancy:
A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.
Total Litter Loss: In total, 1 (No. 37), 5 (No. 61, 64, 68, 69 and 71) and 4 (No. 86, 88, 89 and 96) females showed total litter loss from 100, 300 and 1000 mg/kg bw/day dose groups, respectively. A further one female (No. 87) showed evidence of a pregnancy but no litter was observed. Histologically other than the general findings indicated above the only notable differences between control and treated females were fewer animals lactating (linked to lack of live pups) and 3 without implantation sites on the section considered to be linked to the reduction noted.
Premature Decedents: Female 64 had an enlarged liver and spleen, plus a fluid filled thorax at necropsy. There were numerous histopathological changes and septicemia was present. This is considered to be the cause of death. Although the initial cause is not clear it is likely to have originated in the uterus (degeneration was present) and this may have been a complication of pregnancy and parturition. Therefore, this is considered not to be related to treatment. Female 91 was killed due to issues with parturition and showed non-specific changes; most were considered to be agonal and its death could not be unequivocally related to treatment.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Description (incidence and severity):
A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating: No treatment-related effects were detected in mating performance.
One female (No.90) showed evidence of mating on Day 10 of pairing, but subsequently gave birth earlier than expected. With litter parturition dates and with some evidence of mating on Day 1 of pairing it can be assumed that this was when this female mated. A further female (No.94) failed to show any signs of mating but subsequently gave birth to live offspring. With the exception of these two females, the remaining females mated within four days of pairing (i.e at the first estrus opportunity).
Fertility: There were 0, 4, 1 and 1 mated females that failed to achieve pregnancy in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. The pregnancy rate at 100 mg/kg bw/day was lower than anticipated but, in the absence of any dose-response relationship and any similar findings at higher dosages, this was considered to be incidental and unrelated to treatment.
Gestation length: While gestation lengths observed during the study (between 22 and 24 days) were within the expected range, the distribution of gestation lengths suggested a slight, but statistically significant, shift towards longer gestation length at 300 and 1000 mg/kg bw/day. The anticipated gestation lengths of the early decedent females were both 24 days and together with the process of parturition may have contributed to the deterioration in health.
Litter Responses: There were a total of 12, 8, 11 and 11 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. At 1000 mg/kg bw/day, one pregnant female was not observed to give birth to a litter and a further female was killed in extremis during late gestation due to signs of parturition difficulty; live born offspring from this litter were also euthanized on the same day. In total there were 1, 5, and 4 littering females at 100, 300 and 1000 mg/kg bw/day that subsequently failed to successfully rear their offspring to Day 5 of age. The following assessments of litter response is generally based on the 12, 7, 6 and 5 litters successfully reared to Day 5, although data from all females with live offspring at the point of observation was considered where appropriate.
The mean number of corpora lutea for pregnant females was lower than control for all dosage groups; however, the mean values for treated animals were all similar, showing no dosage relationship and failed to attain statistical significance. A comparison of individual values showed 3, 5 and 4 females with corpora lutea counts less than ten compared to only one in the control. When mean values were calculated using only females that successfully reared offspring to Day 5 of age, mean values for corpora lutea at both 100 and 300 mg/kg bw/day were only slightly lower than control. However mean values at 1000 mg/kg bw/day remained clearly lower than control, although differences for all dosage groups failed to attain statistical significance.
At 1000 mg/kg bw/day, mean pre-implantation loss was higher than control for females that successfully reared offspring to Day 5 of age and, to a lesser extent, for all pregnant females of this dose group, although differences failed to attain statistical significance. However these differences from control were primarily influenced by one female (number 90) that showed particularly high pre-implantation loss. Only one other female showed pre-implantation loss at this dosage and this finding was, therefore, considered most likely to be incidental and unrelated to treatment.
At 100 and 300 mg/kg bw/day, differences from control in mean pre-implantation loss for pregnant females and females that successfully reared offspring to Day 5 of age showed no dosage relationship and were considered to reflect normal biological variation.
Despite the number of implantations for treated females being lower than control, mean post-implantation loss was higher than control for littering females and, to a greater extent, females that successfully reared offspring to Day 5 of age at 100, 300 and 1000 mg/kg bw/day. These differences from control did not attain statistical significance. Additionally at 1000 mg/kg bw/day, one pregnant female was not observed to give birth and may represent a total litter loss in utero. This was confirmed by the presence of implantation sites. There was a lower mean litter size at birth at all dosage groups compared to control for both sets of females, although group mean values for all treated groups were similar.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
reproductive performance
other: based on the severity and adaptive response nature of the findings rather than an absence of findings.

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
immune system
Organ:
spleen
thymus
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Surface righting reflex on Day 1 of age appeared unaffected by maternal treatment at 100, 300 and 1000 mg/kg bw/day.
There was a higher incidence of clinical signs (small, cold, weak and no milk visible in stomach) and increased offspring mortality indicating that the offspring were not thriving at the dosages of 300 and 1000 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, offspring viability on Day 1 was slightly lower than control for littering females but not for females that successfully reared offspring to Day 5 of age. This was due to one female that showed total litter loss at or shortly after birth; only one other female at this dosage showed offspring mortality at that time. At 100 and 1000 mg/kg bw/day no pup mortality was observed between birth and Day 1 of age. One 1000 mg/kg bw/day female showed evidence of pregnancy but there was no litter observed. It can be assumed that this female had a total litter loss shortly after birth. Overall there was considered to be no clear effect on offspring survival around the time of parturition at 100, 300 or 1000 mg/kg bw/day.
At 300 and 1000 mg/kg bw/day, there was a clear decrease in mean offspring viability between Days 1 to 4 of age resulting in a further lowering of litter size on Day 4 of age compared to control for females that successfully reared offspring to Day 5 of age. Differences from control for Day 4 litter size attained statistical significance. The lower offspring viability was more pronounced when litters for females that showed total litter loss between Days 1 and 4 of lactation was included; with the differences from control attaining statistical significance.
At 100 mg/kg bw/day, mean offspring viability between Days 1 to 4 of age for females that successfully reared offspring to Day 5 of age was similar to control. The lower litter size at Day 4 at this dosage was considered to reflect differences already established prior to this time point. One female at this dosage did show total litter loss between Days 1 and 4 of lactation. This female only had a litter size of two. As it is not particularly unusual for such small litters not to be maintained by the female and mortality rate among remaining litters was very low (only one offspring died from birth to day 4 of age), this single litter loss cannot be attributed to the administration of the treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring body weights for each sex on Day 1 appeared unaffected by maternal treatment at 100, 300 and 1000 mg/kg bw/day. These initial body weights were slightly higher than control; probably reflecting the lower mean litter size at these dosage and, at 300 and 1000 mg/kg bw/day, the slightly longer gestation period. Mean litter weights on Day 1 were lower than control at 100, 300 and 1000 mg/kg bw/day as a consequence of the lower litter size at these dosages compared to control while the reduction in litter weights on Day 1 post partum did not attain statistical significance. Surface righting reflex on Day 1 of age appeared unaffected by maternal treatment at 100, 300 and 1000 mg/kg bw/day.
At 300 and 1000 mg/kg bw/day subsequent offspring body weight gain to Day 4 of age was statistically significantly lower than control, resulting in slightly lower mean body weight on Day 4, although differences from control fail to attain statistical significance. There was a higher incidence of clinical signs (small, cold, weak and no milk visible in stomach) and increased offspring mortality indicating that the offspring were not thriving at these dosages. Mean litter weights on Day 4 were statistically significantly lower than control at these dosages reflecting the lower weight gain and lower litter size.
At 100 mg/kg bw/day body weight gain to Day 4 of age was slightly lower than control; however differences fail to attain statistical significance and resulting mean body weight on Day 4 remained similar to control. The incidence of clinical signs was low and did not indicate any effect of maternal treatment. Mean litter weight on Day 4 was statistically significantly lower than control reflecting the lower litter size at this dosage.
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:
effects observed, treatment-related
Description (incidence and severity):
There were higher instances of small, weak and no milk in stomach in offspring from litters of the high and intermediate dose groups.
One male from a control litter had bruising on the dorsal and ventral surface and one female from a 300 mg/kg bw/day litter had scab formation on the left hind limb. These findings were considered to be incidental and a result of physical injuries.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, sex ratio was lower than control for litters derived from females that successfully reared offspring from birth to Day 4 of age, with differences attaining statistical significance. No similar difference in sex ratio was apparent at birth for those litters derived from littering females on Day 1. While these differences may suggest a selective effect on offspring sex distribution or survival, in the absence of any similar finding at 1000 mg/kg bw/day, this finding was considered most likely to be incidental and unrelated to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
body weight and weight gain
gross pathology
Remarks on result:
other: based on the severity and adaptive response nature of the findings rather than an absence of findings

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the reproductive effects in rats (repeated dose oral toxicity effects are discussed in the respective section).
In total 12, 8, 11 and 11 females were found to be pregnant from the control, 100, 300 and 1000 mg/kg bw/day, respectively and no relationship with infertility to the substance treatment was established. For the control, 100, 300 and 1000 mg/kg bw/day groups, 12, 7, 6 and 5 litters were maintained to Day 5. A treatment related adverse effect on litter maintenance/offspring survival was present at 300 and 1000 mg/kg bw/day.
For all dose groups, group mean values for corpora lutea and implantations were lower when compared to controls, with no dose relationship observed and no statistical significance attained.
There was a statistically significant reduction in viable offspring from litters from 300 or 1000 mg/kg bw/day dose groups on Day 4 post partum as a number of females from these dose groups showed total litter loss prior to Day 5 post partum.
The gestation length for females receiving dosages greater than 300 mg/kg bw/day was statistically significantly longer which may account for the increased Day 1 post partum body weights. The initial body weights were slightly higher from all dose groups in relation to controls which would reflect the lower mean litter size at all dose groups. All treatment groups showed generally lower body weight changes between Days 1 to 4 post partum when compared to controls, statistical significance was attained for male and female offspring from 300 or 1000 mg/kg bw/day litters. There was a higher incidence of offspring that were missing or found dead, no milk in stomach, weak and smaller offspring from litters in the 1000 and 300 mg/kg bw/day dose groups.
All individual values on pre- and post-implantation loss were within the background control ranges and no dose relationship was present for mean values for either parameter. Taking into account of these factors, these effects were considered not to be an effect of treatment.
Offspring of either sex showed a lower group mean body weight change for 100 mg/kg bw/day litters compared to controls, however, individual body weights for offspring of either sex were within the historical background control ranges for offspring of Day 4 post partum age and therefore considered to be biological variation and not treatment related.
So it can be concluded that there is an effect on reproductive performance and offspring development. However, there was also maternal toxicity observed at the same dose levels. E.g., predominantly for the 1000 and 300 mg/kg bw/day dose groups microscopic examinations revealed treatment related changes in the liver, thyroid, pituitary, spleen, thymus, lungs and kidneys. The minimal centribular hepatocellular hypertrophy, minimal follicular cell hypertrophy in the thyroid gland and the mild increased hematopoiesis observed in the 100 mg/kg bw/day dose group were regarded as adaptive responses. In consequence, the No Observed Adverse Effect Level (NOAEL) for both systemic toxicity and reproductive toxicity was considered to be 100 mg/kg bw/day, based on the severity and adaptive response nature of the findings rather than an absence of findings.
According to Regulation 1272/2008, Table 3.7.1(a), Hazard categories for reproductive toxicants, „Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, … Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
In consequence, the test item does not need to be classified as reproductive toxicant.
Executive summary:

Introduction

The present GLP study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations, hematology and blood chemistry evaluations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from the control, low and intermediate dose groups on Day 4 post partum. Due to a high number of total litter losses evident in the high dosage group, four females with litters and four females with a total litter loss were investigated to allow comparison between the two sets of females which were in a different physiological state on Day 4 post partum.

Adult males were terminated on Days 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Mortality: One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum and one female treated with 1000 mg/kg bw/day was killed in extremis due to difficulty during parturition. There were no further unscheduled deaths.

Clinical Observations: There were no clinical signs of toxicity for males or the surviving females treated with 100, 300 or 1000 mg/kg bw/day.

Behavioral Assessment: There were no treatment-related effects detected.

Functional Performance Tests: There were no treatment-related changes in the functional performance.

Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

Body Weight: There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption: There were no adverse effects on food consumption or food conversion efficiency for males throughout the study or for females during pre-mating, gestation or lactation at dose levels of 100, 300 or 1000 mg/kg bw/day.

Water Consumption: Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period. An increase in water consumption was detected for animals of either sex from all treatment groups for the majority of the treatment period.

 

Reproductive Performance

Mating: No treatment-related effects were detected in mating performance. The majority of females showed evidence of mating within four days of pairing (i.e at the first estrus opportunity).

Fertility: Four low (100 mg/kg bw/day), one intermediate (300 mg/kg bw/day) and one high (1000 mg/kg bw/day) dose females (No's 40, 42, 44, 47, 67 and 95, respectively) were found to be non-pregnant following positive evidence of mating. A dose relationship was not established.

Gestation Lengths: The distribution of gestation length was between 22 and 24 days.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: The females from all dose groups receiving the test item showed a reduction in corpora lutea, implantation sites and litter size when compared to controls while no dosage relationship was noted and no statistical significance was attained. These females also showed an increase in pre and post implantation loss with no dose relationship. At 300 mg/kg bw/day, the sex ratio (% male pups) for this group was statistically significantly lower than controls.

A number of females showed total litter loss prior to Day 5 post partum; with one, five and four females treated with 100, 300 or 1000 mg/kg bw/day, respectively, showing total litter losses while the single litter loss in the low dose group cannot be attributed to the substance treatment with statistical significance.

The viability of the litters were generally similar to controls on Day 1 however, on Day 4 post partum litters from females receiving 300 or 1000 mg/kg bw/day the offspring viability was statistically significantly lower when compared to controls.

Offspring Growth and Development: On Days 1 post partum the offspring body weights from the treated female litters were generally exceeded when compared to controls and on Day 4 post partum there was a reduction in the remaining offspring body weights and litter weights when compared to controls. Body weight gain over Days 1 to 4 post partum for the surviving pups across all treatment groups was generally lower in relation to controls and a dose relationship between the intermediate and high dose group for the offspring body weights or body weight changes.

There was no effect on surface righting reflex on Day 1 post partum.

There was a higher incidence of offspring going missing, found dead, no milk in stomach, weak and small from litters in the 1000 and 300 mg/kg bw/day dose groups. There were no macroscopic abnormalities detected at terminal kill.

 

Laboratory Investigations

Hematology: Treated males from all groups showed a statistically significant reduction in hemoglobin, hematocrit and erythrocyte count; a dose relationship was present in all cases. With the exception of one male from the high dose group, all individual values were within the background control ranges. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in neutrophil count when compared to controls attaining statistical significance. No toxicologically significant effects were detected in females treated with 300 or 100 mg/kg bw/day.

Blood Chemistry: Males from all dose groups showed a statistically significant increase in cholesterol when compared to controls but without a dose relationship. At 1000 or 300 mg/kg bw/day females showed a statistically significant increase in total protein when compared to controls with a dose related response.

 

Pathology

Necropsy:

Early decedents: One female treated with 300 mg/kg bw/day that was found dead on Day 3 post partum showed an enlarged liver and spleen and a fluid filled thoracic cavity. A further female treated with 1000 mg/kg bw/day that was killed in extremis during parturition had black coloured contents in the caecum, colon, duodenum, ileum and jejunum. The brain, liver, mammary gland and pancreas were also pale.

Scheduled necropsy : At 1000 mg/kg bw/day seven males had enlarged livers of with one of these appearing mottled. Three females from this dose group and two females treated with 300 mg/kg bw/day were also observed with enlarged spleens. There were higher instances of small, weak and no milk in the stomach in offspring from litters of the high and intermediate dose groups.

 

Organ Weights

Liver: Animals of either sex from all treatment groups showed a dose related increase in liver weight both absolute and relative to terminal body weight. Statistical significance was achieved excluding females treated with 100 mg/kg bw/day.

Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increase in kidney weights both absolute and relative to terminal body weight with a dose related response.

Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weights both absolute and relative to terminal body weight with a dose related response.

Thyroid/Parathyroid: Animals of either sex from all treatment groups showed statistically significant increases in thyroid/parathyroid weight both absolute and relative to terminal body weight. Females receiving 100 mg/kg bw/day did not attain statistical significance.

Spleen: Females from all treatment groups showed an increase in spleen weights both absolute and relative to terminal body weight.

 

Histopathology

Liver: Centrilobular hepatocellular hypertrophy, moderate or mild was present in all males treated with 1000 mg/kg bw/day and in eight females from this treatment group. Both sexes treated with 100 or 300 mg/kg bw/day showed from minimal to mild centrilobular hepatocellular hypertrophy and showed a dose dependant trend. Diffuse vacuolation (fat type), minimal or mild was present in 4/8 males and 5/9 females at 1000 mg/kg bw/day with periportal vacuolation being evident in another female. It was also present at a minimal level in both sexes at 100 or 300 mg/kg bw/day. Hematopoiesis was present in 3/9 1000 mg/kg bw/day females; 4/5 100 mg/kg bw/day females and 4/6 300 mg/kg bw/day females. The severity of hematopoiesis was increased at 300 mg/kg bw/day compared to 100 mg/kg bw/day.

Thyroid Gland: Mild follicular cell hypertrophy was present in all males and 8/9 females treated with 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day it was present at minimal or mild severity in males and females.

Pituitary: There was a minor increase in the amount of vacuolation in pars distalis (anterior lobe) of the pituitary gland in males at 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.

Spleen: Increased hematopoiesis was present in 7/9 females at 1000 mg/kg bw/day (mild to marked); 6/6 females at 300 mg/kg bw/day females (mild to marked) and in 4/5 females at 100 mg/kg bw/day (mild or moderate).

Thymus : There was a reduction in the amount of involution noted in treated females from all dose groups when compared to control animals. Involution/atrophy is seen as a normal background change in pregnant and lactating animals therefore not generally recorded thus this may be linked to lower numbers of animals producing and feeding litters.

Lungs: There was a minor increase in the incidence of alveolar macrophage accumulation (focal) in the lungs of females at 1000 mg/kg bw/day only.

Kidneys: Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment. Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change.

Non-Pregnancy: A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.

Total Litter Loss: Several animals in the study showed total litter loss. Histologically other than the general findings indicated above the only notable differences between control and treated females were fewer animals lactating (linked to lack of live pups) and 3 without implantation sites on the section considered to be linked to the reduction noted. There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

 

Conclusion

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproductive toxicity was considered to be 100 mg/kg bw/day, based on the severity and adaptive response nature of the findings rather than an absence of findings.