Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-[(5-amino-4-methylpentyl)amino]-3-(2-methylphenoxy)-propan-2-ol
Cas Number:
1617528-43-4
Molecular formula:
C16H28N2O2
IUPAC Name:
1-[(5-amino-4-methylpentyl)amino]-3-(2-methylphenoxy)-propan-2-ol
Constituent 2
Chemical structure
Reference substance name:
1,1'-[(2-Methyl-1,5-pentanediyl)diimino]bis[3-(2-methylphenoxy)-propan-2-ol]
Cas Number:
1617528-45-6
Molecular formula:
C26H40N2O4
IUPAC Name:
1,1'-[(2-Methyl-1,5-pentanediyl)diimino]bis[3-(2-methylphenoxy)-propan-2-ol]
impurity 1
Chemical structure
Reference substance name:
Mephenesin
EC Number:
200-427-4
EC Name:
Mephenesin
Cas Number:
59-47-2
Molecular formula:
C10H14O3
IUPAC Name:
3-(2-methylphenoxy)propane-1,2-diol
impurity 2
Chemical structure
Reference substance name:
1,3-bis(2-methylphenoxy)propan-2-ol
Cas Number:
17181-49-6
Molecular formula:
C17H20O3
IUPAC Name:
1,3-bis(2-methylphenoxy)propan-2-ol
impurity 3
Chemical structure
Reference substance name:
2-methylpentane-1,5-diamine
EC Number:
239-556-6
EC Name:
2-methylpentane-1,5-diamine
Cas Number:
15520-10-2
Molecular formula:
C6H16N2
IUPAC Name:
2-methylpentane-1,5-diamine
impurity 4
Chemical structure
Reference substance name:
3,3’-(5-(2-hydroxy-3-(o-tolyloxy)propylamino)-4-methylpentylazanediyl)bis(1-(o-tolyloxy)propan-2-ol)
Molecular formula:
C36H52N2O6
IUPAC Name:
3,3’-(5-(2-hydroxy-3-(o-tolyloxy)propylamino)-4-methylpentylazanediyl)bis(1-(o-tolyloxy)propan-2-ol)
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: Approximately 8 weeks on arrival, approximately 10-11 weeks at start of treatment
Females: Approximately 11 weeks on arrival, approximately 13-14 weeks at start of treatment
- Housing:
During the pre-pairing phase: in groups of three (non-recovery) or five (recovery) by sex in polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding
During the pairing phase (Non-Recovery): one male:one female basis within each dose group in polypropylene cages with stainless steel mesh lids and grid flooring suspended over polypropylene trays lined with absorbent paper.
Following the pairing phase (Non-Recovery): Males will be re-housed in their original pre-pairing cages. Females will be individually housed in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding for the gestation, birth and lactation phases of the study.
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item will be formulated in Polyethylene glycol 400. A known amount of test item will be added to an appropriate amount of vehicle and stirred/shaken well to give an acceptable solution at the required concentration. The method of preparation and the vehicle has already been shown to be appropriate as in previous toxicity work (Envigo study number: KP08NC).

VEHICLE
- Concentration in vehicle: 6.25, 18.75 and 37.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males: 44-45 d
females: Day 14 post partum (non-recovery animals); day 72 (recovery animals)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
reduced to 100 mg/kg bw/d in week to due to body weight losses
No. of animals per sex per dose:
12 (+ 5 animals in control and high dose for recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels are based on available toxicity data including a Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number: KP08NC).
- Rationale for selecting satellite groups: The recovery groups will be used for evaluation of reversibility of toxic findings but will not be used for evaluation of reproductive effects.
- Post-exposure recovery period in satellite groups: 28 d
Positive control:
no

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations will be performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. Recovery groups will be observed once daily during the treatment-free period.
- Mortality: Twice daily, early and late during the working period.

Detailed clinical observations will be performed on all non-recovery test and control group animals before the first exposure to the test item and approximately once weekly thereafter. Observations will also be performed on mated females on Day 4, 11 and 18 of presumed gestation and for littering females on Day 4 and 12 of lactation. These observations will be performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any effects of treatment are identified.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights will be recorded for non-recovery males on Day 1 and then weekly until termination. Individual body weights will also be recorded at terminal sacrifice.
For non-recovery females, individual body weights will be recorded on Day 1 and then weekly until pairing. During the pairing phase, females will be weighed daily until mating is confirmed (these data may be presented in the final report only if required). Mated females will be weighed on Day 0, 7, 14 and 20 post coitum and body weights for females which give birth will be recorded on Days 1, 4, 7 and 14 post partum. Body weights will also be recorded at terminal sacrifice.
Body weights for recovery group animals will be recorded on Day 1, then weekly throughout the treatment and recovery period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Non-recovery male dietary intake will be recorded weekly prior to and after pairing. Non-recovery female dietary intake will be recorded weekly prior to pairing. Weekly food efficiency (body weight gain/food intake) will be calculated retrospectively for non-recovery males and non-recovery females prior to pairing, and for males after the pairing phase. Food consumptions will not be performed during the pairing phase.
Dietary intake for mated females will be recorded on Days 0-7, 7-14 and 14-20 post coitum. Food consumptions for females with live litters will be recorded between Days 1-4, 4-7 and 7-14 post partum.
Dietary intake for recovery group animals will be recorded weekly throughout the treatment and recovery period. Weekly food efficiency will be calculated retrospectively for the treatment and treatment-free periods of the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored daily by visual inspection of water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 (males) and Day 13 post partum (females)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: five males and five females
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Prothrombin time (CT), Platelet count (PLT), Activated partial thromboplastin time (APTT), Reticulocyte count (Retic)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 43 (males) and Day 13 post partum (females)
- Animals fasted: No
- How many animals: five males and five females
- Parameters: Blood 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
- sensory reactivity to different stimuli (e.g., auditory, visual and proprioceptive), grip strength (fore and hind limb) and motor activity (using a 44 photobeam unit) will be measured, in the five selected non recovery males per group, once during the final full week of treatment, at least two hours after dosing. Five selected non-recovery females per group will be similarly evaluated on Day 12 post partum.

IMMUNOLOGY: No

OTHER:
- Thyroid Hormone Assessment:
Where possible, blood samples (for serum) will be collected into Greiner Minicollect tubes, allowed to clot, centrifuged, and the serum from each blood sample stored frozen at lower than -60 °C for possible evaluation of thyroid hormones. Blood samples taken to produce plasma will be collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60 ºC for possible evaluation of thyroid hormones. Samples will be taken as follows:
Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring are of the same sex, samples from the same litter will be pooled). If eight or less offspring are present in a litter, then no offspring from that litter will be sampled on Day 4 post partum.
Where possible from each litter, serum samples from two randomly allocated offspring (one male and one female offspring) on Day 13 post partum. Where possible from each litter, plasma samples will also be taken from two randomly allocated offspring (one male and one female offspring) on Day 13 post partum. It may be necessary to alter the number/sex of offspring sampled depending on the litter constituents.
Where possible, serum and plasma samples will be taken from all adult males at termination.
Where possible, serum and plasma samples will be taken from all adult females at termination.
Initially only the serum from adult males and Day 13 offspring will be analyzed for Thyroxine (T4). Analysis of other samples or for other thyroid hormones (i.e., Thyroxine Stimulating Hormone (TSH)) will be considered once these results have been evaluated.

- Estrous Cycle Assessment (Non-Recovery): Vaginal smears will be taken daily throughout the two week pre-pairing treatment period. The stage of the estrous cycle will be recorded for each day. Additionally, vaginal smears will be taken in the morning on the day of necropsy.

- Pregnancy and Parturition (Non-Recovery): Date of pairing, Date of mating, Date of parturition with approximate time for start and completion (i.e., am, mid-day, pm)

- Litter Data (Non-Recovery):
i. Number of offspring born
ii. Number of offspring alive (recorded daily)
iii. Sex of offspring on Day 1, 4 and 13 post partum
iv. Ano-genital distance for each offspring at Day 1 post partum
v. Clinical condition of offspring from birth to Day 13 post partum
vi. Individual offspring weights on Day 1, 4, 7 and 13 post partum (litter weights will be calculated retrospectively from offspring weights)
vii. Visible nipple count for each male offspring at Day 13 post partum


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150/100 mg/kg bw/day showed increased salivation from Day 6 to 43 (males) and Day 6 to 62 (females). Incidences of noisy respiration were also evident in either sex from this treatment group between Days 1 and 43 (males) and Days 4 and 54 (females). One male and two females treated with 150/100 mg/kg bw/day showed isolated instances of hunched posture and two males from this treatment group had pilo-erection on either one or two occasions. Three females treated with 150/100 mg/kg bw/day showed a decreased respiratory rate on either one or two occasions, a further female had lethargy on one occasion and three males had staining around the snout on one or two occasions. Ten males and nine females treated with 75 mg/kg bw/day showed episodes of increased salivation from Day 8 to 43 (males) and Day 15 to 53 (females). Ten males and eight females from this treatment group also showed noisy respiration from Day 4 to 43 (males) and Day 4 to 53 (females). At 25 mg/kg bw/day, two males and two females showed increased salivation from Day 8 to 41 (males) and Day 58 to 62 (females) and noisy respiration was evident in four males and five females from Day 8 to 43 (males) and Day 12 to 57 (females). Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and reflect the distaste of the formulation or potential difficulty in dosing particular animals on occasions rather than true systemic toxicity.
Incidental findings unrelated to treatment consisted of lethargy and pilo-erection in one female treated with 150/100 mg/kg bw/day around the time of littering, chromodacryorrhea in one female treated with 75 mg/kg bw/day and increased salivation and noisy respiration in a small number of control males and females.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were four premature deaths during the study, however all of these deaths were considered not to be test item related.
One control animal was found dead on Day 13. Prior to death, this animal had increased salivation and noisy respiration on Day 12. At necropsy the lungs were dark and at histopathology there was marked necrosis in the trachea. This death was considered to have been due to a dosing trauma.
One male animal treated with 150/100 mg/kg bw/day was found dead on Day 14. This male showed increased salivation, gasping respiration and noisy respiration prior to death. Acute inflammation was present in the tissue around the esophagus and death was considered to be due to a dosing trauma.
One female animal treated with 75 mg/kg bw/day was found dead on Day 32 and had dark adrenals, liver and lungs, and a thin non-glandular region of the stomach at necropsy. There was marked necrosis in the trachea and death was considered to have been due to a dosing trauma.
One male animal treated with 25 mg/kg bw/day was humanely sacrificed on Day 43 due to the severity of clinical signs noted. This male showed red/brown staining around the snout, respiratory pattern changes, pilo-erection, hunched posture, chromodacryorrhea and was non-weight bearing on the front right limb. At histopathology there was a hole in the esophagus and a mass at the top of the forelimb extending along the thorax. This was found to be an abscess and contributed to the deterioration and death of the animal; this was considered to have been due to a dosing trauma.
There were no further unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the first two weeks of treatment, males treated with 75 or 150 mg/kg bw/day attained a statistically significant reduction in body weight gain (p<0.05 and p<0.01, respectively) in relation to controls in a dose related manner. At 150 mg/kg bw/day marked body weight losses were noted during both weeks and during Week 2 at 75 mg/kg bw/day. Following the reduction of the high dose level from 150 mg/kg bw/day to 100 mg/kg bw/day on Day 15 of treatment, improvement in body weight gains were evident thereafter, with a statistically significant increase (p<0.05 and p<0.01, respectively) achieved during Weeks 5 and 6 of treatment. Body weight gains for males at 75 mg/kg bw/day were generally similar to controls from Week 3 onwards. The initial reductions during Weeks 1 and 2 resulted in overall body weight gains to be 45% and 26% lower for males treated with 150/100 or 75 mg/kg bw/day, respectively, in relation to controls.
Males treated with 25 mg/kg bw/day generally showed similar body weight gains to controls.
During the treatment-free period, males previously treated with 150/100 mg/kg bw/day males generally showed an increase in body weight gain compared to controls, attaining statistical significance (p<0.01) during the second week of the treatment-free period.
All non-recovery female dose groups showed lower body weight gains during the first week of treatment, with females treated with 75 or 150 mg/kg bw/day attaining statistical significance (p<0.05). Recovery was evident thereafter for females treated with 75 or 25 mg/kg bw/day; however females treated with 150/100 mg/kg bw/day showed actual body weight losses during the second week of treatment. Consequently these reductions resulted in dose related lower overall body weight gains of 46% and 87% for females treated with 75 or 150 mg/kg bw/day, respectively, in relation to controls.
Females treated with 150/100 mg/kg bw/day showed a slight reduction in body weight gain during the final week of gestation, however, statistical significance was not achieved and body weight at Day 1 of lactation for these females was comparable to controls, therefore this was considered to be due to the slightly lower litter size evident at this level. Slight reductions in body weight gain were evident at 150/100 mg/kg bw/day during lactation, however, statistical significance was not achieved and body weights throughout lactation for these females were comparable to controls. Body weight gains followed the expected pattern during lactation and therefore the intergroup differences were considered to reflect normal biological variation.
Females treated with 150/100 mg/kg bw/day that were designated as recovery females showed actual body weight losses during the first two weeks of treatment. Significant improvement was evident during Weeks 3 and 4; however, slight body weight losses were noted during Week 5. For the remainder of the treatment period and throughout the treatment free period, body weight gain was comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption and food conversion efficiency for males treated with 150/100 mg/kg bw/day were reduced during the first two weeks of treatment. Following the reduction of the high dose level, improvement was evident thereafter. No such effects were detected for males at 75 or 25 mg/kg bw/day. Dietary intake during the treatment-free period for 150/100 mg/kg bw/day males was similar to controls.
Non-recovery females treated with 75 or 150/100 mg/kg bw/day showed lower dietary intake and food conversion efficiency during maturation. Reductions in food consumption were evident during gestation and lactation at 75 and 150/100 mg/kg bw/day and statistical significance was achieved between Days 0 and 7 of gestation (p<0.01 and p<0.05 respectively), between Days 14 and 20 of gestation for females treated with 75 mg/kg bw/day (p<0.05), between Days 4 and 7 of lactation (p<0.01 and p<0.05 respectively). The intergroup difference during gestation was minimal and during both phases, generally did not show a true dose related response. Therefore, this was considered to reflect normal biological variation and is considered not to be of toxicological significance.
Recovery 150/100 mg/kg bw/day females also showed slightly lower food consumptions during Weeks 1 and 2 of treatment. For the remainder of the treatment period food intake was generally similar to controls. During the treatment-free period 150/100 mg/kg bw/day females generally exceeded the food intake of the controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.
Non-recovery males treated with 25 mg/kg bw/day showed a statistically significant increase in total leucocyte count (p<0.05) which was associated with an increase in lymphocytes (p<0.01) compared to controls. All individual values were within the background control ranges and in the absence of any similar effect at the higher dose levels, this finding was considered to be of no toxicological significance.
Non-recovery females treated 75 or 150/100 mg/kg bw/day showed an increase (p<0.05) in reticulocyte count compared to controls. There was no dose related response evident and all individual values were within the background control range. An increase (p<0.01) in reticulocyte was also noted in both males and females previously treated with 150/100 mg/kg bw/day at the end of the treatment free period, but all individual values were within the background control range. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of any toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.
There was a statistically significant reduction noted for creatinine levels for non-recovery males treated with 75 or 150/100 mg/kg bw/day (p<0.05 and p<0.01, respectively). Non-recovery females from all treatment groups showed a statistically significant reduction (p<0.05) in alkaline phosphatase and non-recovery females treated with 150/100 and 75 mg/kg bw/day showed a statistically significant increase (p<0.05) in bile acids. Following the treatment free period recovery males previously treated with 150/100 mg/kg bw/day showed a statistically significant increase (p<0.05) in cholesterol. All of the individual values were within background control range and a true dose related response was not evident for alkaline phosphatase. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no adverse changes in the behavioral parameters at 25, 75 and 150/100 mg/kg bw/day
A number of animals including controls showed sporadic instances of noisy respiration during behavioral assessments in the treatment period. These observations correlate with the daily clinical observations evident. There were no treatment related changes in functional performance considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.
All treated females achieved a statistically significant reduction (p<0.05) in forelimb grip strength and females treated with 150/100 mg/kg bw/day also attained a statistically significant reduction (p<0.05) in hind limb grip strength. True dose related responses were not evident and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological importance.
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 75 or 150/100 mg/kg bw/day showed a statistically significant increase for both absolute and relative to terminal body weight in the seminal vesicle (including coagulating gland) weight (p<0.01). The increased seminal vesicle weights can be associated with increased secretion (dilation) of the seminal vesicles and coagulating gland at 150/100 mg/kg bw/day.
The 75 or 150/100 mg/kg bw/day males also showed an increase in heart (p<0.05) and liver (p<0.01) weight, both absolute and relative to terminal body weight compared to controls, and the 150/100 mg/kg bw/day also showed an increase in absolute and relative spleen weight. The majority of the individual values were within the background control ranges and without any histopathological correlates, these findings were considered to be of no toxicological significance.
At 150/100 mg/kg bw/day the non-recovery females showed an increase in absolute adrenal weight and a reduction in relative adrenal weight (p<0.05) compared to controls. The individual values were within the background control range and although all values were at the upper limit of the ranges, in the absence of any histopathological correlates these findings were considered to be of no toxicological significance.
Recovery
The recovery 150/100 mg/kg bw/day females showed an increase in absolute and relative kidney weight (p<0.01). The individual values were all within the background control ranges and without any histopathological correlates or similar findings in the non-recovery animals at the end of the treatment period, these findings were considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Non-recovery
Premature decedents
The control male (No.2) which was found dead on Day 13 exhibited dark lungs at necropsy.
One male (No. 29) treated with 25 mg/kg bw/day which was humanely sacrificed on Day 43 exhibited a hole approximately 3 mm x 2mm in the right side of the esophagus, and a mass approximately 30 mm x 20 mm x 5 mm located under the right forelimb extending along the exterior of thoracic cavity. Upon inspection the mass was found to contain a clear colorless liquid and a brown granular solid. This was considered to be due to a dosing trauma.
One female (No. 71) treated with 75 mg/kg bw/day which was found dead on Day 32 exhibited dark adrenals, liver and lungs and the non-glandular region of the stomach was thin.
One male (No. 83) treated with 150/100 mg/kg bw/day which was found dead on Day 14 did not show any macroscopic abnormalities at necropsy.

Terminal sacrifice
No toxicologically significant abnormalities were detected in animals of either sex treated with 25, 75 and 150/100 mg/kg bw/day at the scheduled necropsy.
The following findings were not associated with any treatment-related histopathological correlates and were considered to be incidental and unrelated to treatment.
One male treated with 75 mg/kg bw/day exhibited pallor of both kidneys. One female treated with 75 mg/kg bw/day exhibited increased pelvic space in both kidneys. A further female from this treatment group exhibited a mass on the left horn of the uterus. One female treated with 150/100 mg/kg bw/day exhibited a dark and enlarged spleen, a further female exhibited an enlarged spleen, and one female exhibited increased pelvic space in the right kidney. One recovery male previously treated with 150/100 mg/kg bw/day exhibited a pale spleen; a further male from this treatment group exhibited an enlarged and dark spleen.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Premature decedents
Control male 2 was found dead on Day 13. At necropsy the lungs were discolored and at histopathology there was marked necrosis in the trachea. The cause death was considered to have been due to a dosing trauma.
Male 83 (150 mg/kg bw/day) was found dead on Day 14. There was acute inflammation present in the tissue around the esophagus and the cause of death was considered to be due to a dosing trauma.
Female 71 (75 mg/kg bw/day) was found dead on Day 32. There was dark discoloration of several tissues at necropsy and also there was marked necrosis in the trachea. The cause of death is considered to have been due to a dosing trauma.
Male 29 (25 mg/kg bw/day) was sacrificed in extremis on Day 43. There was a hole in the esophagus and a mass at the top of the forelimb extending along the thorax. This was found to be an abscess, which led to the animal’s deterioration, therefore this death was considered to have been due to a dosing trauma.
Terminal sacrifice
Seminal Vesicles and Coagulating Gland
Increased secretion (dilation, vesicle) in the seminal vesicles was noted in three and eight males treated with 75 and 150/100 mg/kg bw/day, respectively. This change was found to have completely recovered following the treatment free period. Increased secretion (dilation, acinar) in the coagulating gland was noted in four males treated with 150/100 mg/kg bw/day, and complete recovery was evident in the recovery animals. This would correlate with the increased organ weights at necropsy. The significance of the increased secretion (dilation) of these accessory sex glands is unclear, as male fertility was unaffected and the condition was completely reversed in the recovery animals with no degeneration evident this finding was therefore considered to be non-adverse.
Mesenteric Lymph Node
Histiocyte aggregates were present in the mesenteric lymph node of all males and females treated with 150/100 mg/kg bw/day, along with four males and one female at 75 mg/kg bw/day. Recovery was evident, with the exception of one male and one female at 150/100 mg/kg bw/day following the treatment free period. This change is generally considered to be a non-adverse finding.

Non Productive Matings
There were two non-productive matings within the study.
Control male 12 and female 24 – female 24 had atrophy in the ovaries with a lack of recent corpora lutea and this was considered to be the reason for the lack of pregnancy.
At 150/100 mg/kg bw/day male 76 and female 95 showed no findings in the tissues examined which could account for the lack of pregnancy.
There were no test item-related microscopic findings in the reproductive tracts 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 the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 25, 75 or 150/100 mg/kg bw/day.
For all treated adult males the T4 blood levels were statistically significantly lower (p<0.001) than controls in a dose related response. All individual values were within the background control range. In view of the lack of any supporting effect of thyroid weights or histopathological correlation, the observed differences from controls were considered to reflect atypically high control values rather than any effect of treatment.
Details on results:
reproductive findings are described in Iuclid section 7.8.1 Toxicity to reproduction

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects up to highest tested dose level

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Detailed results in tabular form are attached below.

Applicant's summary and conclusion

Conclusions:
The oral administration of CGE-PMDA adduct to rats by gavage, at dose levels of 25, 75 and 150 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption in animals of either sex at 150 and 75 mg/kg bw/day. Following the reduction of the high dose level to 100 mg/kg bw/day on Day 15, recovery was evident in these animals and body weight gains also improved at 75 mg/kg bw/day from Week 3 onwards. Treated animals also showed clinical observations that can be associated with the oral administration of an irritant/unpalatable test item formulation and as such, the initial reduced body weight development at 150 and 75 mg/kg bw/day may be the result of the irritant nature of the test item rather than evidence of true systemic toxicity. The microscopic changes evident in the mesenteric lymph nodes were considered to be non-adverse and although the significance of the microscopic seminal vesicle and coagulating gland changes were unclear, there was no evidence of any degeneration or any effect on fertility and complete reversibility was evident. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day for both males and females.
The `No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of CGE-PMDA adduct on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, 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 29 July 2016). It also assesses the ability of the animals to recover from any toxicity over twenty-eight days following the withdrawal of treatment.

 

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 25, 75 and 150 mg/kg bw/day. After two weeks of treatment at 150 mg/kg bw/day the dose level was reduced to 100 mg/kg bw/day on Day 15 following marked body weight losses in animals of either sex; particularly in the males. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same period. Two recovery groups, each of five males and five females, were treated with the high dose (150/100 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further twenty-eight days.

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

Pairing of non-recovery 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 ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected non-recovery parental females from each dose group on Day 12post partum. Five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to terminal sacrifice. Additionally, blood samples were taken at terminal sacrifice from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from non-recovery 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 non-recovery 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 terminal sacrifice for all non-recovery females.

Surviving adult non-recovery males were sacrificed on Day 44 or 45, followed by the sacrifice of all surviving offspring and surviving adult non-recovery females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was sacrificed around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Following forty-three days of treatment, recovery group animals were maintained without treatment for a further twenty-eight days. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examination of selected tissues was performed.

 

Adult Responses

Mortality

There were no unscheduled deaths that were considered to be related to treatment.

There were four premature deaths during the treatment period (Animal numbers 2, 29, 71 and 83) that were considered to be due to dosing trauma.

 

Clinical Observations

Incidences of increased salivation and noisy respiration were evident in animals of either sex treated with 150/100 mg/kg bw/day throughout the treatment period and to a lesser extent in animals of either sex treated with 75 and 25 mg/kg bw/day. Isolated instances of hunched posture, pilo-erection, decreased respiratory rate, lethargy and stained snout were also evident at 150/100 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 25, 75 or 150/100 mg/kg bw/day.

 

Functional Performance Tests

There were no treatment-related changes in functional performance considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.

 

Body Weight

At 150 mg/kg bw/day males showed body weight losses during Week 1 and 2 of treatment when compared to controls. Following the reduction of the dose level to 100 mg/kg bw/day on Day 15, these animals showed significant improvement in body weight gains. As a consequence of the initial body weight losses, the overall group mean body weight gain was 45% lower than controls.

Males treated with 75 mg/kg bw/day showed reductions in body weight gain during Weeks 1 and 2 of treatment. Improvement was noted from Week 3 onwards and body weight gains were comparable to controls thereafter. The earlier reduction however, resulted in the overall group mean body weight gain being 26% lower than controls.

No such effects were detected for males treated with 25 mg/kg bw/day.

Females treated with 150/100 mg/kg bw/day showed reduced group mean body weight gains during pre-pairing and during the final week of gestation compared to controls. No adverse effects were evident during lactation.

At 75 mg/kg bw/day, females showed reduced body weight gains during pre-pairing. No effects were evident during gestation or lactation.

At 25 mg/kg bw/day, females showed comparable body weight gains with the exception of slightly lower body weight gains during the first week of pre-pairing compared to controls.

 

Food Consumption

Males treated with 150/100 mg/kg bw/day showed a reduction in food intake during the first two weeks of treatment. Improvement was evident thereafter. There were no effects detected for males treated with 25 mg/kg bw/day. During the treatment free period, 150/100 mg/kg bw/day males showed dietary intake that was similar to controls.

Females treated with 75 or 150/100 mg/kg bw/day showed lower food consumptions during pre-pairing. Slight reductions in food consumption were also evident in these females during gestation and lactation. Females at 25 mg/kg bw/day were generally similar to controls. The recovery 150/100 mg/kg bw/day females generally showed similar dietary intake to controls, with the exception of the first two weeks of treatment where food consumption was slightly lower.

 

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

 

Reproductive Performance

Estrous Cycle

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.

 

Mating

No treatment-related effects were detected in mating performance.

 

Fertility

No treatment-related effects were detected in fertility.

 

Gestation Lengths

Gestation lengths were essentially similar to control.

 

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, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.

Litter size at birth and subsequently on Days 1, 4, 7 and 13post partumwas lower for females treated with 150/100 mg/kg bw/day when compared to control litters, without attaining statistical significance. No such effects were noted for offspring from females treated with 75 or 25 mg/kg bw/day.

 

Offspring Growth and Development

As a consequence of the slightly lower litter size at birth in females treated with 150/100 mg/kg bw/day, litter weights were reduced in these females throughout lactation. There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13post partumat any dose level.

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 25, 75 or 150/100 mg/kg bw/day.

 

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

 

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Pathology

Necropsy

Offspring

No treatment-related macroscopic abnormalities were detected in interim death or terminal sacrifice offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Adults

No toxicologically significant abnormalities were detected in animals of either sex treated with 25, 75 and 150/100 mg/kg bw/day.

 

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.

 

Organ Weights

There was an increase in seminal vesicle (including coagulating gland) weights, both absolute and relative to terminal body weights. This correlated with the histopathological findings of increased secretion (dilation), the significance of which is unclear.

There were no further toxicologically significant findings on organ weights measured.

 

Histopathology

The premature death of four animals was considered to be due to dosing trauma and was unrelated to treatment with the test item.

Treatment at 75 or 150/100 mg/kg bw/day resulted in histiocyte aggregates in the mesenteric lymph node of males and females. This showed good reversibility and was considered to be non-adverse.

At 75 or 150/100 mg/kg bw/day, increased secretion (dilation) of the seminal vesicles was evident and at 150/100 mg/kg bw/day this was also evident in the coagulating glands. Complete recovery was evident; however, the significance of this finding is unclear.

 

Conclusion

The oral administration of CGE-PMDA adduct to rats by gavage, at dose levels of 25, 75 and 150 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption in animals of either sex at 150 and 75 mg/kg bw/day. Following the reduction of the high dose level to 100 mg/kg bw/day on Day 15, recovery was evident in these animals and body weight gains also improved at 75 mg/kg bw/day from Week 3 onwards. Treated animals also showed clinical observations that can be associated with the oral administration of an irritant/unpalatable test item formulation and as such, the initial reduced body weight development at 150 and 75 mg/kg bw/day may be the result of the irritant nature of the test item rather than evidence of true systemic toxicity. The microscopic changes evident in the mesenteric lymph nodes were considered to be non-adverse and although the significance of the microscopic seminal vesicle and coagulating gland changes were unclear, there was no evidence of any degeneration or any effect on fertility and complete reversibility was evident. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day for both males and females.

The `No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day.