Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
screening study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 September 2016 to 17 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Thyroid hormone data: During the thyroid hormone analysis, no data for Thyroxine (T4) were obtained due to the failure of the analyser. The OECD 421 test guideline requires T4 assessments to be performed for PND 13 pups and adult males. Microscopic examination of thyroids from PND 13 pups and thyroid and pituitary from adults males were examined to observe for any thyroid changes. As a clear endocrine effect, resulting in male infertility was established in this study, the absence of the T4 data was considered not to have negatively impacted on study integrity, as the omission of this data did not impact on the overall conclusion with regards to reproductive toxicity.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-dimethylaminobenzoate
EC Number:
233-634-3
EC Name:
Ethyl 4-dimethylaminobenzoate
Cas Number:
10287-53-3
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl 4-(dimethylamino)benzoate
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White to off-white powder
- Storage conditions of test material: Sealed container 15 to 25 °C, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) Males: 8 to 10 weeks; females: 9 to 11 weeks. Rats were considered sexually mature.
- Weight at study initiation: (P) Males: 380.3 to 480.2 grams; females: 199.8 to 278.9 grams
- Housing: During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, or post-pairing for the unconfirmed mated female, and with their litter during the lactation phase. Males were returned to group housing after the pairing phase. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips, or European Softwood bedding during gestation and lactation phases. Animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation, nesting materials were also provided as forms of environmental enrichment.
- Diet: Animals had ad libitum access to food
- Water: Water from the main tap supply was provided ad libitum via water bottles
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 % (relative)
- Air changes: Minimum of 15 to 20 per hour
- Photoperiod: Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated.

IN-LIFE DATES
- From: 27 October 2016
- To: 21 December 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared at least weekly. The test material was formulated as a solution in corn oil and stored at room temperature (15 to 25 °C) in a sealed container, protected from light. The formulations were stirred continuously from at least 30 minutes before dosing commenced and throughout dosing.

- DOSE VOLUME: 5 mL/kg (based on most recently recorded body weight)

VEHICLE
- Concentration in vehicle: 10, 35 and 120 mg/mL formulations
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations of the test material, prepared at 10 and 120 mg/mL (in corn oil), were analysed to determine homogeneity and stability. Formulations were determined to be homogeneous and stable for 8 days when stored at 15 - 25 °C.
Formulations prepared for use on Day 1 and Week 6 of dosing were analysed to determine the achieved concentration. Triplicate samples were removed from the middle of the test material formulations and were analysed. A single sample was taken from the middle of control formulations and was analysed.

- Achieved Concentration
For the results generated for the week 6 analysis, it was noted that the USP tailing factor for the tets material was recorded as 1.4, with the acceptance criteria being 0.8 to 1.3. Upon investigation it was found that the tailing factor had marginally failed for 1 of the 3 QC standards upon which the assessment is made. The decision was taken to accept the data as the marginal failure of the Tailing assessment would not have adversely affected the results generated.

- Analytical Procedure
This method involves gas chromatographic (GC) determination, with the dose formulations dissolved and diluted in Toluene with the resultant solutions being assayed directly against standard solutions of the test material.

- Calibration Solutions
Stock Standard Solution 2.5 mg/mL: Accurately weigh 25 ± 0.2 mg of the test material into a 10 mL volumetric flask. Dissolve in and make to volume with toluene, mix well. This stock solution was further diluted to form linearity standards.
Stock Standard Solution 0.2 mg/mL: Accurately weigh 10 ± 0.2 mg of the test material into a 50 mL volumetric flask. Dissolve in and make to volume with toluene, mix well.
Blank: Toluene was used as a blank.

SAMPLE PREPARATION PROCEDURE
Prepare each sample solution with the approximate working concentration of 0.2mg/mL. Prepare a stock sample solution by accurately transferring, by pipette, 1 mL of the dose formulation to be analysed into a 50 mL volumetric flask. Dissolve and dilute to volume using toluene and mix well. Dilute the stock sample solution as follows:
- 10 mg/mL Dose Formulation: Use the stock solution as the solution to be analysed (~0.20 mg/mL)
- 35 mg/mL Dose Formulation: Dilute the stock solution by pipetting 3 mL of the stock solution to a 10 mL volumetric flask, dilute to volume using toluene (~0.21 mg/mL)
- 120 mg/mL Dose formulation: Dilute the stock solution by pipetting 2 mL of the stock solution to a 25 mL volumetric flask, dilute to volume using toluene (~0.19 mg/mL)
When the dose formulation is a concentration not stated above then alternative dilutions may be used as long as the final solution concentration is approximately 0.2 mg/mL.

GAS CHROMATOGRAPHY (GC)
GC system parameters
Column (Analytical): DB5-MS, 30 m x 0.25 mm id, 0.25 µm df
Inlet Liner: Single taper, deactivated split liner with glass wool e.g. Agilent 5183-4647
Carrier Gas: Helium @ 1.0 mL/min Constant Flow
Oven temperature Program: 175 °C held for 1 min; 5.0 °C/min to 200 °C, hold for 1 min; 50 °C/min to 350 °C, hold for 10 mis
Injection volume: 1 µL
Runtime: 20 minutes
Inlet Temperature: 254 °C
Split ratio: 50:1
Detector Type: Flame Ionisation (FID)
FID Temperature: 300 °C
Detector Gas flow rates: Hydrogen 30 mL/min; Air 400 mL/min; Makeup (Nitrogen) 35 mL/min
Injector Wash Solvent: Toluene (3 washes pre and post (injection); 3 Sample washes and 6 sample pumps pilot to injection

CALCULATIONS
- Standard Concentration (mg/mL)
(Standard weight (mg) x Standard purity) / Dilution Factor
Construct a calibration curve of test material response vs standard concentration (mg/mL) using the least squares method to determine the equation of the curve as per y = mx + c, Determine the slope (m) and the intercept (c), and the correlation coefficient (R²).

- QC Solution Recovery (%)
((Mean area QC solution - c)) / m) + QC solution concentration (mg/mL) x 100

- Achieved Concentration of Dose Formulation (mg/mL))
((Area of sample - c) / m) x Dilution factor of sample (mL)

RESULTS
The mean % target range for the preparation of formulations was 90 to 110 % of nominal. Results were within this range. Test material was not detected in Group 1 control samples.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 10 days. Females without evidence of mating after 10 days of pairing were paired for up to 5 days with proven males of the same dose group
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal lavage sample. Upon confirmation of mating, vaginal lavage sampling was discontinued, and the male was removed. The day on which mating was confirmed was designated as GD 0.
- After successful mating each pregnant female was caged: Following mating, females were housed individually during gestation and with their litter during the lactation phase.
- Other: Before females were assigned to the study, oestrous cycle data were reviewed to ensure all females allocated to the study showed regular oestrous cycles. Any female not showing a regular oestrous cycle was replaced with a spare female with a recorded regular oestrous cycle.
Duration of treatment / exposure:
Males were dosed for 53 or 54 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase and until the day before necropsy [post-pairing phase]). Females were dosed for up to 64 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase and until LD 13, inclusive, or Day 25 post-coitum for non-pregnant females). Some females were not dosed on LD 0 if they were observed to be starting or had just completed parturition.
Frequency of treatment:
The test material was administered once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The test material has previously been administered to rats by oral gavage, in a formulation prepared in corn oil, at dose levels of 6, 74, and 900 mg/kg/day for 28 days. Toxic effects were limited to animals administered 900 mg/kg/day. Males showed reduced body weights compared with controls; body weight effects were less pronounced in females, but more marked clinical observations, including hunched posture, lethargy and gasping respiration were noted. Haematology changes in red blood cell parameters, consistent with anaemia and a possible erythropoietic response, and increases in leucocyte counts, specifically in the lymphocyte and neutrophil counts, were also evident. Clinical chemistry changes were confined to an increase in bilirubin levels. Macroscopic examinations showed reduced testes weights and small blue discoloration of the testes in males, and increased incidences of dark coloured spleens for all animals administered 900 mg/kg/day. Microscopic examinations revealed testicular changes consisting of moderate tubular atrophy for all males, with two males showing multinucleated giant cells in the seminiferous epithelium. Most males at this dose had no spermatozoa in the epididymides, with multinucleated giant cells present in two of these males. Haemosiderosis for all animals, and minimal/moderate extramedullary haemopoiesis were observed in the spleens. Minimal extramedullary haemopoiesis was also evident for three males and two females administered 74 mg/kg/day; however, in isolation this finding was considered not to represent an adverse effect.
Similar effects were also observed in a 28-day toxicity study carried out with a structurally-related substance, where testes and epididymides changes in males administered 1000 and 300 mg/kg/day, and splenic changes, extending to the 300 mg/kg/day females, were observed. In addition, in a pre-natal developmental toxicity study with the same substance, no teratogenic effects were observed, but delayed ossification and reduced pup weights were noted at 1000 mg/kg/day. This dose was clearly toxic for the mothers, as was 300 mg/kg/day, which was only slightly toxic to the mothers and considered adverse.
The low dose level of 50 mg/kg/day was selected as it was anticipated to be the no observed adverse effect level (NOAEL) over a dosing phase of approximately twice that in the 28-day study.
The intermediate dose level of 175 mg/kg/day was selected by using a 3.5 spacing factor, as recommended in the OECD 421 test guideline (2- to 4-fold spacing factor among dosages), and in order to obtain information on dose response.
The high dose level of 600 mg/kg/day was selected by using a 3.5 spacing factor and rounded to an entire value. This dose level was chosen in order to obtain information on dose response effects in males, and aimed to avoid severe clinical observations in females.
- Rationale for animal assignment: Animals were assigned to dose groups using a total randomisation procedure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily cage side observations were made for ill health and overt toxicity. Animals were observed daily for the first 3 days of dosing, upon return to the home cage and at approximately 0.5, 1, 2, and 4 hours post-dose. In the absence of any toxicologically significant post-dose observations noted during the first three days of dosing, no further post-dose observations were scheduled.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination daily from the start of dosing, including the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Female body weights were recorded twice pre-dose, weekly prior to pairing and until confirmation of mating, on Gestation Day (GD) 0, 7, 14, and 20 and on LD 1, 4, 7, 13, and 14 (prior to necropsy).

FOOD CONSUMPTION: Yes
-The amount of food consumed was recorded twice weekly prior to pairing. Daily food consumptions were recorded for females from GD 0 to 20 and LD 1 to 13. Consumption was calculated as g/animal/day.

OTHER: Oestrus cycle
Daily vaginal lavage samples were taken from all females during acclimatisation (pre-dose) from 1 week after arrival until the day prior to dosing; the stage of oestrus was recorded and only females with regular 4 to 5 day cycles were included on study. Daily vaginal lavage samples were taken from females from the start of dosing until confirmation of mating.

OTHER: Parturition
Animals were observed three times each day, starting when the first females reached GD 21 and until the last female had littered or until Day 26 post-coitum, whichever was soonest. Females were observed for signs of the start of parturition (for example blood in the cage). The time and date of this observation was recorded, where possible, and marked as the end of gestation; where not observed, the end of gestation was the day when completion of parturition was recorded, or the day prior to when LD 1 observations were made. Abnormal signs of nesting, parturition, or nursing were recorded. Special care was taken to disturb the maternal animal as little as possible during LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

SACRIFICE
Females were sacrificed on LD 14 (those that achieved pregnancy), Day 26 post coitum (for females that did not litter), or Post-Pairing Day 14 (non-confirmed mated female) by isoflurane anaesthesia; once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. Blood sampling took place. After the sacrifices, macroscopic examinations were conducted, and all lesions were recorded. The uterus of each female was immersed in a 10 % ammonium sulfide solution, and implantation sites were counted.

CLINICAL PATHOLOGY
Blood samples for haematology (1 x 0.5 mL [EDTA], nominal) and thyroid hormone analysis (1 x 0.6 mL [Serum Separator Tube], nominal) were withdrawn from the abdominal aorta at necropsy; the sampling was performed at a similar time on each occasion.
Haematology Tests: Haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution, width, haemoglobin distribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume and platelet distribution width.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights were recorded for each adult animal. Bilateral organs were weighed together. Tissues were preserved in 10 % neutral-buffered formalin. The following organs and tissues were weighed (W) or processed and microscopically examined (E): Gross lesions, levator ani plus bulbocavernosus muscle complex (W), mammary gland, vagina (E), ovary (W, E), oviduct (W), pituitary (E), uterus (including cervix, W, E) and ureter.
Tissues denoted by (E) in the previous tissue list from adult animals were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with haematoxylin and eosin. Sections of testes and epididymides were also stained with Periodic Acid-Schiff (PAS). Processed tissues were examined microscopically.
Ovaries and uterine content:
The uterus of each female was immersed in a 10 % ammonium sulfide solution, and implantation sites were counted.
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- Litters were culled to 10 pups/litter (five pups/sex where possible). Runts (pups considered unlikely to survive to weaning) were pre-selected for cull. The remaining pups were selected randomly. Culling was considered to have created a uniformed litter size, reducing differences in pup body weight due to litter size.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Litter sizes and offspring sex were recorded on PND 1, 4, 7, and 13. Daily records of mortality and changes in litter sizes were maintained. Each pup was given a detailed physical examination daily from PND 1. Individual pup body weights were recorded on PND 1, 4, 7, and 13. Ano-genital distance of all pups was recorded on PND 4. The number of nipples/areolae for male pups was counted on PND 13.

SACRIFICE
Surplus pups culled on PND 4, and pups sent to necropsy on PND 13, were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal.
After termination, full macroscopic examinations were conducted for all decedents, surplus pups culled on PND 4, and for two male and two female pups in each litter on PND 13 (including any pups with external abnormalities); all lesions were recorded. Where possible, pups found dead or in a moribund condition were examined macroscopically. Culled pups were examined macroscopically and pooled blood samples were taken for thyroid hormone assessment.
The remaining pups in each litter on PND 13 were examined externally for macroscopic abnormalities, paying particular attention to the external reproductive genitals

CLINICAL PATHOLOGY
Pups culled on PND 4 had blood samples (1 x 0.6 mL) for thyroid hormone analysis withdrawn via decapitation to provide one pooled sample for each litter, where possible.
Pups sacrificed on PND 13 had blood samples (1 x 0.6 mL) for thyroid hormone analysis withdrawn by cardiac puncture to provide two pooled samples for each litter (one sample from two males and one sample from two females, where possible).

HISTOLOGY
The thyroid was removed from one pup/sex/litter on PND 13 and fixed in 10 % neutral-buffered formalin. The tissue was weighed 24 hours post-fixation. Thyroid tissue from Group 1 and 2 pups was embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with haematoxylin and eosin.
Statistics:
Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data for each sex was analysed separately, unless stated otherwise. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant when P ≤ 0.05.
Body weight, body weight gains, food consumption (gestation and lactation phases), absolute organ weights, organ: terminal body weight ratios, terminal body weights and PND 13 pup thyroid hormone data only (male, female and combined) were analysed using analysis of variance.
Male and female mating, fecundity, and fertility indices were analysed using a one-sided lower tail Fisher’s exact test.
Mean number of oestrous cycles and mean cycle length was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Percent pregnant, percent delivering and gestation index were analysed using a one-sided lower tail Fisher’s exact test.
Percent of females with stillborn pups was analysed using a one-sided upper tail Fisher’s exact test.
The duration of gestation; number of implantation sites; number of pups born; number of pups alive on Days 1, 4 (before and after culling), 7 and 13 post-partum; number of pups culled on Day 4 post-partum; percent post-implantation loss; and live birth and survival indices were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Pup body weights (male, female and combined) were analysed using analysis of covariance (ANCOVA), with litter size as the covariate.
Live pups/Litter with live pups were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Ano-genital distance (male pups only) were analysed using analysis of covariance (ANCOVA), with litter size as the covariate.
Seminology data were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Indices:
Mating, fecundity, fertility, percent pregnant, percent delivering, gestation, percent post-implantation loss, live birth and survival indices.
Number of pups born and number of pups alive on Days 1, 4 (before and after culling), 7 and 13 post-partum.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant clinical observations were observed.
Isolated instances of raised hair were noted for four females administered 600 mg/kg/day during the pairing or early gestation phases. This was also noted for one female administered 175 mg/kg/day and one female administered 50 mg/kg/day on GD 8. Another female administered 50 mg/kg/day also had raised hair and was noted as subdued on GD 0. The effects were short-lived, and did not impact on the overall health of animals. As such, these findings were considered not toxicologically important.
The remaining clinical observations included isolated instances of sores, lesion, staining of the skin and/or fur, thin fur, and physical injury to the paw/swollen paw. These observations were noted throughout the dose groups, including the control group; as such, they were considered low incidence findings observed in this species and were not considered test material-related.

No toxicologically significant post-dose observations were observed. Mouth rubbing was observed immediately post-dose on Day 2 for one female administered 175 mg/kg/day and three females administered 600 mg/kg/day, with the observation also noted 0.5 hours post-dose on the same day for one female administered 600 mg/kg/day. No further post-dose observations were noted. This observation is occasionally observed following the oral administration of an unpleasant tasting test material formulation and, as such, was considered not to represent systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weight gain was noted during the first week of dosing for females administered 600 mg/kg/day (approximately 2-fold, compared with controls), although mean values were essentially similar to controls during Week 2.
Reduced body weights were also observed at 175 mg/kg/day during the first 2 weeks of dosing, compared with controls. However, statistical significance was not achieved.
The post-pairing body weight data for females administered 175 or 600 mg/kg/day could not be accurately compared with control due to the difference in pregnancy status.
No adverse effect on body weights was noted following administration of 50 mg/kg/day. Females administered 50 mg/kg/day showed reduced body weight gain during Pre-Pairing Week 2, compared with controls, although overall mean body weight during the study was essentially similar to controls. Gestation and lactation body weights for control females were generally comparable with females administered 50 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects on food consumption were noted.
Post-pairing food consumption data for females administered 175 or 600 mg/kg/day could not be accurately compared with controls due to the difference in pregnancy status.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test material-related changes in haematology parameters were evident for females administered 50 mg/kg/day compared with controls. Due to different pregnancy status, control data could not be accurately compared with data obtained for females administered 175 or 600 mg/kg/day.
The TSH levels for females administered 50 mg/kg/day were 17 % higher than controls on LD 14, although values were within the historical background control range. The TSH data for females administered 175 or 600 mg/kg/day were not comparable with controls due to the different pregnancy status.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
All organ weight and/or organ weight ratio changes, including those statistically significant, were attributed to normal biological variation and were considered not test material related as they were small in magnitude, not dose dependent, due to normal inter-animal variability and/or lacked a microscopic correlate.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test material-related macroscopic findings were noted. Tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in animals of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The ovaries, uteri and vaginae from females administered 600 mg/kg/day showed normal cycling microscopically, compared with control females which had microscopic evidence of previous pregnancy and parturition.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test material-related changes in implantation sites were noted for animals administered 50 mg/kg/day compared with controls.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No test material-related changes in gestation length were noted for animals administered 50 mg/kg/day compared with controls.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Fecundity and fertility were adversely affected following 2 weeks of administration of 175 or 600 mg/kg/day, with no pregnancies evident for any female at these dose levels.
Other effects:
no effects observed
Description (incidence and severity):
No effect of test material administration was noted on the number or length of oestrous cycles during the pre-pairing phase. All females showed regular oestrous cycles of approximately 4 to 5 days in length.
Mating performance was not affected by test material administration (with the exception of two animals, all mated within the first 5 days of pairing).
One pair of 600 mg/kg/day animals did not show positive evidence of mating. The female was then paired with another male, but pregnancy was not achieved in this group. Non-mating is occasionally observed in studies of this type, and in isolation, was considered to be incidental.

No test material-related changes in litter sizes or survival indices were noted for animals administered 50 mg/kg/day compared with controls.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects were noted in maternal animals; failure to achieve pregnancy was attributed to effects on the male reproductive system
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No adverse effect on mean pup body weights was noted from 50 mg/kg/day litters, compared with controls.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test material-related changes in litter sizes were noted for animals administered 50 mg/kg/day, compared with controls.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
No test material-related changes in survival indices were noted for animals administered 50 mg/kg/day, compared with controls.
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical observations were noted for pups from 50 mg/kg/day litters. Incidental findings (i.e., number of decedent pups) were observed in both the 50 mg/kg/day and control group litters and, therefore, were considered unrelated to maternal test material administration.

- Ano-Genital Distance
No adverse effect on ano-genital distance was observed for pups from 50 mg/kg/day litters, compared with controls.

- Nipple/Areole Count
No nipples/areolae were present for any male pup from the 50 mg/kg/day or control litters.

- Thyroid hormone
Thyroid stimulating hormone (TSH) was slightly increased for the PND 4 pooled blood sample from animals administered 50 mg/kg/day, compared with controls (+40 %), however, values were within the historical background control range.
On PND 13, male pups had 50 % higher TSH and female pups had 47 % higher TSH, compared with controls; statistical significance was achieved for the PND 13 females and combined male/female data, however, values were within the historical background control ranges.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed, however no pups were available at higher dose levels

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of Female Fertility and Reproductive Performance

Treatment Group

Control

50 mg/kg

175 mg/kg

600 mg/kg

Total Females

10

10

10

10

Unscheduled Deaths Prior to Cohabitation

0

0

0

0

Females Cohabited

10

10

10

10

Unscheduled Deaths During Cohabitation

0

0

0

0

Females Mated

10

10

10

9

Pregnant Females

10

10

0

0

Non Pregnant Females

0

0

10

10

Matings Per Day Periods Of Cohabitation

Day 1

Day 2

Day 3

Day 4

Day 5

 

2

5

1

1

1

 

0

2

4

4

0

 

0

2

7

1

0

 

2

3

2

2

0

Mating Index %

100

100

100

90

Fecundity Index %

100

100

0#

0#

Fertility Index %

100

100

0#

0#

Mating index % = Mated females/females cohabited (excluding females sacrificed during Cohabitation) x 100

Fecundity Index % = Pregnant females/mated females (excluding females with an undetermined pregnancy status) x 100

Fertility Index % = Pregnant females/females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status) x 100

#Fisher 1-tail Descending Test significant at the 0.001 level

 

Table 2: Parturition and Litter Data Summary

 

Group 1

Group 2

Statistics

Number of females with live pups at Day 13 post-partum

10

10

X

Mean duration of gestation (days)

23.3

22.8

W

Mean number of implantation sites

15.4

15.1

W

Mean number of pups born

14.2

14.6

W

Mean number of pups alive Day 1

13.9

14.0

W

Mean % male pups Day 1

52.4

57.8

W

Mean number of pups alive Day 4 before culling

13.5

13.8

X

Mean number of pups culled Day 4

3.5

3.8

X

Mean number of pups alive Day 4 after culling

10.0

10.0

X

Mean number of pups alive Day 7

10.0

10.0

X

Mean number of pups alive Day 13

10.0

10.0

X

W = Wilcoxon rank sum test

X = not analysed

Table 3: Summary of Pup Survival

 

Group 1

Group 2

Statistics

Post-implantation survival index %

92.3

96.4

W

Live birth index %

97.8

96.1

W

Survival index 1-4 %

97.4

98.8

W

Survival index 4-7 %

100.0

100.0

X

Survival index 7-13 %

100.0

100.0

X

W = Wilcoxon rank sum test

X = not analysed

 

Discussion

Once daily oral gavage administration of 50, 175, or 600 mg/kg/day of test material to male rats for up to 54 consecutive days and to female rats for up to 64 days (pre-pairing, throughout gestation and during the first 2 weeks of lactation) resulted in adverse effects on male fertility following 600 or 175 mg/kg/day administration.

Although no scheduled deaths or clinically observable signs of toxicity were evident, and there was no effect on mating performance, no pregnancies were achieved following administration of 600 or 175 mg/kg/day.

Following seminology assessments, sperm count and sperm motility were greatly reduced at these dose levels, and organ weights for the reproductive tissues were greatly reduced for males administered 600 mg/kg/day compared with controls. The reproductive tract of females administered 600 mg/kg/day demonstrated normal oestrous cycling microscopically, whereas the reproductive tracts from control females all confirmed previous pregnancy/parturition. This correlated with the low sperm counts in males and suggests that the failure to achieve pregnancy in females administered 600 mg/kg/day was attributable to effects on the male reproductive systems rather than an effect on the female reproductive system. Based on sperm count and sperm motility data for males administered 175 mg/kg/day, the same effects on the male reproductive systems rather than an effect on the female reproductive system can be inferred for females treated at 175 mg/kg/day.

Overall, the findings in the tissues examined were suggestive of endocrine effects of the test material involving the male reproductive tract and pituitary-thyroid gland axis. The thyroid/parathyroid weights were variable, not dose-dependent and inconsistent between sexes. Microscopically, colloid depletion/follicular cell hypertrophy was present in males from all dose groups. Independently, colloid depletion causes an overall decrease in thyroid weight and follicular cell hypertrophy causes an increase in thyroid weight, hence no consistent effect on these organ weights can be determined and the thyroid/parathyroid weights cannot be directly correlated microscopically, although the microscopic changes in the thyroid correspond with the increases in TSH.

Due to the findings in this study, dose levels of 600 or 175 mg/kg/day were considered to cause adverse effects on male fertility. No adverse effects were noted following administration of 50 mg/kg/day to males, or the resultant offspring.

The omission of T4 data from this study do not affect the conclusion or the no observed adverse effects level (NOAEL) based on the adverse effects on male fertility.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, dosing of the test material resulted in adverse effects on male fertility, therefore a NOAEL for male reproductive toxicity was established as 50 mg/kg/day.
No adverse effects were noted for females administered 600 or 175 mg/kg/day. The absence of pregnancies at these dose levels was considered to have resulted from an adverse effect on the male reproductive system. As such, a NOAEL for reproductive toxicity was established as 600 mg/kg/day for females.
The NOAEL for offspring development was considered to be 50 mg/kg/day in the absence of any adverse effects noted at this dose level.
Executive summary:

The toxicity of the test material to reproduction was investigated in accordance with the standardised guideline OECD 421 under GLP conditions. The study was to screen for effects of the test material on male and female reproductive performance (i.e. gonadal function, mating behaviour, conception, development of the conceptus and parturition) and offspring growth post-partum.

Four groups of 10 male and 10 female Crl:CD(SD) rats were administered 0, 50, 175 or 600 mg/kg/day test material orally by gavage, at a constant dose volume of 5 mL/kg. The control material (vehicle) was corn oil. Formulations prepared for use during Weeks 1 and 6 were analysed for achieved concentration and mean values were within the specified concentration ranges.

Before the start of dosing, all females were screened for regular oestrous cycles; only females showing regular oestrous cycles were included in the study. Males were dosed once daily for up to 54 consecutive days (two weeks prior to pairing, during the pairing phase and until the day before necropsy) and were sent to necropsy in Week 7. Females were dosed for up to 64 days (two weeks prior to pairing, during the pairing phase, throughout gestation and up to Lactation Day [LD] 13, 25 days post-coitum or after the completion of the pairing phase for any non-mated females, where applicable) and sent to necropsy on LD 14, 26 days postcoitum for females which did not achieve a pregnancy. Any female which failed to mate was also sent to necropsy at the end of the pairing phase. Following 2 weeks of dosing, animals were paired for mating on a one male: one female basis within each dose group. Once mating was confirmed, females were individually housed and males were returned to their home cage.

Assessment of toxicity in adults was based on clinical observations, body weights, food consumption, oestrous cycling, mating, fertility and pregnancy indices and offspring parameters. Pup clinical observations, litter size, sex and body weights were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4 and nipple retention was also recorded for male pups on PND 13. One pup/sex/litter was selected from each dose group for thyroid weight recording and processing for examination.

Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Blood samples were collected at necropsy from all adult animals for haematology assessments and from all adults and selected PND 4 and PND 13 pups for thyroid hormone assessments. Selected organ weights were recorded for all adult males and anatomic (microscopic) pathology assessments were conducted on selected tissues from adults and on the thyroid for PND 13 pups.

No unscheduled deaths occurred, and no toxicologically significant clinical or post-dose observations were observed.

Administration of 600 mg/kg/day resulted in lower body weight gains for males, compared with controls and initial reductions in food consumption. Slight reductions in body weight gain were also noted for females during the first 2 weeks of dosing. Mating performance was not affected; however, no pregnancies were evident for any female following administration of 600 mg/kg/day. Significant reductions in sperm counts and lower sperm motility were noted in males administered 600 mg/kg/day, compared with controls. Macroscopic examinations revealed small testis and epididymis and lower reproductive organ weights (testis, epididymis, prostate, levator ani muscle / bulbocavernosus complex and right bulbo-urethral gland) were noted at this dose level, compared with controls. Microscopic examinations revealed colloid depletion/follicular cell hypertrophy of the thyroid gland for all males and an increased incidence in vacuolation of the pituitary gland was also present for most males administered 600 mg/kg/day, compared with concurrent controls. Thyroid stimulating hormone (TSH) levels were elevated for males administered 600 mg/kg/day, compared with controls. Haematology assessments also revealed lower haemoglobin, red blood cell, packed cell volume, mean cell volume, mean cell haemoglobin concentration, haemoglobin distribution width, platelets, platelet crit and eosinophils for males, compared with controls. Reticulocyte (counts and percentage) and large unstained cells were increased, compared with controls. Due to different physiological status, control data could not be accurately compared with data obtained from females administered 600 mg/kg/day.

Similar findings were noted following 175 mg/kg/day administration. Slightly lower body weights were observed for both sexes during the first 2 weeks of dosing, compared with controls. Mating performance was not affected although no pregnancies were evident for any female at this dose level. Reductions in sperm count and lower sperm motility were noted for males administered 175 mg/kg/day, compared with controls and small testis and small epididymis were recorded macroscopically. Microscopic examination of tissues from 175 mg/kg/day males revealed colloid depletion/follicular cell hypertrophy of the thyroid glands and an increased incidence in vacuolation of the pituitary in some males. Increases in TSH were evident for males administered 175 mg/kg/day compared with controls and haematology assessments also showed a slight reduction in haemoglobin and increase in % reticulocytes, compared with controls. Due to different physiological status, control data could not be accurately compared with data obtained for females administered 175 mg/kg/day.

Animals administered 50 mg/kg/day showed no adverse effect on body weight, food consumption, mating performance, fecundity or fertility, with all females of this group becoming pregnant and bearing live pups. Seminology assessments revealed slight reductions in sperm counts although sperm motility and morphology was not affected, and no test material-related microscopic findings were noted in the testis or epididymis of males administered 50 mg/kg/day. Microscopic changes in the thyroid consisted of colloid depletion/follicular cell hypertrophy of the thyroid gland present in some males administered 50 mg/kg/day.

For the pups delivered from females administered 50 mg/kg/day, no test materialrelated differences in litter size or survival indices were noted. Furthermore, no test material-related clinical observations, pup deaths, adverse effects on body weight or ano-genital distance were noted from 50 mg/kg/day litters and there was no evidence of nipple retention. Thyroid stimulating hormone (TSH) was slightly increased for the PND 4 pups from the 50 mg/kg/day litters, compared with controls, and was also higher for PND 13 male and females, however, there were no correlating microscopic changes in the pup thyroid gland from this dose level.

Under the conditions of this study, dosing of the test material resulted in adverse effects on male fertility following administration of 600 or 175 mg/kg/day. Fertility was not affected following administration of 50 mg/kg/day, therefore a ‘no observed adverse effect level’ (NOAEL) for male reproductive toxicity was established as 50 mg/kg/day.

No adverse effects were noted for females administered 600 or 175 mg/kg/day. The absence of pregnancies at these dose levels was considered to have resulted from an adverse effect on the male reproductive system. As such, a NOAEL for reproductive toxicity was established as 600 mg/kg/day for females.

The NOAEL for offspring development was considered to be 50 mg/kg/day in the absence of any adverse effects noted at this dose level.