2-ethylhexyl 4-(dimethylamino)benzoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A Reproduction dose range-finding Study in rats was available on 2-ethylhexyl 4-(dimethylamino)benzoate. Reproductive performance was affected in males and females. The same results were obtained in the reproduction screening study performed on a close analogue. 

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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

Qualifier:

according to guideline

Guideline:

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

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies because of its reproductive characteristics.

Sex:

male/female

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

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

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.

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.

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

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.

Positive control:

no

Parental animals: Observations and 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: Male body weights were recorded once during acclimatisation, on the first day of dosing, at weekly intervals thereafter, and on the day of necropsy (prior to necropsy). 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 (both sexes) and during the post-pairing phase for males. 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: 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).

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

For each male, sperm number, motility and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from one epididymis and epididymis cauda. Each sample was prepared for microscopic evaluation of sperm morphology.
Slides prepared from Groups 1 and 2 males were examined for morphological changes of the sperm.

Litter observations:

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed on Days 54 or 55 of study (Post-Pairing Day 26 or 27) by isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. Blood sampling was performed. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
- Maternal animals: 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.
Thyroid Hormone Tests: Total T4 (thyroxine) and TSH (thyroid stimulating hormone).

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): Cowper’s gland (bulbo-urethral gland, W), epididymis (W, E), glans penis (W), gross lesions, levator ani plus bulbocavernosus muscle complex (W), mammary gland, vagina (E), ovary (W, E), oviduct (W), pituitary (E), prostate (W), seminal vesicle (with coagulating glands, W), testis (including Tunica albuginea, W,E), thyroid and parathyroid (W, 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.

Postmortem examinations (offspring):

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.

Reproductive indices:

Male and female mating, fecundity, fertility, percent pregnant, percent delivering, gestation, percent post-implantation loss, live birth and survival indices.

Offspring viability indices:

Number of pups born and number of pups alive on Days 1, 4 (before and after culling), 7 and 13 post-partum.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant clinical observations were observed.
Two males administered 600 mg/kg/day and one male administered 175 mg/kg/day were noted with tears in the eye during the pairing phase. Another male administered 175 mg/kg/day was noted with moderate salivation on two occasions during the pairing phase. 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. This was also noted for one control male on one occasion during the pairing phase. 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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gains were noted during the first 2 weeks of dosing for males administered 600 mg/kg/day, compared with controls. Slightly lower body weight gains were also evident thereafter, and overall body weight gain was approximately 29 % lower in this group, compared with controls. Lower body weight gain was also 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 for both sexes administered 175 mg/kg/day during the first 2 weeks of dosing, compared with controls. However, statistical significance was not achieved, and mean values for males were essentially similar to controls thereafter.
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. Slightly lower body weight gain was noted during the first week of dosing (-16 %) for males administered 50 mg/kg/day although values were similar to controls during Week 2. 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 for both sexes. 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):

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects on food consumption were noted. Slightly lower food consumption was noted during the first 5 days of dosing (-10 %) for males administered 600 mg/kg/day, compared with controls. Although this was noted, food consumption was approximately 20 % higher than controls after mating until necropsy, and overall food consumption for the duration of the study was 11 % higher than controls; as such, the initial reduction in food consumption was considered not to represent an adverse effect of test material administration.
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:

effects observed, treatment-related

Description (incidence and severity):

Haematology assessments revealed significantly 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 administrated 600 mg/kg/day, compared with controls. Reticulocyte (counts and percentage) and large unstained cells were significantly higher for males administrated 600 mg/kg/day compared with controls. Males administered 175 mg/kg/day also showed a slight reduction in haemoglobin and increase in % reticulocytes compared with controls. No effect was noted for males administered 50 mg/kg/day compared with controls.
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.

Increases in TSH were evident for males administered 175 mg/kg/day (+29 %) or 600 mg/kg/day (+64 %), compared with controls. Statistical significance was never achieved due to the high degree of individual variability and values were within the historical background control range. 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, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test material-related microscopic findings were noted in the testis or epididymis of males administered 50 mg/kg/day.
No histopathological examination was performed of the testis or epididymis from males administered 175 or 600 mg/kg/day and no histopathological examination was performed of the prostate, levator ani muscle / bulbocavernosus complex or right bulbo-urethral gland of males from any dose group.
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.
In the pituitary, an increased incidence in vacuolation was present in few males administered 175 mg/kg/day and in most males administered 600 mg/kg/day compared with concurrent controls. This was characterised by large, clear vacuoles in the cells of the pars distalis.
In the thyroid gland, colloid depletion/follicular cell hypertrophy was present in one control, in some males administered 50 or 175 mg/kg/day and in all males administered 600 mg/kg/day. This was characterised by a decreased colloid content in the follicles and/or an increase in the size of follicular epithelial cells.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

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.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Seminology assessments revealed significant reductions in sperm counts, with a clear dose relationship; -27, -50, and -85 % for males administered 50, 175 or 600 mg/kg/day, respectively, compared with controls. Summary of seminology data can be seen in Table 2.
Lower sperm motility was also evident following administration of 175 or 600 mg/kg/day (-77 or -86 %, respectively), compared with controls.
Due to the adverse effects noted in males administered 175 or 600 mg/kg/day; sperm morphology was only performed for control and 50 mg/kg/day males. The incidence of abnormal sperm was only marginally higher for males administered 50 mg/kg/day, compared with controls (0.9 compared with 0.5 %); as such, this was considered to be incidental.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating performance was not affected by test material administration (with the exception of two animals, all mated within the first 5 days of pairing), although 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.
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 gestation length, implantation sites or litter sizes or survival indices were noted for animals administered 50 mg/kg/day, compared with controls.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

175 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

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.

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on mean pup body weights was noted from 50 mg/kg/day litters, compared with controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

-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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

175 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Summary of Male Organ Weight %-Differences, Compared with Concurrent Controls - Terminal Sacrifice

			Males
			2M	3M	4M
		Level (mg/kg/day)	50	175	600
			% difference
Testis	group mean body weight ratio		+8.185	-1.280	-48.004
	unadjusted weights ratio		+3.877	-4.128	-51.600
Epididymis	group mean body weight ratio		+0.313	-5.821	-30.432
	unadjusted weights ratio		-3.737	-8.351	-35.195
Muscle, Levator Ani Plus Bulbocavernosus Complex	group mean body weight ratio		-6.912	-2.985	-15.549
	unadjusted weights ratio		-10.508	-4.805	-20.047
Prostate	group mean body weight ratio		+6.025	+6.350	-9.593
	unadjusted weights ratio		+1.370	+4.321	-15.869
Bulbo-urethral gland - R	group mean body weight ratio		-4.189	-10.780	-14.156
	unadjusted weights ratio		-6.349	-11.224	-18.594

Table 2: Summary of Seminology Data

	Group 1	Group 2	Group 3	Group 4	Statistics
Number of males examined	10	10	10	10
Mean total sperm count (M/g)	866.4	633.8*	433.9**	126.6***	K
Mean % motile	89.2	78.8	20.1***	12.3***	K
Mean VAP (µm/s)	179.6	148.9	54.8***	113.6**	K
Mean VCL (µm/s)	300.0	266.6	110.5***	192.2**	K
Mean VSL (µm/s)	131.6	108.2	44.5***	71.0**	K
Mean STR (%)	71.4	72.5	50.6	34.6**	K
Mean abnormal sperm (%)	0.5	0.9	-	-	W

VAP = average path velocity

VSL = straight line velocity

VCL = curvilinear velocity          

STR = straightness (VSL:VAP)

- = not examined

*P = 0.05; **P = 0.01; ***P = 0.001

K  = Kruskal-Wallis, Wilcoxon

W = Wilcoxon rank sum test

Table 3: Summary of Male Fertility and Reproductive Performance

Treatment Group	Control	50 mg/kg	175 mg/kg	600 mg/kg
Total males	10	10	10	10
Unscheduled Deaths Prior to Cohabitation	0	0	0	0
Males Cohabitated	10	10	10	10
Unscheduled Deaths During Cohabitation	0	0	0	0
Males mating with at least 1 female	10	10	10	9
Males impregnating at least 1 female	10	10	0	0
Mating Index (%)	100	100	100	90
Fecundity Index (%)	100	100	0#	0#
Fertility Index (%)	100	100	0#	0#

Mating index % = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100

Fecundity index % = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100

Fertility Index % = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100

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

Table 4: 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/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 5: 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 6: 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.

Conclusions:

Under the conditions of this study the NOAEL for male reproductive toxicity was established as 50 mg/kg/day. The NOAEL for female reproductive toxicity was established as 600 mg/kg/day. 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 post-coitum 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 material-related 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.

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

Dose Range Finding Study of Fertility and Early Embryonic Development

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 December 2017 to

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was not designed for specific guidelines, but was expected to meet the requirements of the relevant agencies. The objective of the study was to determine the systemic and reproductive effects of the test material, following daily oral (gavage) administration to the rats, including 14 days pre-pairing, pairing and post-pairing for males (up to 28 days) and to Gestation Day 12 for females, and to provide the basis for the selection of dose levels for subsequent toxicity studies.

GLP compliance:

no

Remarks:

This study is a non regulatory study for which a claim of Good Laboratory Practice (GLP) compliance will not be made. However, the laboratory procedures used were consistent with international standards of GLP.

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. Background data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males were 11 to 12 weeks of age, while females were 9 to 10 weeks of age.
- Weight at study initiation: Males weighed between 309.1 and 400 g; females weighed between 157 and 203.9 g.
- Fasting period before study: No
- Housing: During the pre-pairing phase, animals were housed in groups of up to three 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. 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.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Animals were acclimatised for at least 10 days prior to initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 – 70 %
- Air changes: 15 to 20 air changes/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.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared weekly.
- The test material was formulated in Corn Oil following dispensary SOPs and the formulation method (Method 8375573_O_01D).
- Formulations were stored refrigerated (2 to 8 °C) in a sealed container, protected from light.
- Formulations were stirred from at least 30 minutes and continuously before dosing commenced and throughout dosing.

VEHICLE
- Corn oil was de-gassed with nitrogen prior to use
- Amount of vehicle: 4 mL/kg. Individual dose volumes were based on individual body weights.

Details on mating procedure:

- M/F ratio per cage: 1:1
- During the pairing procedure, one male was housed for up to 9 days with one female 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 washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed. The day on which mating was confirmed was designated GD 0.
- The female without evidence of mating by Pairing Day 9 (Animal R0702 [Group 4 female]) was paired for up to an additional 5 days with a proven male of the same dose group. The male without evidence of mating by Pairing Day 9 (Animal R0302 [Group 4 male]) was paired with an undosed female for up to 5 days.
- Following mating, females were housed individually during gestation. Males were returned to group housing after the pairing phase.

Analytical verification of doses or concentrations:

no

Remarks:

Study sampling and analysis was not required.

Duration of treatment / exposure:

Males were dosed once daily for 28 days (pre-pairing, pairing and post-pairing, where appropriate), females were dosed for up to 32 days (pre pairing, pairing, and gestation or post-pairing), excluding the day of necropsy.

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The high-dose level of 300 mg/kg/day was intended to demonstrate an effect on male fertility. The intermediate-dose level of 200 mg/kg/day was expected to produce marginal or no effect on male fertility. The low-dose level of 100 mg/kg/day was expected to produce no effect on male fertility.
A structurally related substance was tested previously in the rat in Study WDB 31/89829 and Covance Study 8349417. The test material was previously administered to rats by oral gavage in a formulation prepared in corn oil at the dose levels of 6, 74, or 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 there were more marked clinical signs, including hunched posture, lethargy, and gasping respiration. 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 revealed moderate tubular atrophy for all males, with two males also 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. Haemopoiesis for all animals and minimal/moderate extramedullary haemopoiesis were observed in the spleen.
Covance Study 8349417: Daily oral gavage administration of 50, 175, or 600 mg/kg/day to male rats for up to 54 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 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. This correlated with the low sperm counts in males and the failure to achieve pregnancy in females administered 600 mg/kg/day due to effects on the male reproductive systems.

- The oral route of administration was chosen because it is the possible human exposure route.

- Rationale for animal assignment: Upon arrival, animals were assigned to dose groups using a total randomisation procedure. Animals were individually identified by electronic implant. Following the first full weight collection (Day 1 of the predose phase), group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups. Animal R0101 was replaced with a spare animal (Animal R0400 [male]) to achieve uniform mean body weight between groups, as appropriate.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each male animal was given a detailed physical examination once during acclimatisation, then once weekly from the start of dosing, and on the day of terminal necropsy. Each female was given a detailed physical examination once during acclimatisation, then once weekly from the start of dosing until confirmation of mating, and on GD 0, 3, 6, 10, and 13.
- An individual record of the clinical condition of each animal was maintained.
- Post dose observations were not required. Return to cage observations noted in females from Groups 2 through 4 on Pairing Day 1/GD 0 are maintained within the raw data.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded once during acclimatisation, on the first day of dosing, and at weekly intervals thereafter. Female body weights were recorded once during acclimatisation; on the first day of dosing; at weekly intervals until confirmation of mating; and on GD 0, 3, 6, 10, and 13. The body weights recorded for the non-mated female, Animal R0702 (Group 4 female), during the pairing phase are also reported.

FOOD CONSUMPTION: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes). Daily food consumptions were recorded for females from GD 0 to 13.
- Consumption was calculated as g/animal/day.
- Food consumption was not recorded during the pairing phase for both sexes and post-pairing phase for males.

WATER CONSUMPTION: No

Sperm parameters (parental animals):

Parameters examined in P0 male parental generations: testis weight and epididymis weight

Postmortem examinations (parental animals):

SACRIFICE
Mated Females:
- Mated females were sacrificed on GD 13 by cervical dislocation following isoflurane anaesthesia; death was confirmed by exsanguination. Animals were sacrificed in a controlled randomisation order. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
- The ovaries and uteri were removed and examined, and the following data were recorded: Pregnancy status, Number of corpora lutea, Number and intrauterine position of implantations subdivided into: Live embryos, Early intrauterine deaths and Late intrauterine deaths. Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue, in addition to placental tissue. Implantations were allocated numbers relating to their position in utero: Left horn, L1 to L10, Cervix, R11 to R20, Right horn.
- Following examination, all maternal tissue was discarded.
- The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.

Non-Mated Female
- The non-mated female (Animal R0702 [Group 4 female]) was sacrificed by isoflurane anaesthesia on Post-Pairing Day 5. Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal. The uterus of the Animal R0702 (Group 4 female) was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

Males
- Males were sacrificed by isoflurane anaesthesia on Day 1 after the 14-day pairing period. Animals were sacrificed in a controlled randomisation order. Once a suitable deep plane of anaesthesia was established, the major blood vessels were severed to exsanguinate the animal. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

GROSS NECROPSY
- Animals were weighed before necropsy.
- The following organs from all animals were dissected, freed from fat and other contiguous tissue, and weighed before fixation: Epididymides, ovary, pituitary, prostate (including coagulating gland), seminal vesicle, testis, thyroid and parathyroid.
- The following tissues from each animal were retained in 10 % neutral buffered formalin, unless otherwise indicated: Animal identification, epididymides (Modified Davidsons fixative), gross lesions, ovary, pituitary, prostate (including coagulating gland), seminal vesicle, testis (Modified Davidsons fixative), thyroid and parathyroid and vagina.

Statistics:

Data from test material-treated animals were compared with control data. Statistical analyses were performed, where appropriate.
Data for each sex were analysed separately, unless stated otherwise. Only data collected on or after the first day of dosing will be analysed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P = 0.05. By default, significant results were reported as * P = 0.05, + P = 0.01, and/or # P = 0.001.
The following data were analysed using Pristima:
- Body weights - Procedure I (ANOVA) - Pristima.
- Body weight gains- Procedure I (ANOVA) - Pristima.
- Food consumption (gestation) - Procedure I (ANOVA) - Pristima.
- Absolute organ weights and organ to terminal body weight ratios - Procedure I (ANOVA).
- Male and female mating, fecundity, and fertility indices - Procedure IV (one sided lower tail).
- The number of corpora lutea, implantation sites, early resorptions, and percent pre- and post-implantation loss - Procedure III.

Reproductive indices:

- Female mating index = (Mated females / Females cohabitated (excluding females sacrificed during cohabitation)) x 100
- Male mating index = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) x 100
- Female fecundity index = (Pregnant females / Mated females (excluding females with an undetermined pregnancy status)) x 100
- Male fecundity index = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) x 100
- Female fertility index = (Pregnant females / Females cohabitated (excluding females sacrificed during cohabitation or with an undetermined pregnancy status)) x 100
- Male fertility index = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) x 100
- % pre-implantation loss = [( Number of corpora lutea - number of implantations) / Number of corpora lutea] x 100
- % post-implantation loss = [(Number of implantations - number of live embryos) / Number of implantations] x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No adverse clinical observations were noted. However, thinning of the fur was observed for one male administered 300 mg/kg/day (Animal R0301), and thinning/staining of the fur was noted for three females administered 100 mg/kg/day (Animals R0502, R0503, and R0505), one female administered 200 mg/kg/day (Animal R0603), and four females administered 300 mg/kg/day (Animals R0701, R0703, R0704, and R0706). These findings during gestation period, at least for females administered 100 mg/kg/day, along with body weight gain and food consumption decreases, indicate general lack of grooming and is assumed to be indirectly associated to test material administration.
- Additionally, on one occasion most mated females administered 100 to 300 mg/kg/day had mouth rubbing post dose administartion. This finding mostly associated with taste of test material was transient and was not considered adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weight increased for all groups as the study progressed. Overall mean body weight gain (Pre-paring Day 1 to Pairing Day 14) was lower by 20 % for males administered 300 mg/kg/day that was attributed to lower body weigh gains during first week of pre-pairing and second week of pairing. Mean body weight gain was lower by 7 % during pre-pairing phase (Pairing Days 1 to 15) for females administered 200 and 300 mg/kg/day, and statistically significantly lower by 14 % during the gestation phase (Gestation Day 0 to 13) for females administered 100 mg/kg/day, compared with controls. These findings were considered test material-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Mean food consumption was not affected for males.
- For females, food consumption was not affected during the pre-pairing phase, but lower food consumption was noted for most of the days during Gestation period for females administered 100 mg/kg/day and attained statistical significance on GD 12 to 13, compared with control. This finding was attributed to the test material.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- All males mated successfully. All females mated within 4 days, except one female administered 300 mg/kg/day (Animal R0702), which did not mate despite changing the mating partner from Pairing Day 9. However, its male mating partner administered 300 mg/kg/day (Animal R0302) successfully mated with an untreated female (Animal R0799) when cohabited from Pairing Day 9.
- Mating index for males was 100 % in all groups, including controls and all test material treated groups. Mating index was 83 % for females administered 300 mg/kg/day, compared with 100 % for control females and females administered 100 or 200 mg/kg/day. The lower mating index for females was due to an unmated female administered 300 mg/kg/day (Animal R0702). This was an isolated female and its male partner administered 300 mg/kg/day (Animal R0302) mated successfully; as such, the finding was considered incidental in nature.
- The fertility and fecundity indices were 0 % for animals administered 200 or 300 mg/kg/day, compared with 100 % for controls and animals administered 100 mg/kg/day. This finding was attributed to a test material-related effect on male reproductive performance.

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive performance)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive performance)

Effect level:

< 100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Number of implantation sites, number of live embryos, and the percentage of pre-implantation loss were affected in females administered 100 mg/kg/day and were considered adverse.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- Caesarean section data were only available for controls and females administered 100 mg/kg/day. For females administered 100 mg/kg/day, the mean number of implantation sites were lower (8 versus 12 in controls), the percentage of pre implantation loss was substantially higher (36.6 versus 7.9 % in controls), and the mean number of live embryos was lower (7 versus 10 in controls). These findings were considered test material related.
- Early and late resorptions and percentage of post-implantation loss were not affected.

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Remarks:

The study was performed to determine the systemic and reproductive effects of the test material following daily oral (gavage) administration to the rats, including 14 days pre-pairing, pairing and post-pairing for males (up to 28 days) and to Gestation Day 12 for females, and to provide the basis for the selection of dose levels for subsequent toxicity studies.

Critical effects observed:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this study, 100 mg/kg/day is considered the reproductive no observed adverse effect level (NOAEL) for males, while a NOAEL could not be determined for females.

Executive summary:

The objective of the study was to determine the systemic and reproductive effects of the test material, following daily oral (gavage) administration to the rats, including 14 days pre-pairing, pairing and post-pairing for males (up to 28 days) and to Gestation Day 12 for females, and to provide the basis for the selection of dose levels for subsequent toxicity studies.

Four groups of six Crl:WI(Han) rats/sex/group were administered 0 (control material [vehicle: Corn Oil, de-gassed with nitrogen prior to use]), 100, 200, or 300 mg/kg/day by oral gavage at a volume of 4 mL/kg. Males were sacrificed on Day 1 after the 14 day Pairing period and females on Gestation Day (GD) 13, respectively; macroscopic examinations were conducted and organs weight (testis, epididymides, seminal vesicle, prostate with coagulating glands, ovaries, and pituitary and thyroid/parathyroid glands) were measured. The uterus was examined for pregnancy status, number of corpora lutea, and number and intrauterine position of implantations (live embryos, early intrauterine deaths, and late intrauterine deaths).

Assessment of toxicity was based on clinical observations, body weights, food consumption, mating, fertility, and pregnancy indices. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights were conducted as indicated.

No mortality or adverse clinical observations were noted.

Overall mean body weight gain was 20 % lower for males administered 300 mg/kg/day, compared with controls. Mean body weight gain was reduced by 7 % during pre-pairing phase for females administered 200 and 300 mg/kg/day, and statistically significantly reduced by 14 % during the gestation phase for females administered 100 mg/kg/day, compared with controls. Lower food consumption was noted for most of the days during Gestation period for females administered 100 mg/kg/day and attained statistical significance on GD 12 to 13, compared with controls. These finding were considered test material-related.

The fertility and fecundity indices were 0 % for animals administered 200 or 300 mg/kg/day, compared with 100 % for controls and animals administered 100 mg/kg/day. These were attributed to a test material-related effect on male reproductive performance.

For females administered 100 mg/kg/day, the mean number of implantation sites were lower (8 versus 12 in controls), the percentage of pre implantation loss was substantially higher (36.6 versus 7.9 % in controls), and the number of live embryos was lower (7 versus 10 in controls). These findings were considered an adverse effect of test material.

At necropsy, macroscopic observations observed in some test material-treated males were slight/severe small thyroid, slightly pale/mottled kidney, dark focus on the thymus and lungs, slightly small testis, pale and raised area on the epididymis, and slightly large seminal vesicle. The toxicological relevance was not completely clear in the absence of microscopic examination of lesions.

A test material-related effect on organ weights was observed for both sexes. Statistically significantly lower absolute and relative (to terminal body weight) testis weights (approximately 20 %) and slightly lower prostate and thyroid weights were noted for males administered 300 mg/kg/day. For females administered 100 mg/kg/day, the absolute ovary weight was statistically significantly lower (18%).

In conclusion, daily oral gavage administration of the test material at 100, 200, or 300 mg/kg/day to groups of male and female Crl:WI (Han) rats during the pre pairing, pairing, and gestation phases (up to GD 12) showed test material-related effects. Body weight gain was lower during pre-pairing phase for females administered 200 and 300 mg/kg/day, and statistically significantly lower, with correlating decreases in food consumption, during the gestation phase for females administered 100 mg/kg/day. Body weight gain was also lower throughout the study for males administered 300 mg/kg/day. Statistically significantly lower testis weights were observed for males administered 300 mg/kg/day, and statistically significantly lower ovary weights were observed for females administered 100 mg/kg/day. Lower prostate and thyroid weights were also observed for males administered 300 mg/kg/day. Reproductive performance was affected in males administered 200 or 300 mg/kg/day as evident by lower fertility and fecundity indices. Number of implantation sites, number of live embryos, and the percentage of pre-implantation loss were affected in females administered 100 mg/kg/day and were considered adverse.

Under the conditions of this study, 100 mg/kg/day is considered the reproductive no observed adverse effect level (NOAEL) for males, while a NOAEL could not be determined for females.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study was performed to a standardised guideline under GLP conditions, therefore the quality of the database is considered to be good.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproduction screening study (OECD 421) in analogue substance (Read-across)

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 post-coitum 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 material-related 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.

 

Reproduction dose range-finding Study on EHA (Holalagoudar, 2018)

The objective of the study was to determine the systemic and reproductive effects of the test material, following daily oral (gavage) administration to the rats, including 14 days pre-pairing, pairing and post-pairing for males (up to 28 days) and to Gestation Day 12 for females, and to provide the basis for the selection of dose levels for subsequent toxicity studies.

Four groups of six Crl:WI(Han) rats/sex/group were administered 0 (control material [vehicle: Corn Oil, de-gassed with nitrogen prior to use]), 100, 200, or 300 mg/kg/day by oral gavage at a volume of 4 mL/kg. Males were sacrificed on Day 1 after the 14 day Pairing period and females on Gestation Day (GD) 13, respectively; macroscopic examinations were conducted and organs weight (testis, epididymides, seminal vesicle, prostate with coagulating glands, ovaries, and pituitary and thyroid/parathyroid glands) were measured. The uterus was examined for pregnancy status, number of corpora lutea, and number and intrauterine position of implantations (live embryos, early intrauterine deaths, and late intrauterine deaths).

Assessment of toxicity was based on clinical observations, body weights, food consumption, mating, fertility, and pregnancy indices. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights were conducted as indicated.

No mortality or adverse clinical observations were noted.

Overall mean body weight gain was 20 % lower for males administered 300 mg/kg/day, compared with controls. Mean body weight gain was reduced by 7 % during pre-pairing phase for females administered 200 and 300 mg/kg/day, and statistically significantly reduced by 14 % during the gestation phase for females administered 100 mg/kg/day, compared with controls. Lower food consumption was noted for most of the days during Gestation period for females administered 100 mg/kg/day and attained statistical significance on GD 12 to 13, compared with controls. These finding were considered test material-related.

The fertility and fecundity indices were 0 % for animals administered 200 or 300 mg/kg/day, compared with 100 % for controls and animals administered 100 mg/kg/day. These were attributed to a test material-related effect on male reproductive performance.

For females administered 100 mg/kg/day, the mean number of implantation sites were lower (8 versus 12 in controls), the percentage of pre implantation loss was substantially higher (36.6 versus 7.9 % in controls), and the number of live embryos was lower (7 versus 10 in controls). These findings were considered an adverse effect of test material.

At necropsy, macroscopic observations observed in some test material-treated males were slight/severe small thyroid, slightly pale/mottled kidney, dark focus on the thymus and lungs, slightly small testis, pale and raised area on the epididymis, and slightly large seminal vesicle. The toxicological relevance was not completely clear in the absence of microscopic examination of lesions.

A test material-related effect on organ weights was observed for both sexes. Statistically significantly lower absolute and relative (to terminal body weight) testis weights (approximately 20 %) and slightly lower prostate and thyroid weights were noted for males administered 300 mg/kg/day. For females administered 100 mg/kg/day, the absolute ovary weight was statistically significantly lower (18%).

In conclusion, daily oral gavage administration of the test material at 100, 200, or 300 mg/kg/day to groups of male and female Crl:WI (Han) rats during the pre pairing, pairing, and gestation phases (up to GD 12) showed test material-related effects. Body weight gain was lower during pre-pairing phase for females administered 200 and 300 mg/kg/day, and statistically significantly lower, with correlating decreases in food consumption, during the gestation phase for females administered 100 mg/kg/day. Body weight gain was also lower throughout the study for males administered 300 mg/kg/day. Statistically significantly lower testis weights were observed for males administered 300 mg/kg/day, and statistically significantly lower ovary weights were observed for females administered 100 mg/kg/day. Lower prostate and thyroid weights were also observed for males administered 300 mg/kg/day. Reproductive performance was affected in males administered 200 or 300 mg/kg/day as evident by lower fertility and fecundity indices. Number of implantation sites, number of live embryos, and the percentage of pre-implantation loss were affected in females administered 100 mg/kg/day and were considered adverse.

Under the conditions of this study, 100 mg/kg/day is considered the reproductive no observed adverse effect level (NOAEL) for males, while a NOAEL could not be determined for females.

Effects on developmental toxicity

Description of key information

A development toxicity study in rat (OECD 414) is available on 2-ethylhexyl 4-(dimethylamino)benzoate. Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 1998 to 19 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HsdCpb:WU

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Versuchstierzucht, in 33178 Borchen/Paderborn
- Age at study initiation: 11 weeks old at start of mating
- Weight at study initiation: 191 g (min. and max. values 173 and 208 g)
- Housing: The rats were housed in cages in air conditioned rooms. Softwood granulate specially manufactured for housing of animals served as the bedding material.
- Diet: Standard diet ad libitum
- Water: Drinking water ad libitum
- Acclimation period: 6 days before the start of mating

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 22 °C
- Humidity: 53 to 75 % (relative)
- Air changes: Not specified; rooms were air conditioned
- Photoperiod: 12 hours of light per day (6:00 a.m. to 6:00 p.m.).

IN-LIFE DATES
From: 16 June 1998
To: 15 July 1998

Route of administration:

oral: gavage

Vehicle:

other: Miglyol 812 neutral oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The preparations of the test material were produced once a week at the testing facility central dispensary. The preparations were provided in amber glass bottles. The preparations were stored at room temperature in the dark. After the contents of the bottles had been mixed thoroughly, the daily rations were filled into brown flasks provided with a magnetic rod. For administration, the flasks were put on a stir bar and stirred continuously.
- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

According to stability tests, the test material is at least stable for 14 days in the preparation used.
The concentration and homogeneity of test material in the preparations administered were determined once during the study. The results can be seen in table 1.
During preparation of the samples for analysis, homogenisation was not performed correctly. This led to the insufficient concentrations in the analysis. For this reason the values are not considered to be valid. It is to be assumed that the animals received the required amounts of the test material.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 M: 4 F
- Length of cohabitation: Overnight. Groups of 4 sexually mature, virgin females were left overnight with one stock stud.
- Proof of pregnancy: On the following morning co-housing, vaginal smears were taken to check for the presence of sperm. The day when sperm was detected was defined as gestation day 0 (GD 0)

Duration of treatment / exposure:

14 consecutive days from GD 6 to 19

Frequency of treatment:

Once daily

Duration of test:

20 days

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose-finding study 5 rats each were treated with oral doses of 100, 300 and 1000 mg /kg of test material from gestation days 6 to 19. At the dose level of 100 mg/kg a slight maternal toxicity could not be excluded. 300 mg had a weak maternotoxic effect: rooting in the bedding and a lower terminal body weight were observed. 1000 mg/kg had a clear maternotoxic effect. In addition to the findings observed at the low and mid doses, reduced food consumption, lower body weight gain and lighter foetuses were observed at this high dose. Based on these results the dose of 40 mg/kg was chosen as the low dose for the main study, at which no toxic effect was expected to occur. 1000 mg/kg was selected as the high dose, as this dose had led to a clear maternotoxic effect, which was expected to occur at the high dose level in the present study as well. The mid dose of 200 mg/kg is the geometric mean of the low and high doses.
- Animal assignment: Using the randomisation generator VS APL 4.0 on an IBM computer.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked included: behaviour and appearance

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6 and then daily until GD 20.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 6, 10, 15 and 20.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Every 3 days

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # GD 20
On GD 20 all rats were killed with halothane in random sequence. Their abdominal cavities were opened and their ovaries removed together with the uteri. The bodies without uteri and foetuses were weighed and recorded as terminal body weights.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
The uteri (containing the foetuses) were weighed. The number of corpora lutea in both ovaries was determined. After opening the uteri the numbers of live and dead foetuses and the numbers of resorptions, as well as their distribution to the left and right uterine horns, were recorded.

The following types of resorption were differentiated:
- Complete resorptions: Resorptions that had occurred shortly after implantation and were detected after staining of the uteri with ammonium sulphide.
- Early resorptions: Diameter of up to 0.5 cm. No macroscopic differentiation between foetal and placental residues possible.
- Late resorptions: Diameter of usually more than 0.5 cm, clear macroscopic differentiation between foetal and placental residues possible.
The total number of implantations was determined by staining the uteri with ammonium sulphide. Females without implantation sites were recorded as non-pregnant.

Blood sampling:

no

Fetal examinations:

- External examinations: Yes: all per litter were examined macroscopically for abnormal deviations and weighed. Their sex was determined on the basis of the anogenital distance and additionally by inspection of the gonads.
- Soft tissue examinations: Yes: foetuses from a randomised half of females were scheduled for transverse section. The foetuses were fixed in formalin and dissected transversely (method after Wilson, 1964) and inspected for organ malformations (modified method after Barrow and Taylor 1967).
- Skeletal examinations: Yes: foetuses from the other half of the females were eviscerated, fixed in alcohol and treated according to the double staining method of Whitaker and Dix (1979); i.e. the soft parts were cleared with potassium hydroxide solution, the skeletons were stained with Alizarin Red S, and the cartilage with Alcian Blue. The foetuses were then examined for skeletal abnormalities and malformations.

Statistics:

Body weight and body weight gain of females, terminal body weight (body weight without uteri and foetuses, Uterus weights (containing the foetuses), food and water consumption and foetus weights were evaluated using a two-sided Dunnett test.
Corpora lutea, implantations were evaluated using a two-sided Fisher-Pitman Permutation test. Live foetuses, dead foetuses, resorptions, malformed foetuses, % live foetuses (related to implantations) and % post-implantation loss (resorptions and dead foetuses related to implantations) were evaluated using a one-sided Fisher-Pitman Permutation test.

Historical control data:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

As a reaction to treatment, 12/25 rats of the mid dose group and 22/25 rats of the high dose group showed salivation after dosing for about 15 minutes on different days. Rooting in the bedding material for about 15 minutes after dosing on different days was seen in all rats of the mid and in all rats of the high dose group.
Furthermore, hair loss was observed in two rats of the control, low and high dose groups, and in 4 rats of the mid dose group. In the mid and high dose groups one rat each had a scabby skin wound. In the mid dose group one rat had a blood crusted eye. These single findings are not regarded as being treatment related.

Mortality:

no mortality observed

Description (incidence):

None of the animals died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development in the low dose group corresponded to that in the control, but it was very slightly (not significantly) lower in the mid dose group.
The body weight in the high dose group was significantly reduced from GD7 until necropsy on GD 20 (at GD20 -10% when compared to the controls) associated with lower boduweight during during all the treatment period (at GD20: -28% when compared to the controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the low and mid dose groups did not differ significantly from the control, but in the high dose group it was found to be significantly reduced after start of dosing until necropsy.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

The water consumption in the low dose group did not differ significantly from the control. It was slightly (significant only for GD 12 to 15 and 18 to 20) increased in the mid dose group and significantly increased in the high dose group from GD 6 until the animals were killed.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine 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, treatment-related

Description (incidence and severity):

The terminal body weight in the low and mid dose groups did not differ significantly from the control, but was significantly lower in the high dose group.

The absolute and relative uterus weights in the low and mid dose groups were similar to the control, but were lower (absolute weight significantly) in the high dose group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormal findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The frequency of the numbers of implantations in the low, mid and high dose groups corresponded to that in the control.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The resorption rates in the low, mid and high dose groups did not differ significantly from that in the control.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The resorption rates in the low, mid and high dose groups did not differ significantly from that in the control.

Dead fetuses:

no effects observed

Description (incidence and severity):

The frequencies of live foetuses in the low, mid and high dose groups were not statistically different form the control. One dead foetus was seen each in the control and low dose group.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The numbers pregnant per group were 24, 23, 23 and 21 per group for the control, low, mid and high dose groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

The frequency of the numbers of corpora lutea in the low, mid and high dose groups corresponded to that in the control.

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

other: no test item-related effect observed at this dose

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The weights of the male and female foetuses in the low dose group were similar to the control. The foetuses in the mid dose group were slightly lighter when compared with controls (-3/5%), but the weights did not differ significantly from the control. In the high dose group, the foetuses were significantly lighter in comparison to the controls (-25% in males and females).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The frequencies of live foetuses in the low, mid and high dose groups were not statistically different form the control. One dead foetus was seen each in the control and low dose group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The ratio of male to female foetuses in the low, mid and high dose groups did not differ significantly from the control.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The ossification status of the foetuses correlated with their body weight. Corresponding to the low body weight, the sternum, coccygeal vertebrae, head bones and extremities in the high dose group were less ossified. A trend to a slightly lower ossification was also seen in the mid dose group. A larger number of kinked ribs was observed in the high dose group as well.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

No teratogenic effect was found.

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no test item-related effect observed at this dose

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Conclusions:

Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.

Executive summary:

The potential for the test material to cause developmental toxicity via the oral route in the rat was investigated in a study conducted in accordance with the standardised guideline OECD 414 under GLP conditions.

The test material was administered by gavage to pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 40, 200, and 1000 mg/Kg. The control rats in group 1 were treated with the vehicle Miglyol 812 neutral oil. The foetuses were delivered by caesarean section on GD 20 and were examined for macroscopic malformations. Half of them were examined for skeletal malformations and half for organ malformations. The terminal body weights of the dams were recorded.

Rooting in the bedding material was seen in all rats in the mid and high dose groups. alivation was seen in half the animals of the mid and in most animals of the high dose group. None of the animals died. Twenty one to 24 rats out of 25 per group proved to be pregnant.

The body weight gain in the mid dose group was very slightly and in the high dose group clearly reduced. The food consumption was reduced in the high dose group. The water consumption in the mid and high dose groups was dose dependently increased. The terminal body weight and the uterus weight were decreased in the high dose group. The numbers of corpora lutea, implantations and live foetuses were not affected. The number of dead foetuses, complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups. The foetuses in the mid dose group were slightly and in the high dose group clearly lighter than those of the controls.

Despite a trend towards retarded ossification in the mid dose group and a clearly retarded ossification in the high dose group, corresponding to the low foetus weights, the skeletal examinations gave no indication of malformations attributable to the treatment with the test material. The frequency of all malformations was in a usual range.

In conclusion, the dose of 40 mg/kg of test material was systemically tolerated by the rats. The dose of 200 mg/kg led to slight maternotoxic effects, such as rooting in the bedding material, salivation, slightly reduced body weight gain and higher water consumption. The foetuses were slightly lighter and there was a trend towards lower ossification. The dose of 1000 mg/kg led to clear maternotoxic effects, expressed by salivation and rooting in the bedding material, reduced body weight gain and reduced food consumption. The foetuses were clearly lighter and the ossification was retarded at 1000 mg/kg/day.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study was performed to a standardised guideline under GLP conditions, therefore the quality of the database is considered to be good.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity study in rats (1998)

The potential for the test material to cause developmental toxicity via the oral route in the rat was investigated in a study conducted in accordance with the standardised guideline OECD 414 under GLP conditions.

The test material was administered by gavage to pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 40, 200, and 1000 mg/Kg. The control rats in group 1 were treated with the vehicle Miglyol 812 neutral oil. The foetuses were delivered by caesarean section on GD 20 and were examined for macroscopic malformations. Half of them were examined for skeletal malformations and half for organ malformations. The terminal body weights of the dams were recorded.

Rooting in the bedding material was seen in all rats in the mid and high dose groups. alivation was seen in half the animals of the mid and in most animals of the high dose group. None of the animals died. Twenty one to 24 rats out of 25 per group proved to be pregnant.

The body weight gain in the mid dose group was very slightly and in the high dose group clearly reduced. The food consumption was reduced in the high dose group. The water consumption in the mid and high dose groups was dose dependently increased. The terminal body weight and the uterus weight were decreased in the high dose group. The numbers of corpora lutea, implantations and live foetuses were not affected. The number of dead foetuses, complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups. The foetuses in the mid dose group were slightly and in the high dose group clearly lighter than those of the controls.

Despite a trend towards retarded ossification in the mid dose group and a clearly retarded ossification in the high dose group, corresponding to the low foetus weights, the skeletal examinations gave no indication of malformations attributable to the treatment with the test material. The frequency of all malformations was in a usual range.

In conclusion, the dose of 40 mg/kg of test material was systemically tolerated by the rats. The dose of 200 mg/kg led to slight maternotoxic effects, such as rooting in the bedding material, salivation, slightly reduced body weight gain and higher water consumption. The foetuses were slightly lighter and there was a trend towards lower ossification. The dose of 1000 mg/kg led to clear maternotoxic effects, expressed by salivation and rooting in the bedding material, reduced body weight gain and reduced food consumption. The foetuses were clearly lighter and the ossification was retarded at 1000 mg/kg/day.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for maternal toxicity and for developmental toxicity was 200 mg/kg/day. At 1000 mg/kg/day, a lower maternal bodyweight was associated to the lower foetuses bodyweight and retarded skeletal ossification. No teratogenic effect was found in this study.

Justification for classification or non-classification

Based on the available data, 2-ethylhexyl 4-(dimethylamino)benzoate should be classified for reproduction toxicity as Category 1B (H360F; May damage fertility) according to the Regulation (EC) No 1272/2008.