Benzyltoluene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 25 August 2020 to 26 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN
According to the OECD 421 Guideline, the post-natal day 13 (PND 13) is considered the last day of dosing in which the parental females and the pups are killed. However, in this study the females were dosed until day 21 post partum (Day 21 p.p.) inclusive and the pups were directly dosed for 2 weeks after weaning from Day 22 to 35 p.p., inclusive. This study design was set up to provide information on dose selection for a possible OECD 443 (test guideline) longer-term study.

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltoluene

EC Number:

248-654-8

EC Name:

Benzyltoluene

Cas Number:

27776-01-8

Molecular formula:

C14H14

IUPAC Name:

1-benzyl-2-methylbenzene; 1-benzyl-3-methylbenzene; 1-benzyl-4-methylbenzene

Test material form:

liquid

Details on test material:

Container: Transparent plastic container
Storage condition: At room temperature
No specific handling conditions

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD®(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age on the first day of dosing: 10 weeks old
- Weight on the first day of dosing: males mean body weight of 407 g (range: 360 g to 456 g); females mean body weight of 273 g (range: 232 g to 323 g)
- Females were nulliparous and non-pregnant
- Fasting period before study: not specified
- Housing: parental animals (F0) were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages with stainless steel lids and containing autoclaved sawdust. Toward the end of gestation and during lactation, autoclaved wood shavings were provided to females and their litter as nesting material. From Day 22 p.p., the offspring were group housed (by 2 or 3 per sex) in polycarbonate cages with stainless steel lids containing autoclaved sawdust. Each cage contained rat hut and nylabone for environmental enrichment.
- Diet: ad libitum, SSNIFF rat/mouse pelleted maintenance diet, batch No. 57266070 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water: ad libitum, tap water from bottles (filtered with a 0.22 µm filter).
- Acclimation period: males were acclimated for a period of 8 days; females were acclimated for a period of 3 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS (TARGET)
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 September 2020 To: 26 November 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) Carboxymethylcellulose (400-800 cps) + 0.5% (w/v) Tween 80 in drinking water treated by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test item formulation was an emulsion in the vehicle. The test item formulation was prepared according to Study No. 48404 VAS (Magaud, 2021) (homogeneity and stability testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study. The frequency of the test item formulation was based on test item dose formulation stability Study No. 48404 VAS (Magaud, 2021) and vehicle expiry. The control dose formulation preparation frequency was identical to that of the test item formulation. The formulations were stored and administered at room temperature.

VEHICLE
- Justification for use and choice of vehicle: based on previous experimental work and/or preliminary study(ies). The vehicle was considered appropriate for the preparation of stable and homogeneous test item formulations. In addition, the vehicle was considered non-toxic to animals.
- Concentration in vehicle: 6, 20 and 60 mg/mL for the low-, mid- and high-dose groups.
- Amount of vehicle: 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurs or 14 days have elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as Day 0 p.c.
- Any female with no evidence of mating after 14 days was euthanized 24 to 26 days after the end of the mating period (unless delivery occurs). If the female delivers, both the female and litter were retained for the lactation period.
- Further matings after two unsuccessful attempts: no
- Pre-coital time was calculated for each female

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method, based on Gas Chromatography with FID detection (GC-FID), was developed and validated in a separate study (No. 48404 VAS, Magaud, 2021) prior to dose formulation analysis.
The determination of test item concentrations in dose formulations was performed on three occasions in the study (on Day 1 and in Weeks 4 and 8). A sample was taken from control and test item dose formulations and analyzed using the validated method. The acceptance criterion for the measured concentration was ± 15% of the nominal concentration.

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:
- males: for 2 weeks before mating, during the mating period (until evidence of mating), until euthanasia (at least 4 weeks in total);
- females: for 2 weeks before mating, during the mating period (until evidence of mating), during gestation, during lactation until Day 21 p.p. inclusive, until euthanasia for females with no delivery.
- F1 offspring: for 2 weeks after weaning (Day 22 to 35 p.p., inclusive).
Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

low-dose group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected on the basis of the results of a previous toxicity study (Study No. 48405 TSR (Haag, 2020)) performed in the same species, in which the test item was administered daily by gavage to five males and five females at the dose level of 100, 300, 600 or 800 mg/kg bw/day for 14 days.
In this study, signs of poor clinical condition (mainly thin appearance, erected fur and/or hunched posture) were observed in males and females at the two highest dose levels after a few days of treatment; these signs were limited to the first week of treatment at 600 mg/kg bw/day or lasted until study termination at 800 mg/kg bw/day. They led to the premature euthanasia of one 800 mg/kg bw/day female on Day 9 (cold to the touch, decreased activity, half-closed eyes, pallor and ventral recumbency were also noted in this female before death). No other premature or unscheduled deaths were recorded.
At 300 mg/kg bw/day, thin appearance was recorded occasionally in males and females, associated with hunched posture in few females.
At 100 mg/kg bw/day, except thin appearance in one female, only non-relevant clinical signs were reported. Hypersalivation was observed, mainly from 300 mg/kg bw/day onwards.
These signs were accompanied by effects on body weight and food consumption in both sexes when compared to controls.
In males, body weight losses or lower body weight gains were recorded at 300, 600 or 800 mg/kg bw/day mainly on Days 1-4 (+6, -13 and -25 g, respectively, vs. +18 g in controls) and to a lesser extent at 800 mg/kg bw/day on Days 4-8 (+5 g vs. +25 g in controls), leading to lower terminal body weight at 300, 600 and 800 mg/kg bw/day (-5, -9 and -13%, respectively, vs. controls). Lower food consumption was recorded, mainly on Days 1-4 at 300, 600 or 800 mg/kg bw/day (-24, -47 and -53%, respectively) and to a lesser extent at 800 mg/kg bw/day on Days 4-8 (-27%). At 100 mg/kg bw/day, lower body weight gain was noted only at treatment initiation.
In females, body weight losses were recorded at 100, 300, 600 or 800 mg/kg bw/day on Days 1-4 (-8, -20, -31 or -23 g, respectively, vs. +4 g in controls), at 600 mg/kg bw/day on Days 8-11 (-8 g vs. +11 g) and at 300, 600 or 800 mg/kg bw/day on Days 11-14 (-3, -17 and -7 g vs. +3 g), leading to lower terminal body weight at 300, 600 and 800 mg/kg bw/day (-7, -13 or 12%, respectively, vs. controls). Lower food consumption was recorded, mainly on Days 1-4 at 100, 300, 600 or 800 mg/kg bw/day (-15, -45, -80 and -55%, respectively), and to a lesser extent at 600 or 800 mg/kg bw/day on Days 4-8 (-10 and -30%, respectively) and at 300, 600 or 800 mg/kg bw/day on Days 11-14 (-14, -48 or -29%, respectively).
Test item-related macroscopic findings were observed in the forestomach, liver, kidneys and spleen.
Findings suggestive of irritation were observed in the forestomach of 2/5 and 3/5 males at 100 and 800 mg/kg bw/day, respectively, and in one female at 800 mg/kg bw/day. Increased liver weight sometimes correlated with macroscopic enlargement was observed at = 100 mg/kg bw/day in males and at = 300 mg/kg bw/day in females. In the kidneys, abnormal color was noted in both sexes at 600 mg/kg bw/day and enlargement was present in a single male at 800 mg/kg bw/day. The spleen was reduced in size in one male at 800 mg/kg bw/day. An equivocal decrease in heart weights was observed in both sexes at =600 mg/kg bw/day.
The doses of 600 and 800 mg/kg bw/day were considered to exceed the maximum tolerated dose in males and females.
Therefore, 300 mg/kg bw/day was selected as the high-dose level for the present study. The low dose and mid-dose were selected using a ratio representing an approximately 3-fold interval (i.e. 30 and 100 mg/kg bw/day).
- Fasting period before blood sampling for clinical biochemistry: no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: from arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs. Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period.

CLINICAL SIGNS: Yes
- Time schedule: from arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until euthanasia.
The body weight of each female was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c.(post-coitum) and on Days 1, 4, 8, 13, 17 and 21 p.p. Females euthanized prematurely were weighed before euthanasia.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8, 8-13, 13-17 and 17-21 p.p.
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION: No

PLASMA HORMONES: Yes
- Time of blood sample collection: on the morning of day of termination (on Day 22 p.p. from all F0 females and at termination from all F0 males)
- Anesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all males and females
- Parameters evaluated: Thyroid hormones T4 (by LC MS/MS) and TSH (by Luminex MAP® technology)

OTHER:
- Females were allowed to litter normally and rear their progeny until weaning. Any sign of a difficult or prolonged parturition was recorded. The morning when parturition was completed was designated Day 1 p.p. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

The oestrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- for the 2 weeks before the beginning of treatment (during the pre-treatment period),
- from the beginning of the treatment period during the pre-mating period (2 weeks) and the mating period (until the females were mated),
- on the day before euthanasia.

Sperm parameters (parental animals):

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups. Maximum of 10 pups/litter (5/sex/litter as nearly as possible); whenever necessary, partial adjustment (for example six males and four females) was permitted. No cross-fostering was performed.
- On Day 22 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible one male and one female per litter (10/sex/group). Whenever necessary, partial adjustment (for example two males or two females/litter) was permitted. No cross-fostering was performed.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- The pups were observed daily for clinical signs and abnormal behavior.
- The body weight of each pup was recorded on Days 1, 4, 8, 13, 17, 21, 22, 25, 29, 32 and 36 p.p.
- The quantity of food consumed by each pup was measured during the post-weaning period for the interval Days 22-25, 25-29, 29-32 and 32-36 p.p.
- Physical development measurements were performed in live pups of each litter: anogenital distance (AGD): on Day 1 p.p., normalized to the cube root of body weight recorded on Day 1 p.p.; number of nipples and of areolae in male pups: on Day 12 p.p.

THYROID HORMONES
Blood samples were taken in the morning as follows:
- at termination on Day 4 p.p. from at least 2 pups/litter culled, by decapitation under isoflurane. When
they were insufficient pups in a litter to have 2 culled pups, only one culled pup was used for blood collection if available, otherwise there was no blood collection on Day 4 p.p.
- at termination on Day 36 p.p. from each F1 pup.
The levels of thyroid hormone T4 (by LC MS/MS) and thyroid stimulation hormone (TSH by Luminex MAP® technology) were determined.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, all surviving F0 animals were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.
- Male animals: after the end of the mating period (at least 4 weeks of treatment in total).
- Maternal animals: on Day 22 p.p. or with total litter loss.

Prematurely euthanized animals:
- During delivery, one female of the high-dose group with difficulties of delivery was euthanized by inhalation of carbon dioxide gas followed by cervical dislocation.
- During the lactation period, three prematurely euthanized females of the high-dose group were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination.

GROSS NECROPSY
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals including euthanized prematurely females. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 22 p.p., for the female euthanized on Day 23 p.c. due to difficulties of delivery and for the three females euthanized during the lactation period.

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 22 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in Table 1 were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids (when applicable). See also "Any other information on materials and methods incl. tables" for major details on microscopic examinations.

Postmortem examinations (offspring):

SACRIFICE
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood sampled, followed by exsanguination when the thyroids were sampled:
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- pups not selected on Day 22 p.p.: on Day 22 p.p.,
- surviving pups: on Day 36 p.p.

Prematurely euthanized animals:
- Moribund pups and pups prematurely euthanized because of dying mother were euthanized by an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- Pups not selected on Day 4 or 22 p.p. were discarded without further examination. Pups euthanized on Day 36 p.p., and those found dead and prematurely euthanized were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs and for found dead and prematurely euthanized pups particular attention was paid to whether the pup had been fed (e.g. presence of milk in the stomach) when possible. After sampling of thyroids with parathyroids, they were discarded without any further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
The body weight of each F1 pup euthanized euthanized on Day 36 p.p. was recorded before euthanasia. Thyroids with parathyroids of each F1 pup were weighed after fixation and a microscopic examination was performed. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated. See also "Any other information on materials and methods incl. tables" for major details on microscopic examinations.

Statistics:

Body Weight, Food Consumption and Reproductive Data
- Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer’s exact probability test (proportions).

Hormones, Anogenital Distance, Nipples/Areolae, Live Birth Index(es), Sex-Ratio and Post-Implantation Loss
- CITOX software was used to perform the statistical analysis of these data according to the sequence provided in the attached item named "Statistics 1".

Organ Weight
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence provided in the attached item named "Statistics 2".

Reproductive indices:

- pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] x 100
- post-implantation loss (calculated with Excel): [(Number of implantation sites - Number of live pups) / Number of implantation sites] x 100
- mating index: (Number of mated animals / Number of paired animals) x 100
- fertility index: (Number of pregnant female partners / Number of mated pairs) x 100

Offspring viability indices:

- gestation index: (Number of females with live born pups / Number of pregnant females) x 100
- live birth index: (Number of live born pups on Day 1 p.p. / Number of delivered pups) x 100
- viability index on Day 4 p.p.: (Number of surviving pups on Day 4 p.p. (before culling) / Number of delivered pups) x 100
- lactation index on Day 21 p.p.: (Number of surviving pups on Day 21 p.p. / Number of surviving pups on Day 4 p.p. (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The incidence of the main clinical signs observed in surviving control and test item-treated animals during the treatment period are reported in Table 2.
Signs of poor clinical condition related to the test item treatment were observed in some females given 100 or 300 mg/kg bw/day, as follows:
- pre-mating period: at 100 or 300 mg/kg bw/day, piloerection, hypoactivity, round back and/or half-closed eyes were noted in 3/10 females in each group. These signs occurred within the first four days of treatment (and also on Day 16 for one high dose female),
- gestation period: at 300 mg/kg bw/day, hypoactivity or emaciated appearance was noted in 2/9 females over a short period in early gestation (Days 3-8 p.c.),
- lactation period: at 300 mg/kg bw/day, round back and emaciated appearance were noted in 1/6 females on Day 12-20 p.p., associated with piloerection on Day 13 p.c.

Ptyalism was observed with a dose-related incidence and frequency in test item-treated males and females given 100 or 300 mg/kg bw/day during the premating, pregnancy and lactation periods. It was also noted in females given 30 mg/kg bw/day, but during the pre-mating period only. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

The other findings reported during the study, i.e. aggressive behavior, reflux at dosing, loud breathing, cutaneous observations on various parts of the body (cutaneous lesions, scabs, spare hair and/or area of hair loss), were considered to be unrelated to the test item as they were present both in control and test item-treated animals and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:

mortality observed, treatment-related

Description (incidence):

No unscheduled deaths occurred in males at any dose level during the study.

The incidence of prematurely euthanized females is shown Table 3.
During the pre-mating period no unscheduled deaths occurred in females at any dose level during the pre-mating period.
During the gestation period one female at 300 mg/kg bw/day was prematurely euthanized due to difficulties to deliver on Day 23 p.c. Signs of poor clinical condition (pallor of extremities, cold to the touch, lateral decubitus and abdominal breathing) were observed on the day of sacrifice. Hypoactivity was observed on Day 5 p.c. and ptyalism was recorded throughout the gestation period. Sixteen dead pups and one live pup were found in the bedding. At necropsy, there were nine and eight scars in the right and left uterus horns, respectively. Translucent liquid was observed in the abdominal cavity, dilated pelvis (right) and granular kidneys were noted, and the wall of the stomach was depressed and presented several black discolorations. This event or premature euthanasia was considered as test item-related given the high mortality of pups observed at this dose level.
During the lactation period at 300 mg/kg bw/day, three females were prematurely euthanized as the pups in their litters died: on Day 1 (2 dead pups at birth), Day 2 (8 dead and 4 live pups at birth) and Day 7 (13 dead and 3 live pups at birth) for each.
Ptyalism was observed in all females on the days before death. Body weight loss (-8%) was recorded for two females between Day 1 p.p. and the day of sacrifice (one female was not weighed before sacrifice), accompanied by reduced food consumption in one female (5 g/day on Days 1-4 p.p. vs. 48 g/day in controls).
There were no relevant necropsy observations in two females. In the third female, thick brown content was noted in the vagina, and both uterine horns contained thick red content.
These premature deaths of litters, leading to the premature sacrifice of the dams, were attributed to the test item treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and mean body weight changes recorded in control and test item-treated animals are summarized in Table 4.

Males
At 300 mg/kg bw/day, when compared to controls, mean body weight gain was zero or significantly lower during most of the treatment period (-35%, p< 0.05 on Days 1-29), leading to slightly lower mean terminal body weight (-6%). These effects on body weight and body weight change were attributed to the test item treatment.
At 30 or 100 mg/kg bw/day, no effects were noted on mean body weight or mean body weight change.

Females
The following test item-related effects on mean body weight or mean body weight changes were noted during the study:
- Pre-mating period: at 100 and 300 mg/kg bw/day, when compared to controls, mean body weight loss or lower mean body weight gain was recorded on Days 1-15 (+4 g, p<0.01 and -5 g, p<0.001, respectively, vs. +26 g in controls), leading to slightly lower mean terminal body weight (-8% at both dose levels, p<0.05 at 100 mg/kg bw/day).
At 30 mg/kg bw/day, when compared to controls, a tendency towards slightly lower mean body weight gain was recorded on Days 1-15 (+18 g vs. +26 g, i.e. -31%, not significant), but this did not significantly impact the Day 15 body weight (-2%).
- Gestation period: mean body weight was lower on Day 0 p.c. at 30, 100 and 300 mg/kg bw/day (-3, -7 and -9%, p<0.05, respectively, vs. controls). This was the result of the mean body weight loss/lower mean body weight gain recorded before mating.
At 300 mg/kg bw/day, when compared to controls, lower mean body weight gain was recorded throughout the gestation period (-27%, p<0.001), leading to statistically significant, lower mean terminal body weight (-15%, p<0.001).
At 100 mg/kg bw/day, when compared to controls, lower mean body weight gain was recorded, mainly on Days 14-20 p.c. (-12%), leading to non-statistically significant, lower mean terminal body weight (-8%).
At 30 mg/kg bw/day, when compared to controls, a tendency towards lower mean body weight gain was recorded during the gestation period (-5%), but this did not significantly impact the Day 15 body weight (-4%).
- Lactation period: mean body weight was lower on Day 1 p.p. at 100 and 300 mg/kg bw/day (-8 and -14%, respectively, vs. controls). This was the result of the lower mean body weight gain recorded during the gestation period.
At 300 mg/kg bw/day, when compared to controls, higher mean body weight gain was recorded on Days 1-21 p.p. (+47 g vs. +20 g, p<0.05), with a mean body weight loss on Days 1-4 p.p. (-10 g, p<0.001, vs. +19 g in controls), leading to mean body weight on Day 21 p.p. not significantly different from the mean control value (-4%). The same effect was noted at 100 mg/kg bw/day (+32 g on Days 1-21 p.p.) with lower mean body weight gain on Days 1-4 p.p. (+10 g), without statistical significance. Mean body weight on Day 21 p.p. was also not significantly different from the mean control value (-4%).
At 30 mg/kg bw/day, when compared to controls, body weight evolution did not vary significantly.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption values recorded in control and test item-treated animals during the study are summarized in Table 5.

Males
At 300 mg/kg bw/day, when compared to controls, mean food consumption was lower on Days 1-8 (-18%, p<0.01). This effect on food consumption was attributed to the test item treatment and correlated with the lack of weight gain reported during the same period.
At 30 and 100 mg/kg bw/day, no effects were noted on mean food consumption.

Females
The following test item-related effects on mean food consumption were noted during the study:
- Pre-mating period: at 100 and 300 mg/kg bw/day, when compared to controls, lower mean food consumption was recorded on Days 1-8 (-13%, p<0.05 and -17%, p<0.01, respectively) and to a lesser extent on Days 8-15 (-8 and -12%, respectively).
At 30 mg/kg bw/day, no effects were noted on mean food consumption.
- Gestation period: at 300 mg/kg bw/day, when compared to controls, lower mean food consumption was recorded throughout the gestation period and was more pronounced on Days 0-14 (around -15%).
At 30 and 100 mg/kg bw/day, no relevant effects were noted on mean food consumption.
- Lactation period: mean food consumption was lower on Days 1-8 and 17-21 at 100 mg/kg bw/day (between -21%, p<0.05 and -13%, p<0.05) or throughout the lactation period at 300 mg/kg bw/day (between -54%, p<0.001 and -24%, p<0.001) when compared to controls.
At 30 mg/kg bw/day, no relevant effects were noted on mean food consumption.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid hormone levels (T4 and TSH plasma levels) in F0 animals are presented in Table 6 and Table 7.

In males, when compared to controls, dose-related, lower mean T4 levels were recorded at all dose levels of the test item (p<0.01). This was associated with higher mean TSH levels in males given 300 mg/kg bw/day (not significant).
These changes were attributed to the test item treatment and correlated at 300 mg/kg bw/day with the thyroid follicular cell hypertrophy observed at microscopy.
In females at 300 mg/kg bw/day, when compared to controls, the mean T4 level was lower (p<0.01). This was associated with higher mean TSH levels (not significant).
These changes were attributed to the test item treatment. No microscopic changes were noted in the thyroid glands.
The lower mean T4 levels in females at 30 or 100 mg/kg bw/day were not attributed to the test item treatment as the differences were not dose-related and the majority of individual values remained within the control range (only 3 values at each dose level were slightly below the lowest control value).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were observed in the thyroid glands in F0 males. Minimal diffuse hypertrophy of thyroid follicular cells was noted in 6/10 males at 300 mg/kg bw/day. This change correlated with the lower T4 and higher TSH levels.
There were no test item-related microscopic observations noted at the end of the treatment period in the ovaries or oviducts and in the thyroid glands of F0 females.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycles were determined from 2 weeks before the beginning of the treatment period until the females were mated. The data are presented in Table 9.
No test item-related effects were noted on the estrous cycle parameters at any dose level.
The higher mean cycle length in females given 30 mg/kg bw/day (7.0 vs. 4.7 days in controls) was due to the contribution of three females in which proestrus or estrus was not detected or was detected only on one or two occasions (these females mated on the first day and were pregnant). These isolated cases were considered as incidental and not dose-related.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations noted at the end of the treatment period in the testes or epididymides from F0 males.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

PAIRING, MATING AND FERTILITY
Refer also to Table 10. No effects were observed on the precoital time (time taken to mate) or on mating/fertility indexes.

All females mated within four days and all females were pregnant. Live born pups were recorded in all females, except for two high dose females: one prematurely sacrificed on Day 23 p.c. due to difficulties to deliver and the other prematurely sacrificed on Day 1 p.p. due to the death of its litter.

DELIVERY AND LITTER DATA
Refer also to Table 11. All pregnant females delivered, but one female given 300 mg/kg bw/day was euthanized on Day 23 p.c. due to difficulties to deliver. This isolated occurrence together with the premature sacrifice of another female on Day 1 p.p. due to the death of its litter (two pups), accounted for the lower gestation index at this dose level (80%), considered as test-item-related.
No test item-related effects were observed on the duration of gestation or pre-implantation loss at any dose level.
At 100 and 300 mg/kg bw/day, when compared to controls, statistically significant, lower mean number of implantation sites was recorded (16.4 vs. 18.9, p<0.05), as a consequence the mean number of pups was significantly lower (14.5 and 12.6, p<0.05, respectively, vs. 16.5) and the post-implantation loss significantly higher at 300 mg/kg bw/day (23.1 vs. 12.6 %).
These findings were considered to be test item-related and adverse at 300 mg/kg bw/day. The number of corpora lutea was lower at 100 and 300 mg/kg bw/day (17.0 and 16.6 vs. 19.0 in controls). A relationship to the test item treatment could not be excluded.
At 30 mg/kg bw/day, no effects were observed on the implantation sites, post-implantation loss or number of pups delivered.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

lower T4 level for all dose groups.

2

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: NOAEL determined

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

reproductive performance

Remarks on result:

other: NOAEL determined

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

PRE-WEANING
Clinical signs are summarized in Table 12. At 300 mg/kg bw/day, when compared to controls, an increased incidence of clinical signs was noted in three litters:
- one female had two pups that were cold to the touch and showed generalized pallor from Day 1 p.p. until death on Day 2 p.p.,
• a second female had two pups with emaciated appearance, associated with dehydration and hypoactivity from Day 4 p.p. until death on Day 7 p.p.,
• a third female had nine pups, which showed emaciated appearance from Day 12 p.p. until
Day 22 p.p., associated with dehydration on Days 13/14 p.p. to 19 p.p. and absence of milk in the stomach on Day 13 p.p. or on Days 13-15 p.p. These findings were considered to have resulted from the poor clinical condition of their dam during the same period (round back and emaciated appearance on Days 12-20 p.p. with piloerection on Day 13 p.p.), and therefore to have resulted from the test item treatment.
The above-mentioned clinical signs were considered to be test item related and correlated with the low viability index at 300 mg/kg bw/day.
The other findings reported during the lactation period, including those recorded at 30 or 100 mg/kg bw/day, were not attributed to the test item treatment as they were transient and/or are common observations in rat pups of this strain and age.

AFTER WEANING
At 300 mg/kg bw/day, thin appearance was observed in 2/10 males and 2/7 females on Days 1 to 3/4. This sign correlated with low individual body weights (29-32 g and 25-29 g in males and females, respectively, vs. mean control values of 64 and 63 g, respectively).
Ptyalism, related to the gavage procedure, was observed in 1/10 males and 2/7 females on Day 14. At 30 and 100 mg/kg bw/day, no clinical signs were observed.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

PRE-WEANING
See also Table 13.
At 300 mg/kg bw/day, when compared to controls, the number of pups found dead on Days 1-4 p.p. of the lactation period was higher (65 vs. 7 pups). This was associated with a higher incidence of affected litters (9/9 vs. 3/10). These findings were attributed to the test item treatment and considered as adverse.
At 30 and 100 mg/kg bw/day, no effects were noted on the incidence of pups found dead or cannibalized.

For pup viability data see also Table 14.
At 300 mg/kg bw/day, when compared to controls, the lower live birth and viability indices, correlating with the higher number of pups found dead between Days 1-4 p.p., were attributed to the test item treatment. The lactation index was unaffected.
At 30 or 100 mg/kg bw/day, no effects were noted on pup viability.

AFTER WEANING
No test item-related deaths occurred at any dose level.
Three females given 300 mg/kg bw/day were euthanized prematurely on Day 3 as these animals were found outside the study room.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

PRE-WEANING
Mean body weights and mean body weight changes in control and test item-treated pups are summarized in Table 15. When compared to controls, mean body weight was lower at birth at 100 mg/kg bw/day (-7 and -6% in males and females, respectively, not significant) and at 300 mg/kg bw/day (-25% in both sexes, p<0.001). Throughout the lactation period, mean body weight gain was lower at both these dose levels, resulting in lower mean body weights on Day 21 p.p. at 100 mg/kg/day (-11 and -15% in males and females, respectively, not significant) and at 300 mg/kg bw/day (-25 and -28% in males, p<0.01, and females, p<0.001, respectively). These differences were attributed to the test item treatment and considered as adverse.
At 30 mg/kg bw/day, no effects were noted on mean body weight or mean body weight gain.

AFTER WEANING
Mean body weights and mean body weight changes in the F1 generation are presented in Table 16. At 100 and 300 mg/kg bw/day, when compared to controls, mean body weights were lower on the first day of treatment, i.e. Day 22 p.p. (-17 and -31% in males and -19 and -32 in females, respectively, p<0.01). This was considered to have resulted from the lower mean body weight recorded before weaning. During the treatment period, dose-related, statistically significant, lower mean body weights were continuously recorded due mean body weight gains generally lower than those of controls (-14 and -32% in males and -6 and -20% in females, respectively, for the whole period, p<0.01 except in females at 100 mg/kg bw/day), leading to terminal mean body weights that were still lower than those of controls (-15 and -32% in males and -11 and -27% in females, respectively, p<0.01). These effects on body weight and body weight change were attributed to the test item treatment and considered as adverse.
At 30 mg/kg bw/day, no effects were noted in males or females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

AFTER WEANING
Mean food consumption recorded in control and test item-treated F1 animals are summarized in Table 17. At 100 and 300 mg/kg bw/day, when compared to controls, dose-related, lower mean food consumption was recorded throughout the treatment period (between -50 and -16% at 300 mg/kg bw/day, and between -24 and -8% at 100 mg/kg bw/day). These effects on food consumption were attributed to the test item treatment and correlated with the lower body weight gains noted at these dose levels.
At 30 mg/kg bw/day, no effects were noted in males or females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid hormone levels (T4 and TSH plasma levels) in pups are presented in Table 18.

PRE-WEANING
In F1 pups on Day 4 p.p., when compared to controls, dose-related, lower mean T4 levels were recorded at all dose levels of the test item (p<0.01 at 30 and 100 mg/kg bw/day). This was associated with higher mean TSH levels in animals at 300 mg/kg bw/day (not significant).

AFTER WEANING
In F1 pups on Day 36 p.p., when compared to controls, dose-related, lower mean T4 levels were recorded at all dose levels of the test item (p<0.05 or p<0.01). This was associated with higher mean TSH levels in animals at 300 mg/kg bw/day (p<0.05 in females).
These changes were attributed to the test item treatment. No microscopic changes were noted in the thyroid glands.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item-related effects on the anogenital distance were observed in males or females at any dose level. See also Table 19. The statistically significant lower mean anogenital distance in males at 300 mg/kg bw/day (not corrected) was considered to be related to the lower mean body weight as confirmed by the ratio AGD/BW cube root.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were noted in any male pups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

PRE-WEANING
Macroscopic post-mortem observations recorded in dead pups during the lactation period are presented in Table 20.
At 300 mg/kg bw/day, the absence of milk in the stomach observed in 40 pups from 7 litters was considered to be test item-related and adverse.
The other observations were not attributed to the test item treatment as they were reported with low incidences and can occur spontaneously in untreated rat pups of this strain and age.

AFTER WEANING
Macroscopic observations noted at the end of the treatment period were not considered to be related to the test item administration since they were distributed randomly among groups, had no histologic correlates or correlated with common histologic findings in control rats.

Histopathological findings:

no effects observed

Description (incidence and severity):

AFTER WEANING
There were no test item-related microscopic observations noted at the end of the treatment period in the thyroid glands from F1 pups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The sex ratio (percentage of males) is presented in Table 21.
At 300 mg/kg bw/day, a higher percentage of males was noted on Days 1 and 21 p.p. when compared to controls. Taking into account the lowest number of pups in this group and the differences which were not statistically significant and not accompanied by other correlating changes (e.g. external genital abnormalities, corrected anogenital distance), this finding was not attributed to the test item treatment.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

30

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

lower T4 levels for all dose groups

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

yes

Any other information on results incl. tables

Chemical Analysis of the Dosage Forms: Concentration

The test item concentrations in the administered dose formulations analyzed in Weeks 01, 04 and 08 remained within an acceptable range of variations (-3.7% to +11.9%) when compared to the nominal values. The results of all samples were found to be within or equal to the acceptance criteria of ± 15% of their theoretical concentrations. No test item was detected in control dose formulations.

 

Table 2 - Incidence of the Main Clinical Signs in Surviving Animals

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Pre-mating (females) or whole study (males)
Loud breathing	-	-	-	-	-	-	-	1/10
Piloerection	-	-	-	-	-	-	3/10	2/10
Round back	-	-	-	-	-	-	2/10	1/10
Hypoactivity	-	-	-	-	-	-	3/10	1/10
Half-closed eyes	-	-	-	-	-	-	1/10	-
Aggressive behavior	1/10	-	-	1/10	-	-	-	-
Ptyalism	-	-	3/10	10/10	-	5/10	5/10	10/10
Reflux at dosing	-	-	-	-	-	-	-	1/10
Gestation^a
Emaciated appearance	/	/	/	/	-	-	-	1/9
Hypoactivity	/	/	/	/	-	-	-	1/9
Ptyalism	/	/	/	/	-	-	7/10	9/9
Reflux at dosing	/	/	/	/	-	1/10	1/10	-
Spare hair	/	/	/	/	-	-	-	1/9
Lactation
Round back	/	/	/	/	-	-	-	1/6
Piloerection	/	/	/	/	-	-	-	1/6
Emaciated appearance	/	/	/	/	-	-	-	1/6
Ptyalism	/	/	/	/	-	-	7/10	6/6
Reflux at dosing	/	/	/	/	-	-	1/10	-

-: no findings; /: not applicable.

ª: pre-mating: one female (high-dose group) excluded.

b: lactation: three females (high-dose group) excluded.

 

Table 3 - Incidence of Prematurely Euthanized Females

Sex	Female
Dose level (mg/kg bw/day)	0	30	100	300
Number of females	10	10	10	10
Gestation
Difficulties to deliver	0	0	0	1
Lactation
Dead litter	0	0	0	3
Total surviving females at scheduled euthanasia	10/10	10/10	10/10	6/10

 

Table 4 - Mean Body Weights/Mean Body Weight Changes (g)

Sex

Male

Female

Dose level (mg/kg bw/day)

0

30

100

300

0

30

100

300

Pre-mating (males and females) or whole study

Day 1

407

404

411

407

271

274

269

279

% from controls

-1

+1

0

+1

-1

+3

Day 8

431

432

435

407

286

286

269

273

% from controls

0

+1

-6

0

-6

-5

Day 15

453

459

456

436

297

292

273*

274

% from controls

+1

+1

-4

-2

-8

-8

Days 1-8

+24

+29

+24

0^#

+15

+12

+1**

-6^#

% from controls

+21

0

/

-20

-93

/

Days 8-15

+22

+26

+22

+29

+11

+6

+4

+1

% from controls

+18

0

+32

-45

-64

-91

Days 1-15

+46

+55

+46

+29*

+26

+18

+4**

-5^#

% from controls

+20

0

-37

-31

-85

-119

Day 22

465

478

466

441

/

/

/

/

% from controls

+3

0

-5

Day 29

486

501

488

458

/

/

/

/

% from controls

+3

0

-6

Days 15-22

+12

+19

+10

+5

/

/

/

/

% from controls

+58

-17

-58

Days 22-29

+21

+23

+22

+17

/

/

/

/

% from controls

+10

+5

-19

Days 1-29

+79

+97

+77

+51*

/

/

/

/

% from controls

+23

-3

-35

Gestation

Day 0 p.c.

/

/

/

/

302

293

280

276*

% from controls

-3

-7

-9

Day 7 p.c.

/

/

/

/

339

328

317

300**

% from controls

-3

-6

-12

Day 14 p.c.

/

/

/

/

381

369

356

335**

% from controls

-3

-7

-12

Day 20 p.c.

/

/

/

/

474

457

438*

402^#

% from controls

-4

-8

-15

Day 0-7 p.c.

/

/

/

/

+38

+35

+37

+24

% from controls

-8

-3

-37

Day 7-14 p.c.

/

/

/

/

+42

+41

+39

+35

% from controls

-2

-7

-17

Day 14-20 p.c.

/

/

/

/

+93

+88

+82

+67**

% from controls

-5

-12

-28

Day 0-20 p.c.

/

/

/

/

+173

+164

+158

+127^#

% from controls

-5

-9

-27

Lactation

Day 1 p.p.

/

/

/

/

360

356

333

310**

% from controls

-1

-8

-14

Day 4 p.p.

/

/

/

/

380

368

344*

310^#

% from controls

-3

-9

-18

Day 8 p.p.

/

/

/

/

391

381

361

340**

% from controls

-3

-8

-13

Day 13 p.p.

/

/

/

/

391

390

380

341**

% from controls

0

-3

-13

Day 17 p.p.

/

/

/

/

399

391

374

348**

% from controls

-2

-7

-13

Day 21 p.p.

/

/

/

/

381

369

365

366

% from controls

-3

-4

-4

Days 1-4 p.p.

/

/

/

/

+19

+13

+10

-10#

% from controls

-32

-47

/

Days 1-21 p.p.

/

/

/

/

+20

+13

+32

+47*

% from controls

-35

+60

x2.4

/: not applicable. Statistical significance: *: p<0.05; **: p<0.01; ^#: p<0.001.

 

Table 5 - Mean Food Consumption (g/animal/day)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Pre-mating
Days 1-8	34	35	33	28**	24	23	21*	20**
% from controls		+3	-3	-18		-4	-13	-17
Days 8-15	32	33	32	33	25	24	23	22
% from controls		+3	0	+3		-4	-8	-12
Gestation
Days 0-7	/	/	/	/	32 (27)^a	26	26	23*
% from controls						-19 (-4)^a	-19 (-4)^a	-28 (-15)^a
Days 7-14	/	/	/	/	31	30	29	26*
% from controls						-3	-6	-16
Days 14-20	/	/	/	/	33	32	32	29
% from controls						-3	-3	-12
Lactation
Days 1-4	/	/	/	/	48	47	38*	22^#
% from controls						-2	-21	-54
Days 4-8	/	/	/	/	68	66	59*	47^#
% from controls						-3	-13	-31
Days 8-13	/	/	/	/	77	77	73	50^#
% from controls						0	-5	-35
Days 13-17	/	/	/	/	89	87	81	68^#
% from controls						+2	-9	-24
Days 17-21	/	/	/	/	100	98	87**	73^#
% from controls						-2	-13	-27

/: not applicable. Statistical significance: *: p<0.05; **: p<0.01; ^#: p<0.001.

^a: one female excluded (suspicion of food wastage, individual value of 69 g).

 

Table 6 - Thyroid Hormones in F0 Males

	Thyroid hormones
	F0 males
Dose level (mg/kg bw/day)	0	30	100	300	HCD [min; max]
T4 (ng/mL)	39.52 ± 7.850	26.75** ± 5.791	15.42** ± 3.239	11.75** ± 3.745	[25.6; 54.17]
% from controls	/	-32	-61	-70	/
TSH (pg/mL)	3300 ± 949.6	2537 ± 1569.6	2149 ± 1455.6	4307 ± 1300.0	[627; 4455]
% from controls	/	-23	-35	+31	/

HCD: Historical Control Data for F0 males.

Statistical significance: **: p<0.01. /: not applicable.

 

Table 7 - Thyroid Hormones in F0 Females

	Thyroid hormones
	F0 females, Day 22 p.p.
Dose level (mg/kg bw/day)	0	30	100	300	HCD [min; max]
T4 (ng/mL)	29.02 ± 3.885	24.41 ± 2.540	24.77 ± 4.461	14.48** ± 6.158	[16.4; 28.5]
% from controls	/	-16	-15	-50	/
TSH (pg/mL)	1101 ± 430.2	1076 ± 304.6	1009 ± 453.0	1501 ± 1211.2	[216; 2882]
% from controls	/	-2	-8	+36	/

HCD: Historical Control Data for F0 females.

Statistical significance: **: p<0.01; /: not applicable.

 

Table 8 - Relevant Changes in Mean Final Body Weights and Organ Weights in Treated Groups (F0) (% Changes from Controls)

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg bw/day)	30	100	300	30	100	300
Exam. animals / Num. of animals	10/10	10/10	10/10	10/10	10/10	6/10
- Final body weight	+3	0	-6	-3	-3	-4
- Thyroid glands
.absolute	+12	+18*	+16	-14	-8	+6
.relative	+9	+18	+23*	-12	-6	+10

Statistically significant from controls: *: p<0.05.

 

Table 9 - Estrous Cycle

Sex	Female
Dose level (mg/kg bw/day)	0	30	100	300
Number of females examined	10	10	10	10
Number of cycles	2.7	2.2	3.1	2.6
Cycle length (days)	4.7	7.0	3.9	4.1
Rats cycling normally	8	7	8	9

No statistically significant differences vs. controls.

 

Table 10 - Summary of Mating and Fertility Data

Dose level (mg/kg bw/day)	0	30	100	300
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10+10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	2.4	1.5	2.6	3.5
Number of pregnant females	10	10	10	10
Number of females with live born pup	10	10	10	8^a
Mating index (female, %)	100	100	100	100
Fertility index (%)	100	100	100	100

^a: In high-dose group, two females were prematurely euthanized due to difficulties to deliver and dead litter, respectively.

M: Males; F: Females.

No statistically significant differences vs. controls.

 

Table 11 - Summary of Delivery and Litter Data

Dose level (mg/kg bw/day)	0	30	100	300	HCD
Number of pregnant females	10	10	10	10	30
Number of females which delivered	10	10	10	9	30
Number of females with live concepti	10	10	10	8	30
Gestation index (%)	100	100	100	80	100
Mean duration of gestation (days)	21.3	21.4	21.4	21.3	[22.0-22.2]
Mean number of corpora lutea	19.0	18.1	17.0	16.6	[14.0-16.6]
Mean number of implantation sites	18.9	17.1	16.4*	16.4*	[13.4-14.4]
Mean pre-implantation loss (%)	0.6	4.7	2.6	0.7	[4.0-14.7]
Mean number of pups delivered	16.5	15.7	14.5	12.4*	[10.7-12.3]
Mean post-implantation loss (%)	12.6	8.1	11.0	23.1	[16.7-22.5]

Statistical significance: *: p<0.05.

HCD: Historical Control Data OECD 421-422 (control data collected from three studies covering a period ranging from January 2016 to December 2017), [min.; max.].

 

Table 12 - Incidence of Clinical Signs in Lactating Pups

Dose level (mg/kg bw/day)	0	30	100	300	HCD
Hematoma on neck, n (L)	3 (2)	-	-	-	-
Hematoma on head, n (L)	2 (2)	1 (1)	2 (2)	-	-
Emaciated appearance, n (L)	2 (2)	-	-	11 (2)	2 (1)
Cold to the touch, n (L)	2 (2)	-	-	2 (1)	1 (1)
Malformed tail, n (L)	1 (1)	1 (1)	-	-	-
Absence of milk in stomach, n (L)	-	-	-	9 (1)	8 (3)
Generalized pallor, n (L)	-	-	-	2 (1)	-
Dehydration, n (L)	-	-	-	11 (2)	2 (2)
Hypoactivity, n (L)	-	-	-	2 (1)	-
Wound on head, n (L)	-	-	1 (1)	-	-
Thickness on the abdomen, n (L)	-	-	-	2 (2)	-
Increase of size of abdomen, n (L)	-	-	-	2 (1)	-

HCD: Historical Control Data OCDE 421-422 (control data collected from three studies covering a period ranging from 2016 to 2018), n (L); n: number of pups, L: number of litter affected; -: no clinical signs.

 

Table 13 - Incidence of Mortality

Dose level (mg/kg bw/day)	0	30	100	300
Number of pups delivered (number of litters) Mean of pups delivered by female	165 (10) 16.5	157 (10) 15.7	145 (10) 14.5	112 (9) 12.4*
Number of pups found dead and/or cannibalized during the entire lactation period (number of litters affected)	7 (3)	9 (4)	10 (8)	65 (9)
Number of pups found dead during lactation period between Days 1 to 4 p.p.	7	6	9	62#
Number of pups cannibalized during lactation period between Days 1 to 4 p.p.	4	3	5	22

Statistical significance: *: p<0.05; ^#: p<0.001.

 

Table 14 - Pup Viability

Dose level (mg/kg bw/day)	0	30	100	300	HCD
Number of pups Number of litters	165 10	157 10	145 10	113 9	347 30
Live birth index (%) ± SD	100.0 ± 0.0	96.7 ± 6.5	99.3 ± 2.3	56.4 ± 37. 4	93.9
Viability index on Day 4 p.p. (%) ± SD	96.0 ± 7.7	94.2 ± 8.6	94.3 ± 4.5	45.2** ± 34.8	93.2
Lactation index on Day 21 p.p. (%) ± SD	100.0 ± 0.0	100.0 ± 0.0	98.0 ± 6.3	98.3 ± 4.1	96.3

Statistical significance: **: p<0.01.

HCD: Historical Control Data OECD 421-422 (control data collected from three studies covering a period ranging from 2016 to 2018), n (L).

 

Table 15 - Pup Body Weight and Body Weight Change

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Mean body weight
Day 1 p.p.	7.3	7.1	6.8	5.5#	6.8	6.5	6.4	5.1#
% from controls		-3	-7	-25		-4	-6	-25
Day 4 p.p.	10.5	10.9	9.4	7.7*	10.2	10.0	8.9	7.8*
% from controls		+4	-10	-27		-2	-13	-24
Day 8 p.p.	20.6	20.8	18.5	15.3#	20.0	19.8	17.8	14.6#
% from controls		+1	-10	-26		-1	-11	-27
Day 13 p.p.	33.5	33.8	30.6	25.2#	32.9	32.4	29.4	23.7#
% from controls		+1	-9	-25		-2	-11	-28
Day 17 p.p.	43.1	43.1	39.6	33.9*	42.8	41.6	38.0	31.8**
% from controls		0	-8	-21		-3	-11	-26
Day 21 p.p.	58.8	58.5	52.4	44.2**	57.9	56.2	49.5*	41.5#
% from controls		-1	-11	-25		-3	-15	-28
Mean body weight gain
Days 1-4 p.p.	+3.2	+3.8	+2.6	+2.1	+3.4	+3.5	+2.5	+2.3
% from controls		19	-19	-34		+3	-26	-32
Days 4-8 p.p.	+10.1	+9.9	+9.1	+7.1^#	+9.8	+9.8	+8.9	+6.8^#
% from controls		-2	-10	-30		0	-9	-31
Days 8-13 p.p.	+12.9	+13.0	+12.1	+9.9	+12.9	+12.6	+11.6	+9.2
% from controls		+1	-6	-23		-2	-10	-29
Days 13-17 p.p.	+9.6	+9.3	+9.0	+8.7	+9.9	+9.2	+8.5	+8.1
% from controls		-3	-6	-9		-7	-14	-18
Days 17-21 p.p.	+15.7	+15.3	+12.7	+10.3**	+15.1	+14.5	+11.6^#	+9.7^#
% from controls		-3	-19	-34		-4	-23	-36

Statistical significance: *: p<0.05; **: p<0.01; ^#: p<0.001

 

Table 16 - Pup Body Weight and Body Weight Change in the F1 Generation

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Mean body weight
Day 22 p.p.	64	62	53**	44**	63	58	51**	43**
% from controls		-3	-17	-31		-8	-19	-32
Day 25 p.p.	81	80	66**	53**	78	71*	64**	48**
% from controls		-1	-19	-35		-9	-18	-38
Day 29 p.p.	111	109	92**	73**	104	96	86**	66**
% from controls		-2	-17	-34		-8	-17	-37
Day 32 p.p.	138	134	115**	92**	122	116	105**	83**
% from controls		-3	-17	-33		-5	-14	-32
Day 36 p.p.	177	171	150**	121**	149	141	133**	109**
% from controls		-3	-15	-32		-5	-11	-27
Mean body weight gain
Days 22-25	+18	+18	+13**	+9**	+15	+13	+13*	+7**
% from controls		0	-28	-50		-13	-13	-53
Days 25-29	+30	+29	+27*	+20**	+25	+25	+22	+19**
% from controls		-3	-10	-33		0	-12	-24
Days 29-32	+26	+25	+23**	+18**	+19	+20	+19	+17
% from controls		-4	-12	-31		+5	0	-11
Days 32-36	+40	+37	+35*	+30**	+27	+26	+28	+26
% from controls		-8	-13	-25		-4	+4	-4
Days 22-36	+113	+109	+97**	+77**	+86	+83	+81	+69**
% from controls		-4	-14	-32		-3	-6	-20

Statistical significance: *: p<0.05; **: p<0.01.

 

Table 17 - Mean Food Consumption in the F1 Generation (g/animal/day)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Days 22-25	8.5	9.2	6.5	5.0*	7.6	8.8	6.7	3.8
% from controls		+8	-24	-41		+16	-12	-50
Days 25-29	14.3	14.0	11.7	9.4**	13.1	12.3	10.7	10.2*
% from controls		-2	-18	-34		-6	-18	-22
Days 29-32	17.5	17.7	15.2	12.6*	15.1	15.3	13.7	12.6
% from controls		+1	-13	-28		+1	-9	-17
Days 32-36	21.6	21.7	18.5	15.9**	17.8	17.3	16.3	15.0*
% from controls		0	-14	-26		-3	-8	-16

Statistical significance: *: p<0.05; **: p<0.01.

 

Table 18 - Thyroid Hormones in the F1 Generation

	Thyroid hormones
	Pups, Day 4 p.p.
Dose level (mg/kg bw/day)	0	30	100	300	HCD [min; max]
T4 (ng/mL)	9.85 ± 1.197	4.75** ± 0.972	3.05** ± 1.388	3.25 ± 0.382	[26.1; 49.9]
% from controls	/	-52	-69	-67	/
TSH (pg/mL)	628 ± 393.8	577 ± 195.6	441 ± 157.2	821 ± /	[421; 4038]
% from controls	/	-8	-30	+31	/
	Male pups, Day 36 p.p.
	0	30	100	300
T4 (ng/mL)	34.81 ± 5.279	28.21* ± 3.470	21.30** ± 8.340	19.70** ± 3.326
% from controls	/	-19	-39	-43
TSH (pg/mL)	1154 ± 297.6	994 ± 400.2	1156 ± 637.9	1763 ± 973.0
% from controls	/	-14	0	+53
	Female pups, Day 36 p.p.
T4 (ng/mL)	28.83 ± 6.040	22.48* ± 4.411	19.06** ± 4.089	16.03** ± 3.816
% from controls	/	-22	-34	-44
TSH (pg/mL)	657 ± 468.8	654 ± 328.4	729 ± 412.3	1346* ± 624.4
% or fold  from controls	/	0	+11	x2

Statistical significance: *: p<0.05; **: p<0.01. /: not applicable.

 

Table 19 - Anogenital Distance Corrected for Body Weight (AGD mm/BW g^1/3)

Sex	Male				Female
Dose level (mg/kg bw/day)	0	30	100	300	0	30	100	300
Number of litters	10	10	10	8	10	10	10	8
Mean AGD	4.15	4.31	3.97	3.59*	2.13	2.30	2.00	1.92
HCD	4.56 (3.78-5.95)				2.57 (1.64-3.90)
AGD/BW^1/3	2.14	2.25	2.09	2.04	1.13	1.23	1.10	1.12
HCD	2.27 (1.81-3.00)				1.32 (0.82-2.06)

HCD: mean (minimum-maximum) Historical Control Data values.

No statistically significant differences vs. controls.

 

Table 20 - Macroscopic Post-Mortem Observation of Dead Pups

Dose level (mg/kg bw/day)	0	30	100	300
Found dead, P(L)	3 (2)	6 (4)	5 (4)	43 (7)
- deformed bilateral forelimbs and hindlimbs, P(L) - autolyzed, P (L)	0 (0) 0 (0)	0 (0) 0 (0)	0 (0) 0 (0)	5 (1) 1 (1)
- absence of milk (stomach), P(L) - no external abnormalities, P(L)	0 (0) 3 (2)	2 (1) 4 (3)	2 (1) 3 (3)	40 (7) 2 (2)

P: pup; L: Litter (number of pups and litters affected).

No statistically significant differences from controls.

 

Table 21 - Sex Ratio (% of Males)

Dose level (mg/kg bw/day)	0	30	100	300	HCD
Total pups delivered (mean)	16.5	15.7	14.5	12.4*	[10.7; 12.3]
Sex ratio on Day 1 p.p. (% of males)	49.7	48.0	46.1	63.5	[41.9; 50.5]
% from controls	/	-3	-7	+28
Mean umber of males on Day 1 p.p.	8	7.3	6.8	5.3	[7; 10.3]
Sex ratio on Day 21 p.p. (% of males)	47.0	51.0	45.5	58.6	/
% from controls	/	+9	-3	+25
Mean number of males on Day 21 p.p.	4.7	5.1	4.4	3.7	/

HCD: Historical Control Data OECD 421-422 (control data collected from three studies covering a period ranging from 2016 to 2018), [range of litter mean min ; maxi].

Statistical significance: *: p<0.05./: not applicable.

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was established at 30 mg/kg bw/day in females based on adverse in life effects (clinical signs, body weight and food consumption) while the Low Observed Adverse Effects (LOAEL) was set at 30 mg/kg bw/day in males based on T4 decrease at all dose levels,
- the No Observed Effect Level (NOAEL) for reproductive performance (mating and fertility) was considered to be 100 mg/kg bw/day based on the lower number of implantation sites and number of pups delivered, and on the post-implantation loss which were considered as adverse at 300 mg/kg bw/day,
- the LOAEL for progeny development was established at 30 mg/kg bw/day based on the lower live birth and viability indices at 300 mg/kg bw/day, the lower pup body weight at 100 and 300 mg/kg bw/day and the effects on thyroids at all dose levels on Days 4 and 36 p.p.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 21 post-partum (p.p.) and, for F1 offspring, from Day 22 to Day 35 p.p.

This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, parturition, and development of the conceptus and pups during lactation and the early post-weaning period.

Methods

In a GLP compliant study, performed in accordance with OECD guideline 421, three groups of ten male and ten female Sprague-Dawley rats received the test item daily by the oral route (gavage) at the dose level of 30, 100 or 300 mg/kg bw/day, at a constant dosage volume of 5 mL/kg bw/day. A control group of ten male and ten female rats received the vehicle only (0.5% carboxymethylcellulose / 0.5% Tween 80) under the same experimental conditions.

F0 Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. F0 females were treated for an overall period of 8 to 10 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks) until Day 21 post-partum (p.p.) inclusive.

Four groups of 10 male and 10 female F1 pups were treated at the same dose levels for 2 weeks after weaning, i.e. Days 22 to 35 p.p. inclusive.

The actual test item concentrations in the dose formulations were determined in Weeks 1, 4 and 8 using a validated GC-FID analytical method.

Animals were checked daily for clinical signs and mortality.

Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from 2 weeks before before the start of treatment until the females had mated and on day before euthanasia.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 21 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible five males and five females per litter.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed at designated intervals. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

For pups selected to be the F1 generation, food consumption was recorded at designated intervals after weaning.

Thyroid hormones (TSH and T4) were determined in males and females at termination and in F1 pups euthanized on Day 4 or 36 p.p.

F0 males were euthanized after completion of the mating period and F0 females were euthanized on Day 22 p.p. Final body weight was recorded and a complete macroscopic post-mortem examination was performed, with particular attention accorded to the reproductive organs.

Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on epididymides, testes and ovaries in the control and high-dose groups and on all macroscopic lesions.

Pups not selected on Day 4 or 22 p.p. were euthanized and discarded without further examination.

A detailed external examination was performed after euthanasia of pups euthanized on Day 36 p.p., and particular attention was paid to the external genital organs.

Results

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variations (-3.7% to +11.9%) when compared to the nominal concentrations.

F0 animals:

No unscheduled deaths occurred in F0 males at any dose level during the study.

One female given 300 mg/kg bw/day was prematurely euthanasied on Day 23 p.c. due to poor clinical conditions at delivery. Three females given 300 mg/kg bw/day were prematurely euthanized between Days 1 and 7 post-partum as the pups died; these deaths were considered to be test item-related.

Clinical signs of poor condition (piloerection, hypoactivity, round back, half-closed eyes, hypoactivity and/or emaciated appearance) were observed in 3/10 females at 100 and 3/10 females at 300 mg/kg bw/day at the beginning of the pre-mating period, in 2/9 females at 300 mg/kg bw/day during early gestation and one female at 300 mg/kg bw/day during part of the lactation period (Days 12, 20 p.p.).

Ptyalism was observed in test item-treated males and females given 100 or 300 mg/kg bw/day throughout the treatment period and in females given 30 mg/kg bw/day during the pre-mating period only. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

In males, lower body weight gain was recorded at 300 mg/kg bw/day during most of the treatment period (-35% on Days 1-29 vs. controls, p<0.05), leading to lower terminal body weight (-6%). These effects were associated with lower food consumption at 300 mg/kg bw/day on Days 1-8 (-18%, vs. controls, p<0.01). No effects were noted at 30 or 100 mg/kg bw/day.

During the premating period in females, body weight loss or lower body weight gain was recorded at 100 and 300 mg/kg bw/day on Days 1-15 (+4, p<0.01 and -5 g, p<0.001, respectively, vs. +26 g in controls), leading to lower terminal body weight (-8%, p<0.05 at 100 mg/kg bw/day and -8% at 300 mg/kg bw/day). These effects were associated with lower food consumption on Days 1-8 (-13%, p<0.05 and -17%, p<0.01, respectively, vs. controls) and to a lesser extent on Days 8-15 (-8 and –12%, respectively). At 30 mg/kg bw/day, a tendency towards slightly lower body weight gain was recorded on Days 1-15 (+18 g vs. +26 g,-31% vs. control), but this did not significantly impact the Day 15 body weight (-2%).

During the gestation period, statistically lower body weight gain was recorded at 300 mg/kg bw/day over the gestation period (-27% vs. controls, p<0.001), leading to lower terminal body weight (-15%, p<0.001). At 100 mg/kg bw/day, lower body weight gain was recorded mainly on Days 14-20 p.c. (-12%), leading to lower terminal body weight (-8%, p<0.05). These effects were associated with lower food consumption at 300 mg/kg bw/day throughout the gestation period, more pronounced on Days 1-14 p.c. (around -15%). At 30 mg/kg bw/day, a tendency towards lower body weight gain was recorded during the gestation period (-5%), but this did not significantly impact the Day 20 p.c. body weight (-4%). No relevant effects were noted on food consumption at 30 or 100 mg/kg bw/day.

During the lactation period, at 300 mg/kg bw/day, higher mean body weight gain was recorded on Days 1-21 p.p. (+47 g vs. +20 g in controls, p<0.05,) with a body weight loss on Days 1-4 p.p. The same effect was noted at 100 mg/kg bw/day (+32 g on Days 1-21 p.p.) with lower body weight gain on Days 1-4 p.p.

At 30 mg/kg bw/day, no effects were noted on body weight or food consumption.

Estrous cycle was not impacted by the test item treatment.

The mating and fertility indices were unaffected by the test item treatment. All females mated within four days and all females were pregnant.

At 100 and 300 mg/kg bw/day, a lower number of implantation sites, associated with higher post-implantation loss, led to a lower number of pups at birth (14.5 and 12.6, p<0.05, respectively, vs. 16.5 in controls). These findings were considered to be adverse at 300 mg/kg bw/day. The number of corpora lutea was lower at 100 and 300 mg/kg bw/day (17.0 and 16.6 vs. 19.0 in controls). A relationship to the test item treatment could not be excluded.

Thyroid hormone analysis revealed dose-related, lower T4 level at all dose levels in males (p<0.01) and in females at 300 mg/kg bw/day (p<0.01). These changes were associated with higher TSH levels in both sexes at 300 mg/kg bw/day (not significant) and correlated with equivocal increases in thyroid gland weights in males at ≥ 100 mg/kg bw/day and thyroid follicular cell hypertrophy observed at microscopy in males at 300 mg/kg bw/day.

There were no test item-related microscopic observations at the end of the treatment period in the testes, epididymides, ovaries or oviducts.

Pups (Days 1 to 21 post-partum):

The number of pups found dead on Days 1-4 p.p. of the lactation period was higher at 300 mg/kg bw/day (65 vs. 7 pups), associated with a higher incidence of affected litters (9/9 vs. 3/10). These findings accounted for the lower live birth and viability indices. Clinical observations such absence of milk in the stomach, emaciated appearance, dehydration, hypoactivity, cold to the touch and generalized pallor were observed in some high dose pups during the lactation period. Absence of milk in the stomach was also observed in a high number of dead pups (40 pups from 7 litters). Lower pups body weight was recorded at 100 and 300 mg/kg bw/day at birth in males (-7 and -25%, p<0.001, respectively, vs. controls) and females (-6 and -25%, p<0.001, respectively, vs. controls), followed by lower body weight gain at both doses, resulting in a lower body weight on Day 21p.p. (-11 and -25%, p<0.01 in males and -15%, p<0.05 and -28%, p<0.001 in females, respectively). No effects were noted on sex ratio, anogenital distance or the numbers of nipples or areolae.

Thyroid hormone analysis on Day 4 p.p. revealed lower T4 level at all dose levels of the test item, associated with higher TSH level at 300 mg/kg bw/day.

F1 animals (Days 22 to 35 post-weaning):

No test item-related deaths occurred during the treatment period.

Body weights were lower on the first day of treatment at 100 and 300 mg/kg bw/day. This was considered to have resulted from the lower body weight recorded before weaning. Lower body weights were continuously recorded at 100 and 300 mg/kg bw/day during the treatment period due to body weight gains that were generally lower than those of controls (-14 and -32% in males and -5 and -24% in females, respectively, for the whole period), leading to terminal body weights that were lower than those of controls (-15%, p<0.05 and -32%, p<0.01 in males and -11%, p<0.01 and -27%, p<0.01 in females, respectively). These effects were associated with dose-related, lower food consumption throughout the treatment period (between -50%, p<0.05 and -16%, p<0.05  at 300 mg/kg bw/day and between -24 and -8% at 100 mg/kg/day, between Days 22 and 36).

Thyroid hormone analysis on Day 36 p.p. revealed dose-related, lower mean T4 levels at all dose levels of the test item (p<0.05 or p<0.01), associated with higher mean TSH levels in animals at 300 mg/kg bw/day (p<0.05 in females). No microscopic changes were noted in the thyroid glands.

Conclusion

The test item  was administered daily by the oral gavage at the dose level of 30, 100 or 300 mg/kg bw/day to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for the females, throughout gestation and until Day 21 post-partum (p.p.) inclusive. Then, after weaning, three groups of ten male and ten female Sprague-Dawley rat pups (F1 generation) received the test item from Days 22 to 35 p.p. inclusive.

Based on the experimental conditions of this study:

• the No Observed Adverse Effect Level (NOAEL) for parental toxicity was established at 30 mg/kg bw/day in females based on adverse in life effects (clinical signs, body weight and food consumption) while the Low Observed Adverse Effects (LOAEL) was set at 30 mg/kg/day in males based on T4 decrease at all dose levels,


• the No Observed Effect Level (NOAEL) for reproductive performance (mating and fertility) was considered to be 100 mg/kg bw/day based on the lower number of implantation sites and number of pups delivered, and on the post-implantation loss which were considered as adverse at 300 mg/kg bw/day,


• the LOAEL for progeny development was established at 30 mg/kg bw/day based on the lower live birth and viability indices at 300 mg/kg bw/day, the lower pup body weight at 100 and 300 mg/kg bw/day and the effects on thyroids at all dose levels on Days 4 and 36 p.