3,4-dihydroxybenzonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

28 days exposure

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents

Version / remarks:

July 2000

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test material form:

solid: particulate/powder

Remarks:

white to brownish

Details on test material:

Batch 151222

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality). Untreated, nulliparous, non-pregnant females and untreated males were used at the initiation of the study.

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age/weight at study initiation: At initiation of dosing, males were 10 weeks old and weighed between 263 and 312 g and females were 13 weeks old and weighed between 189 and 234 g.
- Fasting period before study: no
- Housing:
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum):
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum):
Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period:
At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: dosing was initiated on 03 May 2017. To: The in-life phase of the study was completed on 05 Jul 2017.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.
The oral route of administration was selected because this is the most likely route of human exposure.
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. (Test Facility Study No. 515456 was used for DCS).

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036 (Merck, Darmstadt, Germany).

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared within 8 days prior to dosing and were homogenized to a visually acceptable level. The formulations were filled out in daily proportions and kept in the refrigerator in the dark until needed. Adjustment was made for the specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice of vehicle was based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 15, 50 and 150 mg/kg
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis were conducted on a single occasion during the treatment phase according to a validated method (Test Facility Study No. 516073, ABL No. 16303).

Immediately after sampling, samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Concentration Analysis
Duplicate middle samples for Groups 1 and 3 and duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
Homogeneity Analysis
Duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (ABL No. 17053) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for ABL No. ABL17053. In this study, stability for at least 5 hours at room temperature, 8 days in the refrigerator and 3 weeks in the freezer was confirmed over the concentration range 1 to 200 mg/mL.

Duration of treatment / exposure:

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days (most females) or 62 days (one female), i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 39, 41 or 43 days.

Routinely, females that are littering are left undisturbed. In this study, female nos. 48 (Group 1), 56, 59 (Group 2), and 64 (Group 3) were not dosed on one occasion as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of an acute oral toxicity study (Test Facility Study No. 515451; treatment-related mortality at 300 mg/kg) and a dose range finder (Test Facility Study No. 515454)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality
F0: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
F1: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical observations
F0: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 15 min) after dosing (on the peak period of anticipated effects after treatment).
The time of onset, grade and duration of any observed sign was recorded.
F1: Clinical observations were performed at least once daily for all pups.
Cohabitation/mating
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
General reproduction data
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.


DETAILED CLINICAL OBSERVATIONS: Yes
Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
F0: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.
F1: Live pups were weighed on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD EFFICIENCY:
/

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
Blood of F1-animals was collected on PND 4 and PND 13-15.
On PND 4 at culling, blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4).
On PND 13-15, from 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH).
- Animals fasted: Yes: F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 5 per sex per treatment group (F0)
- Parameters checked; see Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See Haematology
- Animals fasted: See Haematology
- How many animals: 5 per sex per treatment group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13). These tests were started at least 1 hour (± 15 min) after dosing, after clinical signs observations.
- Dose groups that were examined: all treatment groups
- Battery of functions tested:
sensory activity: hearing ability, pupillary reflex and Static righting reflex
grip strength: Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal
motor activity: Locomotor activity. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.


IMMUNOLOGY: No

OTHER:
BLOOD COAGULATION
Blood samples were processed for plasma, and plasma was analyzed for the parameters Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)
THYROID HORMONE
Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Statistics:

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test (was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test as applied to motor activity data to determine intergroup differences.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Salivation seen after dosing for all animals at 150 mg/kg (up to moderate degree) and incidentally across the other groups (slight degree) was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).

Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

One female at 50 mg/kg (no. 69) was sacrificed on Day 1 of lactation due to total litter loss

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights of treated males and females remained in the same range as controls over the entire treatment period.
Statistically significantly lower mean body weight gain was noted in females at 150 mg/kg on Day 13 of the lactation period. As mean body weights were similar as controls on Day 13 of lactation, the slightly lower body weight gain was not considered to be toxicologically relevant.
Statistically significantly higher mean body weight gain was noted in treated males at the end of the treatment period, i.e. Day 15 of the mating period. As the differences from controls showed no dose-related trend, they were considered to be unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption were noted by treatment up to 150 mg/kg.
In females treated at 150 mg/kg, mean food consumption (before and after allowance for body weight) was slightly higher during the pre-mating period. As the changes were only minimal, not statistically significant and remained within the range considered normal for rats of this strain and age, these changes were not considered to be toxicologically relevant.
In males, food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters were considered not to be affected by treatment.
Isolated statistically significant variations noted (higher platelets in males at 15 mg/kg and lower mean corpuscular volume (MCV) in females at 50 mg/kg) were considered unrelated to treatment due to the absence of a dose-related response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a dose-related increased mean concentration of bile acids was noted, which was statistically significant at 150 mg/kg (relative difference of 88% compared to controls). This was considered to be partly caused by one male (no. 34) showing a notably high value of 56.5 µmol/L, that was above the P95 value of available historical control data. Other individual bile acid values of treated males remained in the range considered normal for rats of this age and strain.
The other statistically significant difference noted in clinical biochemistry parameters (i.e. lower urea in females at 15 mg/kg) was considered to be unrelated to treatment due to the absence of a dose-related response.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Increased liver weight (males only) and uterus weight were noted at 150 mg/kg, reaching statistical significance. Relative differences of 10% (liver) and 35% (uterus) were noted compared to controls. These changes were considered not to be related to treatment with test item due to lack of a dose-related pattern, general overlap and variability in individual values and absence of any correlating macroscopic or microscopic finding. Moreover, all values remained within the range of available historical control data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups.
Findings of note were lower mean values for total movements and ambulations in males at 150 mg/kg (relative differences from controls: 28 and 19%, respectively) and higher values in females at 150 mg/kg (relative differences from controls: 43 and 74%, respectively). Statistical significance was not achieved, there were no corroborative clinical signs or changes in other parameters in the neuromuscular domain (including gait, air righting reflex and grip strength), and all values at 150 mg/kg remained within the range of available historical control data. In combination with the inconsistent direction of the differences in males (lower values) and females (higher values), these differences in motor activity were considered not to represent a test item-related effect.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item‑related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item‑related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

No effect on T4 serum levels or blood coagulation observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.
The high dose of 150 mg/kg was considered to be the highest feasible dose in this type of study as in the acute oral toxicity study (Test Facility Study No. 515451), treatment-related hunched posture and mortality was observed for all animals within two hours after receiving 300 mg/kg.

Executive summary:

Title

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of CH02906 in rats by oral gavage.

Guidelines

The study was based on the following guidelines:

·     OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

·     OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

·     OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

·     OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

·     EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

·     OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

·     OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Rationale for dose levels

Based on the results of a dose range finder in which no dose limiting effects were noted at 150 mg/kg (Test Facility Study No. 515454) and acute oral toxicity data (oral LD50 between 50 and 300 mg/kg, Test Facility Study No. 515451), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 15, 50 and 150 mg/kg.

Study outline

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days (most females) or 62 days (one female), i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 39-43 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality/viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F₀-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

Results

Accuracy and homogeneity of formulations were demonstrated by analyses.

Parental results:

No parental toxicity was observed up to the highest dose level tested (150 mg/kg).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (150 mg/kg).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (150 mg/kg).

Conclusion

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.

The high dose of 150 mg/kg was considered to be the highest feasible dose in this type of study as in the acute oral toxicity study, treatment-related hunched posture and mortality was observed for all animals within two hours after receiving 300 mg/kg.