Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2012 - 06 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
yes (incl. certificate)
Remarks:
WIL Research Europe B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (4-nonylphenoxy) acetic acid
- Physical state: Yellow to brown viscous liquid
- Analytical purity: 97.4% (GC fingerprint)
- Expiration date of the lot/batch: 22 June 2014
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: approx. 313 g (m) and 204 g (f)
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams 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 left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: A 1:1 ratio of Polyethylene glycol 400 (specific gravity 1.125; Merck, Darmstadt, Germany) and water (Elix, Millipore S.A.S., Molsheim, France).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity or the density of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Amount of vehicle (if gavage): 5ml/kg
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of 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 occurred, the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (11 June 2012), according to a validated method (Project 499642, BASF Project 05Y0836/11X473). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable when the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated when the coefficient of variation was ≤ 10%. Formulations were considered stable when the relative difference
before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 42, 48 (Group 1), 52 (Group 2) and 63 (Group 3) were not dosed during littering.
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. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
Of all males of the control and high dose group and all males suspected to be infertile additional slides
of the testes were prepared to examine staging of spermatogenesis. The testes were processed,
sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The
Netherlands).
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 60, 200 mg/kg body weight
Basis:
actual ingested
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 the 14-day dose range finding study (Project 499639; BASF Project 01R0836/11X416).

Examinations

Parental animals: Observations and examinations:
At dose level allocation, 5 animals/sex/group were randomly selected for functional observations, locomotor activity, clinical pathology, organ weights (full list) and histopathology.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing at no specific time point, but within a similar time period after dosing for the respective animals. 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
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled post mortem examination between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (with a maximum of 24 hours)
- How many animals: the selected 5 animals/sex/group
- Parameters checked: White blood cells, Differential leucocyte count, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration MCHC, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled post mortem examination between 7.00 and 10.30 a.m.
- Animals fasted: Yes
- How many animals: the selected 5 animals/sex/group
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period after clinical signs observations (incl. arena observations, if applicable) and before blood sampling.
- Dose groups that were examined: the selected 5 animals/sex/group.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. 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.
Sperm parameters (parental animals):
Of all males of the control and high dose group and all males suspected to be infertile additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Mortality / Viability, clinical signs, body weights, sex

GROSS EXAMINATION OF DEAD PUPS:
yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
All males and females were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
- Females which delivered: on Lactation Days 6-7.
- Females which failed to deliver (nos. 60, 66): Post-coitum Days 27-28 (females with evidence of mating)
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
- selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.
- All remaining males: Epididymides, Testes

HISTOPATHOLOGY:
Samples of the following tissues and organs from all animals were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and Female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Of all males of the control and high dose group and all males suspected to be infertile additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The stomach of all selected animals of Groups 2 and 3 and the thyroids of all selected females of Group 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all animals of Groups 1 and 4 and from all males that failed to sire and all females that failed to deliver healthy pups (20 mg/kg bw/day: nos. 20 and 60; 60 mg/kg bw/day: nos. 26 and 66). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial cell structure.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 6-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
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. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Mating index, Fertility index, Conception index, Gestation index, Duration of gestation
Offspring viability indices:
Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period.
At 200 mg/kg bw/day rales and piloerection were noted for two animals on a single day, and hunched posture and tremor were also noted for individual animals on single days. At the limited incidence observed, these were not considered to be toxicologically relevant. Incidental findings that were noted included chromodacryorrhea of the right eye, dull right eye and alopecia. These findings 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. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weight gains were slightly decreased in males and females during the pre-mating and/or mating period.
Males at 200 mg/kg bw/day had slight but significantly lower body weight gain on Day 8 of the mating period, while females lost a small amount of weight on Day 8 of the premating period and had slightly lower weight gain on Day 1 of the mating period.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Relative food consumption was slightly lower for females at 200 mg/kg bw/day during the pre-mating period and over Days 4-7 and 14-20 on the post coitum period (only significantly different on Days 14-17). However, the difference from controls was only slight and this was not considered to be toxicologically relevant.
At 20 mg/kg bw/day relative food consumption was significantly higher than controls on lactation Days 1-4. The difference was attributable to high means for female nos. 52 and 59, and was not treatment related or toxicologically relevant.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Red blood cells and haematocrit values were significantly lower for males at 60 and 200 mg/kg bw/day and reticulocytes and red blood cell distribution width (RDW) were significantly higher for females at 200 mg/kg bw/day. However, in the absence of any relevant effects on any other related parameter, these were not considered to be toxicologically relevant.

CLINICAL CHEMISTRY
Total thyroxine (T4) concentrations were significantly higher for animals of both sexes at 200 mg/kg bw/day, and cholesterol was significantly higher for females at this dose level. The statistically significant changes seen for males at 20 (increased aspartate aminotransferase [ASAT] and lower inorganic phosphate) and 60 mg/kg bw/day (lower inorganic phosphate) occurred in the absence of a treatment related distribution and were not considered to be toxicologically relevant.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex (except for the dull eye in one rat), static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not have a relationship to treatment. Females at 200 mg/kg bw/day had lower total movements and ambulatory counts compared to controls (not statistically significant). This was mostly attributable to lower activity for these females over blocks 4-7. In the absence of any relevant clinical signs like lethargy, this was not considered to be toxicologically relevant.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
Males at 200 mg/kg bw/day had significantly higher absolute and relative liver, thyroid and kidney weights (relative liver weights were higher for females as well). Absolute and relative thyroid weights were higher for females at 60 and 200 mg/kg bw/day (not significantly higher at 200 mg/kg bw/day). High dose females also had significantly increased relative uterine weights compared to controls.
Absolute spleen and kidney weights were statistically significantly higher in males at 60 mg/kg bw/day. Since there was no dose-dependency or the difference to control values was minor, these findings were not considered to be toxicologically relevant. All other organ weights and organ to body weight ratios were similar to control levels.

GROSS PATHOLOGY
Several reddish or dark red foci on the stomach glandular mucosa were noted for four males and irregular surface of the forestomach was noted for one male and one female at 200 mg/kg bw/day. These were likely related to the acidic nature of the test substance. At 60 mg/kg bw/day male no. 26 had reduced size of both testes and epididymides. This male’s infertility was confirmed at the microscopic examination. This was not considered to be treatment related. Macroscopic findings noted for control and treated animals included a greenish soft nodule on the left epididymis, pelvic dilation of the kidneys, enlarged mandibular lymph nodes, yellowish hard nodule on the epididymal adipose tissue, alopecia, several tan foci on the left clitoral gland, ectopic splenic tissue and an enlarged spleen. These remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related trend. These findings were therefore not considered to be toxicologically relevant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were present in the stomach (both sexes) and thyroid gland (200 mg/kg bw/day, females)
Stomach:
- Hyperplasia of the squamous epithelium was recorded at 60 mg/kg bw/day in 1/5 males and 2/5 females (minimal) and at 200 mg/kg bw/day in 4/9 males (1 minimal, 2 slight and 1 moderate) and in 5/5 females (1 minimal, 3 slight and 1 moderate).
- Hemorrhage(s) of the glandular stomach (minimal-slight) was recorded in 4/9 males at 200 mg/kg bw/day.
Thyroid gland (females):
- Diffuse follicular hyperplasia/hypertrophy was recorded in 2/5 females of Group 1 (minimal), 1/5 of Group 2 and 3 (minimal) and 5/5 females of Group 4 (3 minimal and 2 slight).
In the stomach a minimal degree of hyperplasia of the squamous epithelium was considered to be within the normal background level and therefore not considered treatment-related. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed.

Gestation
The gestation index and duration of gestation were unaffected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Systemic Toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility were observed

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There were six pups in the 200 mg/kg bw/day group that died or went missing in the first days of lactation. In contrast, no pups in any other group died or went missing. Missing pups were most likely cannibalized. It is not uncommon for a few pups to be lost during the first days of lactation. The incidence was higher than seen for the other groups in the study. However, the deaths were limited to 1-2 pups per litter and the mean incidence for postnatal loss (0.6) was within the historical control range (95% = 1.0). Furthermore, the number of living pups was slightly higher at 200 mg/kg bw/day
compared to the other groups.

EARLY POSTNATAL PUP DEVELOPMENT
The number of living pups at first litter check, viability index and sex ratio were unaffected by treatment, and clinical signs and external macroscopy did not reveal treatment-related findings.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups included pale appearance and black discoloration of the tail apex. These were the only two clinical signs seen and were noted for individual animals. These were incidental in nature and were not treatment related or toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights were significantly lower for female pups at 200 mg/kg bw/day on Day 1 of lactation. The difference from controls was not statistically significant by lactation Day 4, though pup body weights were slightly (not significantly) lower for pups of both sexes on lactation Days 1 and 4. This was not considered to represent significant developmental toxicity.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach. Incidental macroscopic findings among surviving pups included black discoloration of the tail apex (seen for a single pup at 200 mg/kg bw/day) and no milk in the stomach seen for all pups in a single
control litter (no. 44). Due to the limited incidence observed these signs were not considered to be toxicologically relevant.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 200 mg/kg bw/day was derived.
Executive summary:

A GLP compliant combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in male and female Wistar Han rats at dose levels of 20, 60 and 200 mg/kg bw/day. Animals of the control group received the vehicle, a 1:1 ratio of Polyethylene glycol 400 and water, alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-56 days). The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Males: Week 4; females: end of lactation), body weight and food consumption (at least at weekly intervals), clinical pathology (Males: Week 4; females: end of lactation), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Daily administration of test material at the dose of 200 mg/kg bw caused slightly reduced body weight gains in males and females. Liver and thyroid gland weights were increased in both sexes. Kidney weights were slightly increased in males. Macroscopically, foci and irregular surface of the stomach were observed. Histopathological examinations revealed slight to moderate hyperplasia of the squamous epithelium of the stomach in males and females and minimal to slight hemorrhages of the glandular stomach in males. In the thyroid of females minimal to slight diffuse follicular hypertrophy was observed. Examination of thyroid hormones revealed increased T4 levels in both sexes. No toxicologically relevant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, and clinical laboratory investigations). No compound-related adverse effects were observed at 60 mg/kg bw/day. No reproduction toxicity and no developmental toxicity was observed up to the highest dose level tested (200 mg/kg bw/day).

In conclusion, treatment with the test article by oral gavage in male and female Wistar Han rats at dose levels of 20, 60 and 200 mg/kg bw/day revealed parental toxicity at 200 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) was determined at 60 mg/kg bw/day. No reproductive or developmental toxicity was observed with treatment up to 200 mg/kg body weight/day. Based on these results, a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 200 mg/kg bw/day was derived.