Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Mar 2016 - 03 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Jul 2000)
Principles of method if other than guideline:
In addition, this study enhanced the guideline recommendations by parameters which are part of the following test guidelines:
- EPA Health Effects Test Guidelines, OPPTS 870.3800: Reproduction and Fertility Effects (Aug 1998)
- OECD Guidelines for Testing of Chemicals; No. 416 (22 Jan 2001)
- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 34 - 36 days old and females: 27 - 29 days old
The 48 male and 48 female animals included in the study were 40 - 42 days (males) and 12 weeks (females) old at the beginning of treatment
- Weight at study initiation: (P) Males: 139.4 g - 167.3 g; Females: 185.9 g - 220.3 g
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop- Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 21 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy).
- Water: supplied from water bottles (ad libitum)
- Acclimation period: 6 days (males) and 8 weeks (females)

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity (%): 30-70
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 08 Mar 2016 To: 04 Aug 2016 (sacrifice of rearing animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance suspensions in 0.5% Carboxymethylcellulose suspension in drinking water with 5 mg/100 mL Tween 80 were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% Carboxymethylcellulose suspension in drinking water with 5 mg/ 100mL Tween 80 and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1, 3, 10 g/100ml
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until sperm were detected in the vaginal smear, maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80 for a period of 7 days at room temperature were carried out prior to the start of the study. At the beginning, in the mid and towards the end of premating, once during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample was retained.

Results of the various analyses:
• The stability of test substance in 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100mL TWEEN 80 was demonstrated for a period of 7 days at room temperature
• The results of the homogeneity analysis of the high dose as well as concentration control analysis of the mid and high dose were well within the specification limits. This is generally also true for the low dose; two samples of the low dose were measuered just outisde the specification range. Since all other values taken at the same time point were well within the specification range, these two departed samples were regarded as outliers. A technical error during sample preparation, processing and measurement cannot be excluded.
Duration of treatment / exposure:
The duration of treatment covered a 10 weeks premating period in males, 2 weeks premating in the females, 2 weeks mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as up to 30 days of the lactation period (corresponding to 21 days lactation and up to 9 days postweaning) and 35 days post-mating (for sperm negative females).
Frequency of treatment:
daily
Details on study schedule:
The females were allowed to deliver and rear their pups until PND 4 (standardization), 13 or 21.
Before weaning of the F1 generation pups on PND 21, 10 male and 10 females per group (1 per sex per litter) were selected by lot to become the F1 rearing animals. Surplus F1 generation pups were sacrificed after weaning. The F1 rearing animals remained untreated. The selected F1 rearing animals were reared until sexual maturity was reached (developmental landmarks) and were sacrificed after completion of the sexual maturity. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.
On study day 37, a functional observational battery and motor activity measurement were carried out in five male animals per group.
On study day 101, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group.
Blood samples were taken from all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14.
The male and female animals were sacrificed 84/85 and 118/126 days, respectively, after the beginning of the administration of the male animals, and examined.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a pre-test
Positive control:
no

Examinations

Parental animals: Observations and examinations:
MORTALITY
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals and the F1 rearing animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10, 13, 17 and 21.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION
Generally, food consumption was determined once a week for male and female parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10, 10 - 13, 13 - 17 and 17 - 21.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14; time point of collection: in the morning
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes (males), no (females)
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14; time point of collection: in the morning
- Animals fasted: Yes (males), no (females)
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes

Functional observation battery
A functional observational battery (FOB) was performed in the first five male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
Behavior on removal from cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypy, Gait, Activity/arousal level, Feces (consistency/color) within 2 minutes, Urine (amount/color) within 2 minutes, Rearing within 2 minutes, Other findings

Sensory motor tests/Reflexes:
The animals were removed from the open field and subjected to following sensory motor or reflex tests:
Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity measurement
The Measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

IMMUNOLOGY: No

OTHER:
Thyroid Hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adult males were fastened and the dams were not fastened before the blood sampling. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4). All generated serum samples were frozen at -80°C until measurement. T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period. Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female
parental rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male parental animals sacrificed on schedule:
Sperm motility, Sperm morphology, Sperm head count (cauda epididymis), Sperm head count (testis)
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups or 2 preferably female pups per litter were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

PARAMETERS EXAMINED
• All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
• A check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-13 and 14-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 13, and 21. Furthermore, viability and lactation indices were calculated.
• The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
• The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 13 and 21. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the
morning) and on PND 4 immediately before standardization of the litters. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
• Anogenital distance was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.
• All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. The number of nipple/areola anlagen was counted.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups were evaluated by the methods, which are described in detail in section “Postmortem examinations” below.

SEXUAL MATURATION (F1 REARING ANIMALS)
• Vaginal Opening: All female F1 rearing animals (10/group) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.
• Preputial separation: All male F1 rearing animals (10/group) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.
Postmortem examinations (parental animals):
SACRIFICE
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Bulbourethral gland (Cowper’s gland), Cauda epididymis, Epididymides, Glans penis, Musc. levator ani together with Musc. bulbocavernosus (males only), Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY
Histopathological examinations were performed according to the table listed in "any other information on materials and methods incl. tables"

Postmortem examinations (offspring):
Pup necropsy observations
• On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
• On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The thyroid glands of all control group 00 and test group 03 male animals were processed histotechnically to HE-stained slides and assessed by light microscopy.
• On PND 21, the surplus F1 generation pups that were not used as F1 rearing animals were likewise be sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Necropsy observations (F1 rearing animals)
After completion of the sexual maturity, the F1 rearing animals were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all F1 rearing animals were examined externally and eviscerated, and their organs were assessed macroscopically.
All F1 rearing animals that died or were sacrificed in a moribund state were examined externally, eviscerated and their organs were assessed macroscopically.
All F1 rearing animals without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
• Food consumption, body weight and body weight change,gestation days, anogenital distance, anogenital index: DUNNETT-test (two-sided)
• Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
• Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment
• % live male day x, %live female day x: WILCOXON test (two-sided)
• Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, Blood parameters, Weight parameters pathology: KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided)
• Spermanalysis parameters: WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one-sided) without adjustment were used.
Reproductive indices:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index,
Offspring viability indices:
Live birth index, Postimplantation loss, Viability index, Lactation index, Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period. The statistically significantly decreased body weight change in the mid-dose females during PND 7 - 10 was considered as spontaneous in nature and not treatment related as no doseresponse relationship occurred.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of the F0 males and females in all dose groups (100, 300 and 1000 mg/kg bw/d) was not influenced by the treatment throughout the entire study period. The statistically significantly increased food consumption in the mid-dose F0 females during PND 4 – 7, high-dose F0 males during premating days 63 - 70 and high-dose F0 females during PND 17 - 21 was considered as spontaneous in nature and not treatment-related.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. In females of test group 2 (300 mg/kg bw/d) cholesterol levels were significantly lower compared to controls, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. There were no test substance-related findings in male and female animals of all test groups regarding sensorimotor tests/reflexes. No test substance-related impaired parameters were observed in male animals of all test groups in quantitative parameters. The landing foot-splay test (FST) in high-dose females was statistically significantly below the concurrent control values (about 26%). The individual values of the high-dose females ranged from a minimum of 7.5 cm to a maximum of 9.8 cm and laid well within the individual value range of the historical control data (range of 6.9 – 13.5 cm for 6 studies in the last 4 years. In these high-dose females, no corresponding clinical findings were observed and no other parameter in FOB or MA was changed, thus giving no indication of neurotoxicity. The mean values of the low- and mid-dose females were not affected. Therefore, the decrease of the high-dose females in FST was assessed as incidental and not-treatment related. The observed parameters rearing and grip strength for forelimbs and hindlimbs in females of all dose groups did not show any test substance-related or spontaneous impairments.

Motor activity measurement
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed in the parental male and female animals. All findings occurred either individually or were biologically equally distributed over control and high dose treated animals. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The stages of spermatogenesis in the testes of males of the high dose were comparable to those of the controls.
The female animals Nos. 110, 111, and 145, and the male mating partners No. 10, 11, and 45 showed no histopathological findings that could explain the impaired fertility. The male No. 45 had a slight prostatic lymphoid infiltrate. However, this change belongs to the spectrum of background lesions that do not impact fertility.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
T4 medians in parental males of test group 3 (1000 mg/kg bw/d; T4 – 17%) were lower compared to controls but not statistically significant. No histopathological correlate or any alteration of the thyroids was found.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.92 and 3.97 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
MALE REPRODUCTION DATA
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for control male No. 10 paired with control female No. 110. Thus, the male mating index was 91.7% in the control and 100% in the low-, mid- and high-dose groups.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two control males (0 mg/kg bw/d - Nos. 10 and 11) and one high-dose male (1000 mg/kg bw/d - No. 45) did not generate pregnancy. Thus, the male fertility index ranged between 83.3% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross lesions.

FEMALE REPRODUCTION DATA
The female mating index calculated after the mating period for F1 litter was 91.7% in test group 00 and 100% in test groups 01 - 03.
The mean duration until sperm was detected (GD 0) varied between 2.6 / 1.9 / 4.5* / 2.8 [*=p<=0.05] test groups 00 - 03, respectively). The statistical significant increase in test group 2 was not related to dose and was within the historical control range (1.6 – 5.8 days). Therefore, it is assessed as incidental and not treatment-related.
All female rats delivered pups or had implants in utero with the following exceptions:
• Control female No. 110 (mated with male No. 10) did not become pregnant (sperm-negative)
• Control female No. 111 (mated with male No. 11) did not become pregnant (sperm-positive)
• High-dose female No. 145 (mated with male No. 45) did not become pregnant (sperm-positive)
The fertility index varied between 90.9% in test group 00, 100% in test groups 01 and 02 and 91.7% in test group 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
None of the non-pregnant females had any relevant gross lesions.
The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.2 days). The gestation index was 100% in all test groups 0 - 3.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.8 / 12.1 / 12.6 and 12.3 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.3 / 11.2 / 12.3 and 11.8 pups/dam in test groups 00 - 03, respectively)
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 98.4% / 99.3% / 99.3% and 99.2% in test groups 00 - 03. Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the test substance Octabenzone did not adversely affect reproduction and delivery of the F0 generation parental females.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 LITTER DATA/PUPS
There were no test substance-related, adverse clinical signs observed in any of the F1 generation pups of the different test groups. For one male pup (No. 3) of control dam No. 107 a distended abdomen (grade: severe) and a paresis of both hindlimbs were observed on PND 11.

F1 REARING ANIMALS
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F1 rearing animals in any of the groups during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
PUP NUMBER
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

PUP VIABILITY
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 100% / 98.5% and 100% in test groups 00 - 03 without showing any association to the treatment. The lactation index indicating pup mortality on PND 4 - 21 varied between 73.8% / 73.8% / 75% and 75% (because of sacrificed male and female pups on PND 13 for hormone measurements) in test groups 00 - 03 without showing any relation to the treatment.

F1 REARING ANIMALS
There were no test substance-related or spontaneous mortalities in any of the groups
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 LITTER DATA/PUPS
Mean body weights and body weight change of all male and female pups of all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control values throughout the entire lactation period. The statistically significantly increased body weight change in the low-dose male pups during PND 1 - 4 was considered as spontaneous in nature and not treatment related as no dose-response relationship occurred. One male and one female runt was seen in the control, one male and two female runts were seen in test group 01 and one male and one female runt was seen in test group 02.

F1 REARING ANIMALS
The body weights and body weight change of all test substance treated male and female rats (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control values throughout the entire study. The statistically significantly increased mean body weights in the low- and high-dose males and in the low-dose females on study day 0 and the statistically significantly increased body weight change in the low-dose males during study days 14 - 21 were considered to be spontaneous in nature and not treatment-related as they were not dose-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 REARING ANIMALS
Food consumption of all test substance treated male and female rats (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
F1 LITTER DATA/PUPS
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 male and female pups (test groups 01 - 03 [100 - 1000 mg/kg bw/d]).
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. However, owing to the high background rate in the control animals on this day, all animals were habitually rechecked for nipples/areolae on PND 20, one day prior to weaning. During the re-examination no areolae were detected in all male pups of all test groups (01 - 03).

F1 REARING ANIMALS
• Vaginal opening:
Each female F1 rearing animal was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 34. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups amounted to 29.3; 31.8** (**p≤0.01); 29.8 and 31.4* (*:p≤0.05) days. The mean body weight on the day, when vaginal opening was recorded, amounted to 89.0 g, 102.8** g, 93.9 and 101.9** g in test groups 00 - 03 [10 - 13]. Both parameters of the low- and high-dose group showed statistical significance whereas the mid dose group showed none. However, there was no dose relationship since the lowest test group showed the highest value. In addition, all values were close to the mean of the historical control range of the test facility (see supplement). Therefore, the changes were assessed as incidental and not treatment-related.
• Preputial separation:
Each male F1 rearing animal was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 48. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups amounted to 41.4; 40.8; 39.9 and 41.0 days. The mean body weight on the day, when preputial separation was recorded, amounted to 175.6 g, 174.4 g, 169.0 and 180.1 g in test groups 00 - 03 [10 - 13].
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 LITTER DATA/PUPS
A few F1 pups showed spontaneous findings at gross necropsy, such as distended urinary bladder, dilated renal pelvis, hydronephrosis, hydroureter and discolored thymus (red). These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

F1 REARING ANIMALS
One male low-dose F1 rearing animal showed the spontaneous finding discolored thymus (red) at gross necropsy. This finding occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, this finding was not considered to be associated to the test substance.
Histopathological findings:
no effects observed
Description (incidence and severity):
The thyroid gland of PND13 male animals of test group 03 were comparable to those of the control group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones
At PND 13 in male pups of test groups 11 and 13 (100 and 1000 mg/kg bw/d) T4 levels were significantly lower compared to controls. The T4 median in test group 11 was not dose-dependently changed and, therefore, this alteration was regarded as incidental and not treatment-related. The T4 median in test group 13 was 16% lower compared to controls. T4 medians in F1 females at PND 13 of test group 13 (1000 mg/kg bw/d; T4 -10%) as well as in F0 males of test group 3 (1000 mg/kg bw/d; T4 – 17%) were also lower compared to controls but not statistically significant. In F0 males and in F1 PND13 male pups, no histopathological correlate or any alteration of the thyroids was found. Therefore, the T4 statistically significant decrease of T4 in male PND13 pups of test group 13 was regarded as treatment-related but not adverse.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

 

Female animals
Test group (mg/kg) 01
(100)
02
(300)
03
(1000)
Kidneys 96% 106%* 102%
Liver 87%* 108% 106%

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of the liver were significantly decreased (statistically significant changes printed in bold):

  Male animals Female animals
Test group (mg/kg) 01
(100)
02
(300)
03
(1000)
01
(100)
02
(300)
03
(1000)
Spleen 122%* 103% 113%      
Liver       85%** 106% 102%

*p <= 0.05; **p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Octabenzone was 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was 1000 mg/kg bw/d.
Executive summary:

The test item was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (12 male and 12 female Wistar rats) were dosed daily with the vehicle only (0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100mL Tween 80). The duration of treatment covered a 10 weeks premating period in males, 2 weeks premating in the females, 2 weeks mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as up to 30 days of the lactation period (corresponding to 21 days lactation and up to 9 days postweaning) and 35 days post-mating (for sperm negative females). Pups of the F1 litter were selected (F1 rearing animals) for specific post-weaning examinations. The study was terminated with the terminal sacrifice of the rearing animals. After 10 or 2 weeks of premating treatment in males or females, respectively, the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents and F1 rearing animals was determined once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13, 13 - 17 and 17 - 21. Body weights of F0 parents and F1 rearing animals were determined once a week. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20 and on postnatal days (PND) 1, 4, 7, 10, 13, 17 and 21. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7, 13 and 21. Their viability was recorded. At necropsy on PND 21, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 rearing animals was recorded. All F1 rearing animals were assessed macroscopically. The various analyses demonstrated the stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100mL TWEEN 80 over a period of 7 days in a refrigerator, confirmed the homogeneous distribution of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100 mL TWEEN 80 and verified correct concentrations of the test substance preparations.

The following test substance-related adverse effects/findings were noted:

F0 PARENTAL ANIMALS, all dose groups

No test substance-related adverse findings during clinical examinations, no effects on reproductive performance and no findings during clinical pathology and pathology

F1 PUPS, all dose groups

No test substance-related adverse findings during clinical examinations and no gross findings

F1 REARING ANIMALS, all dose groups

No test substance-related adverse findings during clinical examinations, sexual maturation and no gross findings

In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was 1000 mg/kg bw/d.