Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.11.2014 - 29.06.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
Test system:
All animals were free from clinical signs of disease at the start of the study. The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.

Reason for species selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Age (at supply); sex:
10 - 11 weeks (males/females)

Supplier:
Charles River Laboratories, Research Models and Services, Germany GmbH

Identification:
The rats of the parental generation (F0 animals) were identified clearly by ear tattoo. All live pups were identified by skin tattoo on PND 1.

HOUSING AND DIET:
During the study period, the rats were housed individually in Polycarbonate type M III cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, was added for environmental enrichment.
The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
The animals were housed in a fully air-conditioned room. Central air-conditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity and 15 air changes per hour. The day/night cycle was 12 hours (12 hours light from 06.00-18.00h, 12 hours dark from 18.00-06.00h). There were no or only minimal deviations from these limits.
The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week with water containing an appropriate disinfectant.
The food used was ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Azo Initiator VA044 was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced daily.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water over a period of 4 hours at room temperature was proven before the start of the study.
Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. Given that the test substance is completely miscible with drinking water, solutions were considered to be homogenous and no homogeneity analyses were carried out.
Duration of treatment / exposure:
30 days for males
63 days for females
Frequency of treatment:
once daily
Details on study schedule:
Date Phase of study /examination Study day
25 Nov 2014 Study initiation date/ signature of study plan -13
02 Dec 2014 Supply of animals/beginning of acclimatization period/experimental starting date -6
08 Dec 2014 End of acclimatization period and beginning of administration period 0
21 Dec 2014 First mating of parental animals for the formation of F1 generation pups 13
05 Jan 2015 Urinalysis of parental males 28
06 Jan 2015 FOB1; MA1 of male parental animals 29
07 Jan 2015 End of administration period of males 30
08 Jan 2015 Blood sampling/sacrifice of parental males 31
from 12 Jan 2015 Parturition of F1 generation pups postnatal day (PND) 0 35
from 16 Jan 2015 Sacrifice of F1 litter PND 4 39
04 Feb 2015 FOB2; MA2 of female parental animals 58
06 Feb 2015 Urinalysis of parental females 60
09 Feb 2015 End of administration period of females 63
10 Feb 2015 Blood sampling/sacrifice of parental females 64
29 Jun 2015 Experimental completion date (draft report to QAU)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
F0 generation parental animals and their progeny:
On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1.

Examinations

Parental animals: Observations and examinations:
Parental animals:
Mortality:
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations:
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal. The littering 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 public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering 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:
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:
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

Food consumption:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

Water consumption:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• 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.
• Females without a litter and without positive evidence of sperm in the vaginal smear or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume. Therefore, these values are only documented in the Individual Tables (PART II).

Functional observational battery:
A functional observational battery was performed in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started 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; during this period 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:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
6. Other findings
Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. behavior on removal from the cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupilsize
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements/stereotypes
14. gait abnormalities
15. activity/arousal level
16. feces excreted within 2 minutes (appearance/consistency)
17. urine excreted within 2 minutes (amount/color)
18. rearing within 2 minutes
19. other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory
motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. touch sensitivity (touch response)
3. vision (visual placing response)
4. pupillary reflex
5. pinna reflex
6. audition (auditory startle response)
7. coordination of movements (righting response)
8. behavior during handling
9. vocalization
10. pain perception (tail pinch)
11. other findings
12. grip strength of forelimbs
13. grip strength of hindlimbs
14. landing foot-splay test

Motor activity assessment:
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. 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 numbers of beam interrupts was counted over 12 intervals of 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 rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and 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.

Oestrous cyclicity (parental animals):
Female reproduction and delivery data:
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

Sperm parameters (parental animals):
Male reproduction data:
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Litter observations:
Pup number and status at delivery:
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.

Pup viability/mortality:
In general, 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 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations.

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

Pup clinical observations:
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data:
The pups were weighed on the day after birth (PND 1) and on PND 4. 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). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. “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.

Postmortem examinations (parental animals):
CLINICAL PATHOLOGY:
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were
evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group:

Hematology:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameters and methods:
Parameter Unit Method References
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry Operator’s Guide for Advia 120 System
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation: MCV x erythrocytes
Mean corpuscular volume (MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular hemoglobin (MCH) fmol calculation: hemoglobin erythrocytes
Mean corpuscular hemoglobin
concentration (MCHC) mmol/L calculation: hemoglobin hematocrit
Platelet count (PLT) giga/L flow cytometric laserlight scattering
Differential blood count % and giga/L cytochemistry coupled with flow cytometry
Reticulocytes (RET) % cytochemistry coupled with flow cytometry
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Parameter and method:
Parameter Unit Method References
Prothrombin time (Hepato Quick’s test) (HQT) seconds citrated blood with calcium thromboplastin Fischer, M. and Falkensammer, Ch., Klin. Wschr. 86, 577-583 (1974)

Clinical chemistry:
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
Parameters:
Enzyme (systematic name and system number):
Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase)
Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase)
Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase)
γ-Glutamyltransferase (GGT) (γ-glutamyl) peptide: aminoacid-γ-glutamyl-transferase)
Blood Chemistry Parameter:
Sodium
Potassium
Chloride
Inorganic phosphate
Calcium
Urea
Creatinine
Glucose
Total bilirubin
Total protein
Albumin
Globulins
Triglycerides
Cholesterol
Bile acids

Urinalysis:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Parameters:
pH
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Blood
Specific gravity
Sediment
Color, turbidity
Volume

PATHOLOGY:
Necropsy:
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Female animal No. 139 of test group 3 (1000 mg/kg bw/d) was sacrificed in a moribund condition on study day 42. The animal was necropsied and assessed by gross pathology as soon as possible.

Organ weights:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski’s method; see section 3.8.1.10)
48. Vagina
The ovaries of the intercurrently sacrificed female were fixed in 4% neutral-buffered formaldehyde solution.

Histopathology:
A special stain for fat (Oil-Red-Oil) was performed in animal Nos. 31 and 131 of test group 3 (1000 mg/kg bw/d).
Female animal No. 139 of test group 3 (1000 mg/kg bw/d), which was sacrificed in a moribund state, was processed histotechnically and assessed like control animals with all organs listed above.
Special attention was given on the stages of spermatogenesis in the testes.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
Pup necropsy observations:
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Summary clinical observations for males and females (except gestation and lactation periods):
Male animals Nos. 33, 35 and 37 of test group 3 (1000 mg/kg bw/d) showed soft feces during premating days 7 and 8. A relation to
treatment was not assumed because of the transient and short occurrence of the finding. No other clinical findings were observed for male and female animals of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d).

Summary clinical observations for females during gestation:
Female animal No. 139 of test group 3 (1000 mg/kg bw/d) showed poor general condition, piloerection and a distended abdomen between gestation days 40 to 42. The animal was sacrificed in a moribund condition on gestation day 42. These findings were considered to
be incidental or spontaneous in origin and without any relation to treatment.
No other clinical findings were observed for female animals of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d).

Clinical observations for females during lactation:
Female animal No. 120 of test group 1 (100 mg/kg bw/d) showed a palpable mass through skin from lactation days 9 to 15. On lactation days 16 to 19 a skin lesion on the left flank was observed also in this animal. Female No. 129 of test group 2 (300 mg/kg bw/d) showed sparse fur in the abdominal region from lactation day 21 onwards until sacrifice.
The findings in both animals were assessed to be incidental and not related to treatment.
No other clinical findings were observed for female animals of test groups 0-3 (0, 100, 300
and 1000 mg/kg bw/d).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Female animal No. 139 of test group 3 (1000 mg/kg bw/d) was sacrificed moribund on gestation day 42.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight of male animals of test group 3 (1000 mg/kg bw/d) was significantly reduced on premating day 13, but not on mating day 8 or on post-mating day 0. The significant deviation to the control group was most likely related to the temporarily reduced food consumption. Some individuals of this test group lost weight between premating days 0 to 7, others did not. All animals recovered and gained weight from premating day 7 onwards. Therefore, these changes were regarded to be related to treatment but not adverse.
No significant deviation in mean body weights were observed for female animals of test group 3 (1000 mg/kg bw/d) during the entire study period. The same was true for mean body weights of male and female animals in test groups 1 and 2 (100 and 300 mg/kg bw/d), which were not affected.
Mean body weight change values in male animals of test group 3 (1000 mg/kg bw/d) were significantly lower between premating days 0-7 and 0-13. In female animals, body weight loss could be observed between premating days 0-7. However, no significant deviations
were observed between premating days 7-13 and 0-13 because the animals recovered. During gestation and lactation, mean body weight change values were comparable to the control group. Therefore, the temporary stagnation in body weight development of male
and female animals was assessed to be treatment-related but not adverse because of the fast occurring recovery.
Mean body weight change values of male and female animals in test groups 1 and 2 (100 and 300 mg/kg bw/d) were not affected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in males and females of test group 3 (1000 mg/kg bw/d) was significantly decreased between premating days 0 to 7. The finding could have been related to temporary irritation of the upper digestive tract. Both, male and female animals of test group 3 (1000 mg/kg bw/d) got used to the application after the first week of administration and food consumption values turned to normal values typical for rats of this strain and age. Therefore, the temporarily reduced food consumption was assessed to be related to treatment but not to be an adverse sign of systemic toxicity. No comparable observation were made in any other test group of the study.
Note: During the gestation period, food consumption between GD 14-20 was accidently not determined for single female animals of all test groups including the controls. This deviation to the study plan did neither influence the toxicological assessment nor the validity of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. After the administration period in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) hematocrit values were higher compared to controls (not significantly in test group 3), but the means were within the historical control range (hematocrit 0.392-0.428 L/L; PART III, Supplement). In females of test group 2 (300 mg/kg bw/d) red blood cell (RBC)
counts were increased and in males of test group 1 (100 mg/kg bw/d mean corpuscular hemoglobin content (MCH) was decreased, but the values were not dose-dependently changed. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. In males of test group 2 (300 mg/kg bw/d) γ-glutamyl transferase (GGT) activities were decreased, but the changes were not dose-dependent. In females of test group 3 (1000 mg/kg bw/d) triglyceride values were increased. This was the only changed parameter in these individuals. Therefore, the GGT activity decrease in males of test group 2 was regarded as incidental and not treatment-related, whereas the higher triglyceride levels in females of test group 3 were assessed as treatment-related but not adverse.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed. In males of test group 3 (1000 mg/kg bw/d) urine pH values were decreased. This alteration per se without any change among kidney parameters was regarded as treatment-related but not adverse.
Behaviour (functional findings):
not examined
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):
A centrilobular vacuolation of hepatocytes was seen in males and females of test group 3 (1000 mg/kg bw/d). A special stain for fat (Oil-Red-Oil) was negative revealing the absence of fat in those vacuoles. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Fertility:
The female animal (No. 139), showed a massively dilated uterus and cervix, as a consequence of a severe purulent inflammation in the uterus, which explained the nonpregnant status of this animal. The male mating partner (Nos. 39) showed no histopathological changes.
Deceased animal:
Animal No. 139 was sacrificed in a moribund state showing a severe purulent uterus inflammation with consequent massive uterine and cervical dilation, reactive plasmocytosis in the iliac, renal and mediastinal lymph nodes. Other reactive responses to the severe inflammation were a severe myeloid hyperplasia in the bone marrow and moderate extratramedullary hematopoiesis of myeloid type in the spleen. Additionally, the thymus showed severe atrophy most likely as a consequence of the bad state of this animal. All of these finding explained the infertility. They were not considered to be treatment-related but incidental.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single rats only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.

Motor activity measurement:
There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group.
Regarding single intervals in male animals of test group 3 (1000 mg/kg bw/d) a significantly reduced activity was observed in interval 11. As no other single intervals as well as the overall motor activity showed significant deviations to the control values, the finding was assessed to be incidental and not related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Female fertility index:
All sperm positive rats delivered pups with one exception in test group 3 (1000 mg/kg bw/d). Female animal No. 139, which was mated with male No. 39, had sperm in vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 100% in test groups 0-2 and 90% in test group 3. These values reflected the normal range of biological variation inherent in the strain of rats.
The mean duration of gestation was similar in all test groups, i.e. 22.4 days (test group 0), 22.5 days (test group 1), 22.2 days (test group 2) and 22.1 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Male fertility index:
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
Male animal No. 39 of test group 3 (1000 mg/kg bw/d), which was mated with female No. 139, did not generate F1 pups. Thus, the male fertility index was 90% in test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data
Reproductive performance:
no effects observed
Description (incidence and severity):
Male mating index:
The male mating index was 100% in all test groups.

Female mating index:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.6 days for test groups 0, 2 and 3 and 2.5 days for test group 1. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Gestation index:
The gestation index was 100% in all test groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One pup of test group 1 (No. 117-10) was not properly nursed on PND 3 and 4. All other surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during lactation (PND 0-4) was 100% in test groups 0, 2 and 3 and 99.2% in test group 1. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. One pup of test group 0 (control) was found stillborn and 3 pups of test group 0 were cannibalized. One pup of test group 1 was found dead. A relation to treatment was excluded.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
2 male runts were found in test group 0 and 2 female runts were found in test group 1, one female runt was found in test group 2. Two female runts occurred in test group 3.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female pup of test group 1 (No. 117-10) showed a large heart, a small discolored liver and an empty stomach. One male pup of test group 3 (No. 132-05) showed a dilated renal pelvis (right). These findings were assessed as being spontaneous in nature and without toxicological relevance.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of the present Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration of Azo Initiator VA044 by gavage to male and female Wistar rats did not result in signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.
Executive summary:

Analyses

The various analyses confirmed

• the stability of the test substance in drinking water over a period of 4 hours at room temperature,

• the overall accuracy of the prepared concentrations.

Effects

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

Test group 3: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

• No test substance-related, adverse findings were noted.

F1 PUPS

Clinical Examinations/ Gross Findings

• No test substance-related, adverse findings were noted.

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

• No test substance-related, adverse findings were noted.

F1 PUPS

Clinical Examinations/ Gross Findings

• No test substance-related, adverse findings were noted.

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

• No test substance-related, adverse findings were noted.

F1 PUPS

Clinical Examinations/ Gross Findings

• No test substance-related, adverse findings were noted.