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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (Jul 2000)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
EC Number:
938-781-3
Cas Number:
117527-94-3
Molecular formula:
C21H21N304.C16H11N304.Cr unspecified.C12-C14 chain unspec.
IUPAC Name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
Details on test material:
- Physical state: solid/black
- Storage condition of test material: room temperature, avoid humidity

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: (P) Males: 305.0-337.7 g; Females: 199.7-236.9 g
- Housing: individually in Makrolon type M III cages (floor area of about 800 cm²), with the following exceptions: During overnight matings, male and female mating partners were housed together in Makrolon type M III cages; Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum from water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jan 2015 To: 19 Mar 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: drinking water containing 0.5% Carboxymethylcellulose and Cremophor
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, the vehicle was filled up to the desired volume, subsequently released with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.4, 2.0 and 10.0/5.0 g/100 ml
- Amount of vehicle (if gavage): 10 ml /kg bw
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 07.00-09.00 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 7 days in the refrigerator was proven before the start of the study. Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control. In samples of the middle concentration only concentration control analyses were performed. As the dose level was reduced from 1000 to 500 mg/kg bw/d, additional concentration control and homogeneity analyses were performed for the highest concentration 7 days as well as 8 days after beginning of the administration period.
The various analyses confirmed the stability of the test substance in drinking water over a period of 7 days in the refrigerator and the overall accuracy of the prepared concentrations (with the exception of a single test substance preparation in the high dose group, where a dose of about 125 mg/kg bw was applied on study day 7; this deviation did not affect the validity of the study and did not have an influence on the determination of the no observed adverse effect level).
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily (exception: no administration to animals being in labor)
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 200 and 1000/500 mg/kg bw. The high dose level was reduced from 1000 to 500 mg/kg bw/d from study day 7 onwards
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As requested by the sponsor
- Rationale for animal assignment: distributed randomly according to weight among the individual test groups, separated by sex

Examinations

Parental animals: Observations and examinations:
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: Yes
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.

BODY WEIGHT: Yes
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.

FOOD CONSUMPTION: yes
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: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPY
The eyes of all male animals were examined prior to the start and at the end of the administration period using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY:
In a previously performed repeated-dose 28-day toxicity study in Wistar rats, a (multi)focal to extensive inflammation was observed in the lungs of all high-dose animals (1000 mg/kg bw/d). This was regarded to be caused by the test substance and adverse in nature. Therefore, bronchoalveolar lavage fluid examinations were carried out in the second 5 animals of each test group.
The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. Cytology was examined in bronchoalveolar lavage fluid (BAL). Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. The following parameters were examined: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Epididymides weights, testes weights, staging of spermatogenesis
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.

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. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

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.

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):
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.

Organ weights:
Weight assessment was carried out on all animals. The following weights were determined:
Anesthetized animals, Epididymides, Testes

Histopathology:
The following organs / tissues of all parental animals were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
All gross lesions, Cervix, Coagulating gland, Epididymides (modified Davidson’s solution), Ovaries (modified Davidson’s solution), Oviducts, Prostate gland, Seminal vesicles, Testes (modified Davidson’s solution), Vagina, Uterus.
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed. The following tissues were examined: testes, epididymides, ovaries from all control and high dose animals.
Postmortem examinations (offspring):
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.
Statistics:
The following statistical methods were used:
• Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days: DUNNETT test (two-sided).
• Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), 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, 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).
• Broncho-alveolar lavage fluid (BALF) and lung tissue parameters: WILCOXON-test (one-sided).
• Weight parameters: 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 (two-sided).
Reproductive indices:
male and female mating index, male and female fertility index, gestation index, postimplantation loss
Offspring viability indices:
Live birth index, pup number and status at delivery, viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mortality at 1000 mg/kg bw.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 1000 and 500 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 1000 and 500 mg/kg bw
Histopathological findings: non-neoplastic:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Female animal No. 136 of test group 3 (1000 mg/kg bw/d) showed reduced nutritional and poor condition from premating day 4 onwards and was found dead on premating day 6. In test group 3 (1000 and 500 mg/kg bw/d) black discoloration of eyes and skin was observed. Males and females of test group 3 showed black discolored skin over the entire body from premating day 3 onwards and black discoloration of the eyes was observed from premating day 8 onwards until sacrifice. Male and female animals of test groups 2 and 3 (200 and 1000 as well as 500 mg/kg bw/d) showed black discolored feces from premating day 1 onwards. Grey discoloration of skin over the entire body was observed in male animals of test group 2 (200 mg/kg bw/d) from mating day 2 onwards. Black discoloration of the eyes of males of test group 2 was observed from mating day 10 onwards until sacrifice. Five females of test group 2 (200 mg/kg bw/d) showed grey discolored skin over the entire body from mating day 2 onwards. All findings which were observed in animals of test groups 2 and 3 (200 and 1000 as well as 500 mg/kg bw/d) were assessed to be related to treatment.
During gestation, female animals of test group 3 (1000 and 500 mg/kg bw/d) showed black discoloration of the eyes and the skin. Female animals of test group 2 (200 mg/kg bw/d) showed black discolored eyes and grey discoloration of the skin during the gestation period. These changes were assessed to be related to treatment. No changes were observed for female animals of test group 1 (40 mg/kg bw/d). Female animal No. 128 of test group 2 (200 mg/kg bw/d) was sacrificed moribund on gestation day 29 after showing a hard abdomen and poor general condition. The findings were assessed to be unrelated to treatment.
During lactation, female animals of test group 3 (1000 and 500 mg/kg bw/d) showed black discoloration of the eyes and the skin. Female animals of test group 2 (200 mg/kg bw/d) showed black discolored eyes and grey discoloration of the skin during the lactation period. These changes were assessed to be related to treatment.
No changes were observed for female animals of test group 1 (40 mg/kg bw/d).

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was significantly reduced in male and female animals of test group 3 (1000 mg/kg bw/d) between premating days 0 to 7. Additionally, female animals of test group 3 (1000 and 500 mg/kg bw/d) showed also significantly reduced values between gestation days 0 to 7 and 14 to 20 as well as between lactation days 1 to 4. All changes were assessed to be related to treatment and adverse.

WATER CONSUMPTION (PARENTAL ANIMALS)
No test substance-related findings with regard to water consumption were observed.

BODY WEIGHT (PARENTAL ANIMALS)
Mean body weights of male animals of test group 3 (1000 and 500 mg/kg bw/d) were significantly reduced on premating days 7 and 13 as well as on mating day 8 and on post-mating day 1. In female animals of test group 3 (1000 and 500 mg/kg bw/d), mean body weights were significantly lower on premating day 7, during the entire gestation period and on lactation day 4. These differences to the control animals were assessed to be related to treatment and adverse (Table 10).
Mean body weights of male and female animals in test groups 1 and 2 (40 and 200 mg/kg bw/d) were not affected in a treatment-related manner. Although mean body weight values of male animals in test group 1 (40 mg/kg bw/d) were significantly lower on premating day 13 as well as during mating and post mating periods, the differences to the control animals did not occur in a dose-dependent manner. The changes were assessed as being incidental and not related to treatment.
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 (Table 8, 9). In female animals, body weight loss could be observed between premating days 0-7 with a slight recovery after dose reduction. During gestation, mean body weight change values were lower between GD 14-20. Between lactation days 0-4, body weight loss occurred again. These changes were assessed to be related to treatment and adverse.
Mean body weight change values of male and female animals in test groups 1 and 2 (40 and 200 mg/kg bw/d) were not affected in a treatment-related manner. Although mean body weight values of male animals in test groups 1 and 2 (40 and 200 mg/kg bw/d) were significantly lower between premating days 0-7 and 0-13, the change was transient and did not occur at later stages of the study. The same was true for the single deviation in female animals of test group 1 (40 mg/kg bw/d) between pre-mating days 7-13. The changes observed in test groups 1 and 2 (40 and 200 mg/kg bw/d) were assessed as being incidental and not related to treatment.

OPHTHALMOLOGICAL EXAMINATION (PARENTAL ANIMALS)
Remainders of the pupillary membrane and corneal stipplings were observed in several male animals of all test groups before the beginning of the administration period and on study day 23 (towards the end of the administration period). Those findings were assessed as being incidental in nature since they did not show a dose-response relationship and had incidence levels compared to controls.
Male animals of test groups 2 and 3 (200 as well as 1000 and 500 mg/kg bw/d) showed dark discoloration of the eyes. The examination did not reveal information on the specific location of the dye in the eye balls. Findings at the retina or the cornea were not observed.
REPRODUCTIVE FUNCTION: (MALE PARENTAL ANIMALS)
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. The male fertility index was 100% in test group 3, 90% in test group 0 and 1 and 80% in test group 2. 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 FUNCTION: (FEMALE PARENTAL ANIMALS) (Table 1)
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 1.7, 2.7, 1.9 and 1.6 days in test groups 0-3. 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.

All sperm positive rats delivered pups except 1 female animal of test group 0 control), 2 female animals of test group 1 (40 mg/kg bw/d), 2 female animals of test group 2 (200 mg/kg bw/d) as well as 4 female animals of test group 3 (1000 and 500 mg/kg bw/d) (Table 2).
Female animals Nos. 133, 135, 137 and 138 (test group 3) which were mated with male Nos. 33, 35, 37 and 38 had sperm in vaginal smear and showed implantation sites but delivered no pups. Female No. 125 (test group 2) and female No. 118 (test group 1) mated with animal Nos. 25 and 18 did get sperm in vaginal smear and showed implantation sites but delivered no pups. Female Nos. 111 (test group 1) and 103 (test group 0) mated with animal Nos. 11 and 3 did get sperm in vaginal smear and showed implantation sites but delivered no pups.
The female fertility index was 100% in test group 3, 90% in test groups 0 and 1 and 80% in test group 2.

The mean duration of gestation was similar in all test groups, i.e. 22.3 days (test group 0), 22.5 days (test group 1), 22.2 days (test group 2) and 22.6 days (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.

The gestation index was 100% in test group 0 (control), 88.9% in test group 1 (40 mg/kg bw/d), 100% in test group 2 (200 mg/kg bw/d) and only 55.6% in test group 3 (1000 and 500 mg/kg bw/d) (Table 3). The lower value for test group 1 was assessed to be incidental as no dose-response relationship occurred and the value of 88.9% calculated for test group 1 is almost equal to the lower limit of the historical control range (89% to 100%). The low value of test group 3 was clearly below the normal range of biological variation. The change was assessed to be related to treatment and adverse.

The live birth index was 98.9% in test groups 0 and 1 and 100% in test groups 2 and 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.

The postimplantation loss was 17.1% in test group 0 (control), 23.0% in test group 1 (40 mg/kg bw/d), 8.8% in test group 2 (200 mg/kg bw/d) and 74.2% in test group 3 (1000 and 500 mg/kg bw/d) (Table 4). The high value for test group 1 was assessed to be incidental as no dose-response relationship occurred and is close to the upper limit of the historical control range (0.7% to 18.3%). The very high value of test group 3 was clearly above the normal range of biological variation. The change was assessed to be related to treatment and adverse.

BRONCHOALVEOLAR LAVAGE FLUID (PARENTAL ANIMALS) (Table 12)
At the end of the administration period in males of test group 3 (1000 and 500 mg/kg bw/d) absolute neutrophil cell counts in the bronchoalveolar lavage fluid (BAL) were slightly increased, although total cell counts in the BAL were not changed. Relative neutrophil cell counts in BAL were increased and relative macrophages cell counts decreased (Table 12).

ORGAN WEIGHTS (PARENTAL ANIMALS)
The decrease in terminal body weight in males and females of test group 3 (1000 and 500 mg/kg bw/d) was regarded to be treatment-related (Table 11). The decrease in terminal body weight in males of test group 1 (40 mg/kg bw/d) was interpreted to be incidental and not related to treatment due to a missing dose-response relationship.
The significant increase in relative testicular weight in test group 3 animals (1000 and 500 mg/kg bw/d) was regarded to be a consequence of the reduced terminal body weight. Especially as no histopathologic findings were observed in the testes this was not thought to be a direct effect of the test substance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All animals of test group 3 (1000 and 500 mg/kg bw/d) revealed a grey discoloration of the carcass, the skin, the fatty tissue and all parenchyma. In test group 2 (200 mg/kg bw/d) also all animals were affected, but the discoloration was slighter and not all parenchyma were affected. No discoloration was observed in male and female animals of test group 1 (40 mg/kg bw/d) (Table 13).
Except the discolorations observed for the intestinal contents of some animals, 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.
The female animals (Nos. 103, 111, 125, 128, 136), which were not pregnant as well as the male mating partners (Nos. 3, 25, 28, 36) did not show relevant gross lesions. In the male animal No. 11 of test group 1 (40 mg/kg bw/d) only unilaterally testis and epididymides were present. This could be the cause for not having offspring in this mating pair. A relation to treatment was excluded.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All 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.
The female animals (Nos. 103 and 136), which were not pregnant as well as the male mating partners (Nos. 3 and 36) did not show relevant histopathological findings. Animal Nos. 111, 125, 128 and 11, 25, 28 were not examined histopathologically.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 200 mg/kg bw, animals were passively coloured (test item is a black dye). This is regarded as non-adverse, but treatment related.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Male and female fertility indices within normal range. No histopathology findings on reproductive organs. The NOAEL is based on the Increased postimplantation loss ( = lower gestation index).

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
no effects on live birth indes. Increased postimplantation loss at 500 mg/kg bw. Decreased postnatal viability at high dose group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight change values of female pups as well as males and females together were significantly reduced at the high dose group.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discoloration of stomach and intestinal content as well as liver, heart, lung and kidney (indicates presence of the black dye, not adverse)
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam was equally distributed among groups 0, 1 and 2. However, the absolute as well as the mean number of delivered pups in test group 3 were clearly decreased. The effect was assessed to be treatment-related and adverse.
The viability index indicating pup mortality during lactation (PND 0-4) was 99% (test group 0), 100% (test group 1), 98.2 % (test group 2) and 66.7% (test group 3). The low viability index of test group 3 (1000 and 500 mg/kg bw/d) was clearly below the historical control range and assessed as being related to treatment.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of test groups 1-3 showed a discoloration of the skin what was regarded to be related to treatment. Depending on the test group the skin was discolored grey in test groups 1 and 2 (40 and 200 mg/kg bw/d) as well as black in test group 3 (1000 and 500 mg/kg bw/d). Three female pups of test group 3 (1000 and 500 mg/kg bw/d) showed reduced nutritional condition (no milk in stomach). The finding was assessed to be related to treatment.
Seven male and 6 female pups of test group 2 (200 mg/kg bw/d), all belonging to female animal No. 127, also showed reduced nutritional condition (less milk in stomach). This kind of finding went in parallel to reduced pup weights on PND 1. Although only 1 litter was affected it was assumed that the finding occurred most likely secondary to toxic effects in the dam resulting in lower milk production. One female pup of test group 3 showed a short tail. This finding was assessed to be incidental and not related to treatment.

BODY WEIGHT (OFFSPRING)
Mean body weights and body weight change values of pups of test group 1 (40 mg/kg bw/d) were comparable to test group 0 (control). The same was true for test group 2 (200 mg/kg bw/d) although the litter of female animal No. 127 had several individuals with low body weights. Mean body weights of pups in test group 3 (1000 and 500 mg/kg bw/d) were lower compared to the control but not significantly. Body weight change values of female pups as well as males and females together were significantly reduced (Note: As surviving male pups occurred only in 2 litters no statistical test was performed for this sex).

SEXUAL RATIO (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between control and test groups 1 and 3 but the number of live male and females on PND 4 in test group 2 was significantly changed. As no dose-response relationship occurred the changes were regarded to be incidental and not related to treatment.

GROSS PATHOLOGY (OFFSPRING)
Post mortem autolysis was observed in 1 male pup of test group 0 (control), 1 male pup of test group 1 (40 mg/kg bw/d) and 1 female pup of test group 3 (1000 and 500 mg/kg bw/d). One male pup and 6 female pups of test group 1 showed dilated renal pelvis. These findings were assessed as being spontaneous in nature and without toxicological relevance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL is based on the Increased postimplantation loss ( = lower gestation index) and the reduced postnatal viability.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary Mating Report

  Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 40 mg/kg bw/d Test Group 2/ F 200 mg/kg bw/d Test Group 3/ F 1000/500 mg/kg bw/d
No. of females mated N 10 10 10 9
- Inseminated N 10 f- 10 10 9
Female mating index % 100 100 100 100
-- Pregnant N 9 f- 9 8 9
Female fertility index % 90 90 80 100
No. of males mated N 10 10 10

9

- With inseminated females N 10 f- 10 10 9
Male mating index % 100 100 100 100
- With pregnant females N 9 f- 9 8 9
Male fertility index % 90 90 80 100
Females with defined Day 0 pc N 10 10 10 9
Mating days until Day 0 pc Mean 1.7 x+ 2.7 1.9

1.6

  S.d. 1.1 1.1 1.1 1.1
Days 0 To 4 N 10 10 10 9
  % 100 100 100 100
Days 5 To 9, and 10.14 N 0 0 0
  % 0 0 0 0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics. f=FISHER-EXACT; x=WILCOX

Table 2: Summary Pregnancy Status Report - Reproduction (Sex: Female)

    Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 40 mg/kg bw/d Test Group 2/ F 200 mg/kg bw/d Test Group 3/ F 1000/500 mg/kg bw/d
No. of females at start N 10 10 10 9
No. of females mated N 10 10 10 9
Without evidence of mating N 0 0 0 0
- Pregnant N 0 0 0 0
- Not pregnant N 0 0 0 0
Females with defined Day 0 pc N 10 10 10 9
Pregnant N 9 9 8 9
- sacrificed scheduled N 9 9 8 9
Not pregnant N 1 1 2 0
- sacrificed moribund N 0 0 1 0
- sacrificed scheduled N 1 1 1 0
Pregnant, not delivering N 0 1 0 4
Delivering N 9 8 8 5
-- With liveborn pups N 9 8 8 5
  % 100 100 100 100
-- With all pups stillborn N 0 0 0 0
  % 0 0 0 0

Table 3: Summary Delivery Report (Sex: Female)

    Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 40 mg/kg bw/d Test Group 2/ F 200 mg/kg bw/d Test Group 3/ F 1000/500 mg/kg bw/d
No. of females at start N 10 10 10 9
No. of females mated N 10 f- 10 10 9
  % 100 100 100 100
Pregnant N 9 f- 9 8 9
  % 90 90 80 100
Dead N 0 0 1 0
Without delivery N 1 2 2 4
- Pregnant N 0 1 0 4
- Not pregnant N 1 1 2 0
-- Delivering N 9 f- 8 8 5 *
  % 100 88.9 100 55.6
-- With liveborn pups N 9 f- 8 8 5 *
Gestation Index % 100 88.9 100 55.6
Gestation days Mean 22.3n 22.5 22.2 22.6
  S.d. 0.5 0.5 0.5 0.5
  N 9 8 8 5
-- With stillborn pups N 1 f+ 1 0 0
  % 11.1 12.5 0 0
-- With all pups stillborn N 0 f+ 0 0 0
  % 0 0 0 0

Table 4: Summary Litter Report

    Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 40 mg/kg bw/d Test Group 2/ F 200 mg/kg bw/d Test Group 3/ F 1000/500 mg/kg bw/d
Total Number of Pregnant Females N 9 9 8 9
Total number of litters N 9 8 8 5
With liveborn pups N 9 f- 8 8 5
  % 100 100 100 100
With stillborn pups N 1 f+ 1 0 0
  % 11.1 12.5 0 0
With all pups stillborn N 0 f+ 0 0 0
  % 0 0 0 0
Implantation Sites N 109 101 93 103
  Mean 12.1 x+ 11.2 11.6 11.4
  S.D. 2.00 4.20 1.60 2.20
  N 9 9 8 9
Pups delivered N 92 87 85 27
  Mean 10.2 x- 10.9 10.6 5.4*
  S.D. 3.00 2.20 2.20 3.10
  N 9 8 8 5
Postimplantation Loss Mean% 17.1 x+ 23 8.8 74.2 **
  S.D. 14.4 30.3 12.6 27.6
  N 9 9 8 9
 viability index (PND 0 - 4)  %  99  100  98.2  66.7
 sex ratio  live males  %  40.1  49.8  58.5*  24.0
 PND 4  live females  %  59.9  50.2  41.5*  76.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics. f=FISHER-EXACT; x=WILCOX

Table 5: Summary Clinical Observations female pups, during lactation days 1-4

    Test Group 0/ F 0 mg/kg bw/d Test Group 1/ F 40 mg/kg bw/d Test Group 2/ F 200 mg/kg bw/d Test Group 3/ F 1000/500 mg/kg bw/d
Animals examined N 54 44 35 16
Animals with signs N 0 44 35 16
abnormalities
short tail
N 0 0 0 1
general condition
reduced nutritional condition
N 0 0 6 3
skin                                                     
discolored
N 0 44 35 16
reproduction
not assessed (died)
N 0 0 1 1
normal N 54 0 0 0

Table 6: Summary Clinical Observations male pups, during lactation days 1-4

    Test Group 0/ M 0 mg/kg bw/d Test Group 1/ M 40 mg/kg bw/d Test Group 2/ M 200 mg/kg bw/d Test Group 3/ M 1000/500 mg/kg bw/d
Animals examined N 38 43 50 11
Animals with signs N 1 43 50 11
general condition -- reduced nutritional condition N 0 0 7 0
skin -discolored N 0 42 50 11
reproduction - not assessed (died) N 1 1 0 1
Normal N 37 0 0 0

Table 7: Summary necropsy observations of pups (Number of affected male/female pups per test group)

  Test group 0 Test group 1 Test group 2 Test group 3

(0 mg/kg bw/d)

(40 mg/kg bw/d) (200 mg/kg bw/d) (1000/500 mg/kg bw/d)
Skin discolored 0/0 42/44 50/34  9 / 11
Stomach content discolored 0/0 0/0 4/0 0/3
Intestinal content discolored 0/0  1 / 2 43/33  9 / 10
Liver discolored 0/0  4 /4  16/14  6 / 9
Heart discolored 0/0 0/0  3 /1  3 / 2
Lung discolored 0/0  3 / 1  16 / 9  4 / 6
Kidney discolored 0/0 0/0 0/0 0/6

Applicant's summary and conclusion