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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS of test material (as cited in study report): 4051-63-2
- Substance type: Solid / red
- Physical state: solid
- Analytical purity: 89 area-% by HPLC-analysis at 496nm
- Purity test date: 2011
- Lot/batch No.: 0003908072

- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Cri :WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks
- Fasting period before study: overnight
- Housing: 1 rat per cage, except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

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

IN-LIFE DATES: From: 2012-02-14 To: 2012-04-19

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare the suspension, 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. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least twice a week and were stored at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 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 (Mating attempts were performed for a maximum of 2 weeks).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Pregnant animals and their litters were housed together until PND 4 (end of lactation). Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations (deviations <5%).
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)

Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 - 14 weeks

Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finder study

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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. 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before initial test substance administration and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of F0 parents were determined on study day 0 (start of the administration period) and thereafter once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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, 14 and 20.
Food consumption of F0 females, which gave birth to a litter was determined for PND 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).

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

OTHER:
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.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals
- Maternal animals: all surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Weight assessment was carried out on all animals. The following weights were determined:
Adrenal glands, Anesthetized animals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Spleen, Thymus

The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:
Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve (modified Davidson’s solution), Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Postmortem examinations (offspring):
SACRIFICE
- All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means; Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions; Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Reproductive indices:
Male / female mating indices, male / female fertility indices, gestation index, live birth index, postimplantation loss

Offspring viability indices:
Viability index was calculated using the number of live pups/litter on the day after birth, and on lactation day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

During premating, mating and post-mating red discolored feces was seen in all animals of the test groups 1-3 (100, 300 and 1000 mg/kg bw/d). The sign came up first on study day 7 in test group 1 (100 mg/kg bw/d) and on study day 1 in test groups 2 and 3 (300 and 1000 mg/kg bw/d). The findings were considered to be related to treatment as the substance is a red pigment, but assessed as being non-adverse.

For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed with the exception of male control animal No. 1 and high-dose male No. 31. Thus, the male mating index was 90% in control group and test group 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Three males of control group (animal Nos. 1, 4 and 8 mated with female Nos. 101, 104 and 108, respectively) and 1 male animal of test group 3 (animal No. 31 mated with female No. 131) did not generate F1 pups. Thus, the male fertility index ranged between 70% and 100%. 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 female mating index calculated after the mating period for F1 litter was 90% in control group and test group 3 (0 and 1000 mg/kg bw/d) and 100% in test groups 1 and 2 (100 and 300 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.8, 3.3, 2.3 and 2.6 days in test groups 0-3 (0, 100, 300, 1000 mg/kg bw/d, respectively).

The mean duration of gestation was between 22.0 and 22.4 days and did not show significant differences.

The gestation index reached 100% in all groups including the control.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (control group), 99% (test groups 1 and 2) and 93% (test group 3). Single stillborn pups were seen in test groups 1 and 2 and in one female of test group 3. Additionally, 2 more dams of test group 3 had 2 and 5 stillborn pups.

The viability index as indicator for pup mortality between PND 0 and 4 was 99% for the control, 98.2% for test group 1 (100 mg/kg bw/d), 99.1% for test group 2 (300 mg/kg bw/d) and 93.5 % for test group 3 (1000 mg/kg bw/d).

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The substance did not cause adverse effects on reproductive function in the screening study (OECD 422) in rats at the limit dose.