Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Screening study for reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 99.4 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: cca 8 weeks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Guar Gum in distilled water
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): not mentioned
- Concentration in vehicle: 1 %
- Amount of vehicle (if gavage): 20 ml/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear daily
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 600 and 2400 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Observations of the animals included clinical signs, body weights, and food consumption. All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily throughout the duration of the study. Daily during treatment (approximately 30 to 90 minutes postdose), each animal was removed from the cage and given a detailed clinical examination. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.
Oestrous cyclicity (parental animals):
At initiation of test article administration and until evidence of copulation was observed, or the cohabitation period ended, the females were examined daily by vaginal lavage to establish estrous cyclicity. Vaginal lavage of the females was completed daily by 10:00 A.M. Toward the end of the gestation period, females were examined twice daily for signs of parturition.
Litter observations:
Number of implantation scars/dam, Litter size, Viable pups, Pup sex ratio (% viable males/litter), Stillborn pups, Stillborn index, Pup weights and Pup survival (days 0-4) were observed.
Postmortem examinations (parental animals):
The animals were examined carefully for external abnormalities including masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) along the uterus were counted and recorded.
Body weights and protocol-designated organ weights were recorded for all surviving adult animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weight). Paired organs were weighed together. The right and left testis were weighed separately.
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides for the control and 2400 mg/kg/day group were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
The animals were examined carefully for external abnormalities including masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) along the uterus were counted and recorded. In males, Epididymides, Prostate gland, Seminal vesicles, and Testes were weighed. In females, Ovaries and Uterus with cervix were weighed. Food and water were provided ad libitum during the study.
Statistics:
Data for each sex within a set were analyzed separately. The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using Group Pair-wise Comparisons, Fisher's Exact Test, Arcsin-Square Root or Covariate Analysis.
Reproductive indices:
Mating procedures:
After 2 weeks of treatment, the males were randomly cohabited, one male to one female, from the corresponding treatment or control group. Each female was housed in the cage of a male during pairing. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or sperm present in the vaginal lavage. The day on which positive evidence of copulation was observed was considered GD 0. After evidence of mating was observed, the female was returned to an individual cage. The maximum mating period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia. After the mating period, the males were individually housed and continued on treatment for approximately 2 weeks. At the end of this period, the males were euthanized and necropsied.
Offspring viability indices:
The number of pups born, liveborn, and stillborn, pup sex ratio (% males per litter) in the treated groups was observed. No macroscopic observations were evident in any pups (stillborn, died on study, or LD 4) at necropsy.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Considerable mortality was seen in F at the 600 and 2400 mg/kg/day levels. Several F in the 600 and 2400 mg/kg/day groups died during the gestation and lactation period. Both M and F rats at all treatment levels were observed with discoloured urine.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related effect on food consumption was evident in all treated males and females in comparison to controls,although these reductions were not dose-dependent.Decreased body weights were observed in both males and females at all treatment levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A treatment-related effect on food consumption was evident in all treated males and females in comparison to controls,although these reductions were not dose-dependent.Decreased body weights were observed in both males and females at all treatment levels.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Black or red foci in glandular stomach of F at 600 and 2400 mg/kg/d, erosion/ulcer in pyloric stomach, minimal focal necrosis and moderate periportal vacuolation of hepatocytes, edema, erosion/ulcer in glandular stomach of F at 2400 mg/kg/d; not in males.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic changes were noted in the liver and glandular stomach of F rats at 2400 mg/kg/day, but not in males.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Treatment did produce a lengthening of the estrous cycle; mean estrous cycle length (number of days per cycle) was increased in the treated females in comparison to controls, but was only statistically significant at 600 mg/kg/day.
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
< 150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup survival was poor in the 600 and 2400 mg/kg/day groups. At 150 mg/kg/day, no effect of treatment was evident from parturition data, litter size, or pup survival to LD 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weights were decreased at all treatment levels; mean pup body weights (by sex and sexes combined) in the 150 mg/kg/day group were similar to control at birth (LD 0), but 8-10% lower by LD 4.
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic observations were evident in any pups (stillborn, died on study, or LD 4) at necropsy.
Histopathological findings:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Both male and female rats at all treatment levels were observed with discolored urine, and decreased body weights and food consumption; thus a No-
Observed-Effect-Level (NOEL) was not achieved. Mating, fertility, and fecundity indices (male and female) in all treatment groups were comparable to controls. Likewise, the copulatory interval (days to mating) was unaffected by treatment. No test article-related organ weight changes were noted in rats of either sex. The number of pups born, liveborn, and stillborn in the 150 mg/kg/day group was similar to controls and unaffected by treatment. Pup survival (to LD 4) in the 150 mg/kg/day group was 89% and was comparable to the controls at 97%. Pup body weights were decreased at all treatment levels. Pup sex ratio (% males per litter) in the treated groups was considered comparable to controls and unaffected by treatment.
No macroscopic observations were evident in any pups at necropsy.
Pup survival was poor in the 600 and 2400 mg/kg/day groups. Very few pups survived to LD 4 in the 2400 mg/kg/day group, but no clinical observations were noted for these pups.
Executive summary:

The mating, fertility, and fecundity indices (83.3-100%) for both treated males and females were unaffected by treatment with Anthraquinone and were similar to controls (91.7-100.0%). Likewise, the copulatory interval (days to mating) was unaffected by treatment and ranged from 2.4-3.1 days in the treated groups in comparison to 3.0 in the controls.

No macroscopic observations were evident in any pups at necropsy. At 150 mg/kg/day, no effect of treatment was evident from parturition data, litter size, or pup survival to LD 4. No test article-related organ weight changes were noted in rats of either sex.

Samples (2.0 mL) of the dosing formulations at each concentration were collected from Weeks 1, 3, and 8 to assess the concentration of the test article. The samples were collected from the middle of the container, while stirring, using a syringe, and placed into 20 mL scintillation vials. Recoveries at all levels were relatively low throughout the study period. Recoveries ranged from 66.0-73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-99.5% (Week 8) in the treated groups.