Registration Dossier

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:
2011-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Test material form:
solid: particulate/powder
Details on test material:
CAS No. 68259-05-2
Description: Red powder
Purity: 99.55 wt%
Expiry Date (Retest Date): 18-Jul-2020
Storage Conditions: Room temperature (20 ± 5 °C)

Test animals

Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 315 to 364 g, Females: 182 to 212 g
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-12-15 To: 2012-02-06

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared fresh daily using the test item as supplied by the Sponsor.


VEHICLE
- Concentration in vehicle: as adjusted to dose
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: (1:1)
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing no further mating was performed.
- After successful mating each pregnant female was caged (how): groups of three or four
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle
and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of
concentration and homogeneity. During the last week of the treatment, samples were taken from
the middle to confirm concentration.

Duration of treatment / exposure:
Males: Minimum 4 weeks
Females: Approximately 6 weeks

Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 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: Based on information on a substance of similar structure.
Positive control:
Not required.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twic for mortality)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena.
- Cage side observations : changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g.
lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: daily
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
During histopathology, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Testis weight and epididymis weight were determined.
Litter observations:
STANDARDISATION OF LITTERS
not required

PARAMETERS EXAMINED
The following parameters were examined: 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; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

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

HISTOPATHOLOGY / ORGAN WEIGHTS
As required for a full subacute oral toxicity study.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of apparently non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined.
Statistics:
Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Percentage mating = ( Females mated / Females paired) * 100
Fertility index = ( Females achieving a pregnancy / Females paired) * 100
Conception rate = ( Females achieving a pregnancy / Females mated) * 100
Gestation index = ( Number of females with living pups / Females pregnant) * 100
Birth index = (pups born alive / number of implantations) * 100
post-implantation loss
Offspring viability indices:
Viability index (%) = number of live pups on day 4 after birth/number of live pups on the day of birth x 100

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:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

All animals mated within the first pairing period. The median and mean precoital times were unaffected by treatment with the test item. Mean
precoital times were 4.3, 2.5, 2.8 and 2.8 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3 days in all groups.
Two females in the control group and one female in groups 2, 3 and 4, respectively, were not pregnant. This finding was considered to be incidental. As a result the fertility index in the control group was 81.8% and 90.9% in groups 2 - 4.

Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (15.8, 14.4, 15.3 and 15.6 in order of ascending dose level) and gave no indication of a test item-related effect.

The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.3, 21.6, 21.4 and 21.4 days, in order of ascending dose level.

No effects on implantation rate or post-implantation loss were observed at any dose level.
The mean number of implantations per dam was similar in all groups (13.8, 12.2, 13.9 and 13.1 in order of ascending dose levels). The mean incidence of post-implantation loss as a percentage of total implantations was 4.0, 8.2, 5.4 and 7.6%, in order of ascending dose level.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

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)

No effects on litter size were observed at any dose level.
Mean litter size at first litter check was 13.2, 11.6, 13.1 and 12.1 pups in order of ascending dose levels. No dead pups at first litter check were recorded in any group.
No effects on postnatal loss were observed at any dose level.
Incidentally, 2, 2 and 1 pups were lost in groups 1, 2 and 4, respectively, between day 0 - 4 post partum.

No test item-related abnormal findings were noted at first litter check or during the first 4 days post partum.
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test
item. On day 1 post partum mean pup weights were 5.7, 6.0, 5.8 and 6.0 g in order of ascending
dose level. Also mean body weight gain was similar in all groups and on day 4 post partum no
effects on mean body weights were recorded.
No findings were noted at macroscopic examination of F1 pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
No indication of toxicity to reproduction in rats were observed in the screening study at doses of up to 1000 mg/kg bw .