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Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Screening of reproductive function and early embryonic development.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-March 2002 to 04-Nov-2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study suitable for the screening of reproductive function and early embryonic development.
Qualifier:
according to guideline
Guideline:
other: Japanese MHW (No. 316) Guidelines for Reproductive/Developmental Toxicity Studies of Drugs
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center)
- Age at study initiation: males (8 weeks), females (7 weeks)
- Weight at study initiation: males (267 to 306 g), females (170 to 209 g)
- Fasting period before study: none specified
- Housing: stainless steel cages (same sex during quaranteen and acclimitization; individually during initial dosing; one-to-one for mating)
- Diet (e.g. ad libitum): CRF-1 Oriental Yeast Co, ad libitum
- Water (e.g. ad libitum): tap, ad libitum
- Acclimation period: 14 days following a 5 day quaranteen.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 41 - 61
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (start of administration of test substance to necropsy) : From: 8 April 2002 To: 6-10 May 2002 (females) and 28-31 May 2002 (males)
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: RM-60 was measured out for each concentration and pulverized well using a mortal and pestle. Then PEG 400 was added to RM-60 little by little to make suspensions at specified concentrations. Dosing preparations were made up at least once a week, divided into daily doses, and stored in a storage container placed in the test article storage room of the testing facility under refrigerated, light-shielded conditions until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle:
- Amount of vehicle (if gavage): 10 ml/kg-bw
- Lot/batch no. (if required): WAQ5263
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: one-to-one
- Length of cohabitation: all pairs mated within 5 days.
- Proof of pregnancy: positive vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations and homogeneity of the test article in the dosing preparations were determined by HPLC prior to and after the dosing period. The concentrations were between 100.9 and 104.0% (acceptable range: 90 - 110%), and the coefficient of variance (C.V.) indicating homogeneity was between 0.3 and 2.1% (acceptable values: 5% or lower). Since the values were within the acceptable ranges, there were no problems with the concentrations or homogeneity of the test article. Dosing preparations remaining after administration were discarded.
Duration of treatment / exposure:
Male and females were treated / exposed 14 days prior to pairing, through mating, and females only through early stages of pregnancy.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg-bw
Basis:
other: nominal administered by gavage
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: No effects were noted at 1000 mg/kg either in a repeated dose toxicity study of RM-60 by 2-week oral administration in rats or in a study of RM-60 on embryo-fetal development in rats. Accordingly, the high dosage level in the present study was set, as the limit dose prescribed in the Guidelines for Toxicity Studies of Drugs, at 1000 mg/kg/day. The intermediate and low dosage levels were set at 300 and 100 mg/kg, respectively, by decreasing geometrically by a factor of about 3.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once before and once after administration of dose.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Two times per week.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes, one time per week.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Oestrous cyclicity (parental animals):
Once per day from the first dosing day to the day when copulation was confirmed.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations: sperm count in epididymides, sperm motility, sperm morphology, sperm servivability.
Litter observations:
Mortality of embryos and implantation sites were observed for each dam macroscopically. After being observed, the embryos were fixed and preserved in 10% neutral buffered formalin together with the uterus and placenta removed from respective dams.
Postmortem examinations (parental animals):
The males were sacrificed the day after the last dosing day by bleeding from the abdominal aorta under ether anesthesia and necropsied. The testes and epididymides were removed, and these organs and the caudae epididymidis were weighed. The relative testis and epididymis weights were calculated by dividing the respective absolute organ weight by the final body weight. The removed bilateral testes and bilateral capita epididymidis were fixed in Bouin's solution for 2 - 3 hours and preserved in 10% neutral buffered formalin. The left cauda epididymis was stored frozen (set at -80°C) until the day of examination.

Females which had copulated were sacrificed on Day 13 of pregnancy (13 days after copulation) by bleeding from the abdominal aorta under ether anesthesia and necropsied for pregnancy. Regarding the females whose pregnancy was confirmed, the number of corpora lutes and number of implantation sites were counted, and the ovaries and uterus (with embryos and placenta) were fixed and preserved in 10% neutral buffered formalin. Regarding the females in which implantation was not found, the ovaries and uterus were removed. The uterus was stained with 10% ammonium sulfide, and the absence of implantation scars was re-confirmed. Then the uterus was fixed and preserved in 10% neutral buffered formalin together with the ovaries.

Postmortem examinations (offspring):
Not applicable.
Statistics:
Data were analyzed according to the statistical methods described as follows. Significance tests were performed between the control group and each of the groups treated with RM-60. Probabilities less than 5% (1% for Bartlett's test) were considered statistically significant. Data for non-pregnant females obtained after copulation were excluded from the totals. Group mean values and standard deviations were calculated for body weight (with body weight gain), food consumption, organ weights (relative organ weight included), number of sperms, number of sperms/g of the left cauda epididymis, sperm motility, sperm survivability, abnormal sperm rate, number of estrous cases (until the day before pairing), days till copulation after pairing, number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, number of dead embryos (number of post-implantation losses), dead embryo rate (post-implantation loss rate), and number of live embryos. Subsequently, the data were tested by Bartlett's method for homogeneity of variance. When the variances were homogenous, Dunnett's method was used. When the variances were heterogeneous, a Dunnett-type method using a rank order was used. The fertility index was analyzed by chi square tests.
Reproductive indices:
The number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, number of dead embryos (number of post-implantation losses), dead embryo rate (post-implantation loss rate), and number of live embryos.
Offspring viability indices:
Not applicable.
Clinical signs:
no effects observed
Description (incidence and severity):
Loose stool and transient salivation, but also noted in control group.
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:
not examined
Other effects:
not examined
Description (incidence and severity):
Test substance intake: gavage study
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Compared with the control group, the number of dead embryos (number of post-implantation losses) and dead embryo rate (post-implantation loss rate) were significantly higher in the 100 mg/kg group, but these findings were not dose-dependent changes. No significant differences were seen in the number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, or number of live embryos between any of the groups treated with RIVI-60 and the control group.
Reproductive effects observed:
not specified
Conclusions:
It is concluded that the no observed adverse effect level of RM-60 is >=1000 mg/kg for general toxicity in dams, for reproductive functions of parent animals, and for early embryonic development.
Executive summary:

RM-60 at dosage levels of 100, 300, and 1000 mg/kg was administered orally by gavage to male rats daily before mating and throughout the mating period and to female rats daily before mating and throughout the mating period up to the early stages of pregnancy, and the effects of RM-60 on reproductive functions of male and female animals and early embryonic development were assessed. Regarding the parent animals, loose stool and salivation were noted in both sexes in the groups treated with RM-60, but these findings were also noted similarly in the control group. It is concluded, therefore, that these findings were not attributable to RM-60 but caused by PEG400, the vehicle. No effects of RM-60 were noted in body weight, food consumption, or necropsy findings in either sex. Also, no effects of RM-60 were noted in male organ weights or sperm analysis parameters. In the examination for reproductive functions, no effects of RM-60 were noted in the number of estrous cases, days till copulation after pairing, copulation index, or fertility index. In the observation of embryos, no effects of RM-60 were noted in the number of corpora lutea, number of implantation sites, implantation rate, number of pre-implantation losses, pre-implantation loss rate, number of dead embryos (number of post-implantation losses), dead embryo rate (post-implantation loss rate) or number of live embryos.

From the above results, it is concluded that the no observed adverse effect level of RM-60 is >=1000 mg/kg for general toxicity in dams, for reproductive functions of parent animals, and for early embryonic development.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a Japanese MHW (No. 316) guideline, a NOAEL for reproductive toxicity was determined to be >=1000 mg/kg/day for parental animals (Nihon Bioresearch, 2002). Male and female rats were given daily oral doses ranging from 0 - 1000 mg/kg-bw. In this study, the parental animals exhibited no adverse effects.


Short description of key information:
Not a reproductive hazard

Effects on developmental toxicity

Description of key information
Not a developmental or teratogenic hazard
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to guideline
Guideline:
other: U.S. Food and Drug Administration (FDA), Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, Sept. 22, 1994, Vol. 59, No. 183
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: 5 months
- Weight at study initiation: 2.5 - 4.3 kg (at study assignment)
- Fasting period before study: no
- Housing: units of six stainless steel cages
- Diet (e.g. ad libitum):
^ Approximately 150 g of Certified Rabbit Chow® #5322 (PMI® Nutrition International) was offered to each rabbit until day of first dosage
^ Approximately 180 to 185 g of the chow offered to each rabbit from day of first dosage.
- Water (e.g. ad libitum): Local water passed through Reverse Osmosis filter (R.O. water) then chlorinated.
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 22
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 Apr 2004 To: 28 May 2004
Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) carboxymethylcellulose (CMC) in 0.1% (w/v) Tween® 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Suspensions of test article and vehicle were prepared at least once weekly at the Testing Facility. Prepared suspensions were stored refrigerated.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg-bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis performed to dermine the test article concentration at the start of the study and at the end of the study. Analysis by HPLC.
Details on mating procedure:
The female rabbits were naturally bred by breeder male rabbits of the same source and strain before shipment to the Testing Facility. The rabbits were mated on four consecutive days and shipped to the Testing Facility to arrive on DGs 1, 2, 3 or 4. The day of mating was considered to be DG 0.
Duration of treatment / exposure:
Gestation days 6 through 19
Frequency of treatment:
Daily
Duration of test:
23 days (animals were Caesarean-sectioned on gestation day 29).
Remarks:
Doses / Concentrations:
0, 100, 300, & 1000 mg/kg-bw
Basis:
other: dose administered by gavage
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dosages were selected on the basis of a dosage-range study conducted at the Testing Facility. In that study, dosages of 0 (Vehicle), 100, 300, or 1000 mg/kg/day were administered orally by stomach tube to groups of six timed mated rabbits on DGs 6 through 19. There were no test article-related deaths, clinical signs, or alterations at necropsy. Body weight changes and food consumption values were increased in the does given Tinosorb S in these small samples; however, there was minimal impact on the average body weights. Caesarean-sectioning and litter observations were comparable among the groups or within the ranges observed historically, and no fetal gross external alterations were observed. Based on the results of this study, dosages of 100, 300, and 1000 mg/kg/day were selected for the developmental toxicity study of Tinosorb S in rabbits.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during study, and once daily during the post-dosage stage

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: approx. 60 minutes after dosage

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29
- Organs examined: gross examination of the thoracic, abdominal and pelvic viscera, including liver, lungs, and kidneys.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all fetuses
- Soft tissue examinations: Yes: all fetuses
- Skeletal examinations: Yes: all fetuses
- Head examinations: Yes: all fetuses
Statistics:
Data generated during the course of this study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, The Vivarium Temperature and Relative Humidity Monitoring System, Microsoft® Excel (part of Microsoft® Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate. Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test(18) was used to analyze the data. Count data obtained at Caesarean-sectioning of the does were evaluated using the procedures described above for the Kruskal-Wallis Test.
Historical control data:
January 2002 thru 2004 from 74 studies representing 967 rabbits (932 pregnant).
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The maternal no-observable-adverse-effect-level (NOAEL) of Tinosorb S (FAT 76’884) is was determined to be at least 1000 mg/kg/day; the highest dosage tested. The developmental NOAEL is also at least 1000 mg/kg/day and there were no adverse effects on embryo-fetal development. Based on these data, the test substance should not be identified as a developmental toxicant.
Executive summary:

Eighty timed-mated female Hra:(NZW)SPF rabbits were randomly assigned to four dosage groups, 20 rabbits per dosage group. The test article, Tinosorb S, and/or vehicle, 0.5% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous Tween® 80, were administered once daily on days 6 through 19 of gestation (DGs 6 through 19) at dosages of 0 (Vehicle), 100, 300 and 1000 mg/kg/day. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded before intubation. 

Rabbits were observed for viability at least twice each day of the study. Observations for clinical signs, abortions, premature deliveries and deaths were made before and approximately 60 ± 10 minutes after dosage administration and once daily during the postdosage period. Body weights were recorded on DG 0, the day of arrival at the Testing Facility and daily during the dosage and postdosage periods. Feed consumption values were recorded daily after arrival at the Testing Facility.

Rabbits were sacrificed on DG 29, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of corpora lutea were recorded. The uterus of each rabbit was excised, weighed and examined for pregnancy, number and distribution of implantation sites, live and dead fetuses and early and late resorptions. Placentae were examined for size, color and shape. Fetuses were individually identified, weighed and examined for gross lesions. All fetuses were examined internally to identify sex. Cavitated organs were evaluated in all fetuses by dissection. A single cross-section was made between the parietal and the frontal bones, and the brain was examined in situ. All fetuses were examined for skeletal alterations.

No deaths were caused by Tinosorb S. One doe in the 1000 mg/kg/day dosage group was sacrificed on day 19 of gestation (DG 19) due to its moribund condition as the result of an intubation accident. All other rabbits survived until scheduled Caesarean-sectioning. All clinical and necropsy observations were considered unrelated to Tinosorb S. Body weights, body weight gains, gravid uterine weights, and absolute and relative feed consumption values were unaffected by dosages of Tinosorb S as high as 1000 mg/kg/day. No Caesarean-sectioning or litter parameters were affected by dosages of Tinosorb S as high as 1000 mg/kg/day, nor were any gross external, soft tissue or skeletal fetal alterations (malformations or variations) caused by dosages of Tinosorb S as high as 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The summary of three key studies conclude that the test substance, Bemotrizinol, is not a teratogenic hazard.

Bemtrizinol resulted in an oral maternal toxicity NOAEL of >=1000 mg/kg/day in rabbits (Charles River Doscovery, 2005). Upon examination of maternal toxicity there were no adverse effects seen in body weight gains, gravid uterine weights, and absolute and relative feed consumptions.

Bemotrizinol resulted in a NOAEL of >=1000 mg/kg in maternal and pre- and postnatal offspring rats (Nihon Bioresearch, 2002). Regarding the dams there were no significant changes in their reproductive functions or food consumption and body weight. Regarding the offspring (F1), there were no adverse effects in their number of offspring, sex ratio, number of stillbirths, birth index, viability index 4 days after birth, weaning index, incidence of external abnormalities, general signs, or body weight, as well as any reproductive functions of the F1 generations. The F2 generations experienced no changes in the implantation rate, pre- or postimplantation losses, pre- or postimplantation rates, number of live fetuses, sex ratio of live fetuses, fetal body weight of either sex, incidence of external abnormalities, or incidence of placental abnormalities.

Bemotrizinol did not influence the development of dams, embryos or fetuses up to the dose level of 1000 mg/kg bw/day (RCC, 1998)

Justification for classification or non-classification

The available data on fertility and the three key developmental toxicity studies resulted in a NOAEL of >=1000 mg/kg and no adverse reproductive effects were seen after Bemotrizinol administration.

Bemotrizinol should not be considered a reproductive hazard (Substances which cause concern for human fertility) under the EU DSD classification system (EU Directive 647/548/EEC) or under the EU CLP classification system (EU Regulation 1272/2008).

Additional information