Registration Dossier

Administrative data

screening for reproductive / developmental toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 20, 2014 - December 15, 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed according to GLP and the methods applied are fully compliant with OECD TG 421.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
migrated information: powder

Test animals

other: Crl:CD (SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories Italia s.r.l, Via Indipendenza. 11, 23885 – Calco (Lecco), Italy, on May 9th, 2014, (shipping slip No CEC03523).- Age at study initiation: 10 weeks- Weight at study initiation: (P) Males:347.3-373.9 g; Females250.0-272.9 g- Fasting period before study: -- Housing: single- Use of restrainers for preventing ingestion (if dermal): no- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 2 weeksENVIRONMENTAL CONDITIONS- Temperature (°C): 22°C ± 2°C- Humidity (%): 55% ± 15%,- Air changes (per hr): 20 air changes per hour filtered on HEPA,- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
oral: gavage
other: Methocel KM4 Premium 0.25 % in Milli-Q
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:VEHICLE- Justification for use and choice of vehicle (if other than water):- Concentration in vehicle: 0.25 % in pure water (Milli-Q)- Amount of vehicle (if gavage): 10 mL/kg- Lot/batch no. (if required): DT381554
Details on mating procedure:
- M/F ratio per cage: 1/1- Length of cohabitation: 7 evenings/week with a maximum of 14 evenings- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of the formulated test item was checked twice during the dosing period (i.e., on formulations prepared on one day of the pre mating and post mating periods).The HPLC analytical method was validated. A stability and homogeneity study in 0.25% Methocel® K4M Premium in water was performed.
Duration of treatment / exposure:
F0 Males were treated for 2 weeks before the start of the mating period and throughout the same.Males were further dosed after the mating period until the minimum total dosing period of 28days was completed.F0 Females were treated for 2 weeks before the start of the mating period and throughout thesame. Treatment continued throughout pregnancy and until Day 3 of lactation.A concurrent control group received 0.25% Methocel® K4M Premium aqueous solution with thesame dosing regimens. All groups consisted of 10 males and 10 females.
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
80 (40m / 40f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 28 day study with similar chemical- Rationale for animal assignment (if not random): random- Other:


Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: twice dailyBody Weight and Food and Water Consumption: Yes - Time schedule for examinations:Males and females were weighed on the day before the start of dosing (Day -1 of the study), on the first day of dosing (Day 1 of the study) and then weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (Day 0 of lactation) and on Day 4 of lactation. Weights were reported individually for each adult animal. During the pre-mating period and pregnancy, food and water consumption were measured at least weekly and during lactation on Day 4.OTHER:
Sperm parameters (parental animals):
A detailed qualitative examination of one testis and the equilateral epididymis was done in 10male rats of the control (vehicle) group, as well as in 10 males of the high dose group, taking intoaccount the tubular stages of the spermatogenic cycle. The examination was conducted in orderto identify treatment-related effects such as missing germ cell layers or types, retainedspermatids, multinucleate or apoptotic germ cells and disorganization of the germinal epithelium.
Litter observations:
On Day 0 of lactation, the pups of each litter were identified with a mark made by appropriatelycoloring different sites of the body; this mark was renewed when necessary.Live pups were counted and sexed and litters weighed within 24 hours of parturition (Day 0 oflactation) and on Day 4 post partum.At birth and during the lactation period, all the young were individually observed for:- Live and stillbirths;- Mortality ensuing after live birth, ascertained daily. Whenever possible, any pup found dead was examined externally and internally in an attempt to determine the cause of death;- Sex (on Day 0 of lactation by an external examination and measurement of the ano-genital distance and by internal examination, at sacrifice, on Day 4 of lactation);- External abnormalities at birth;- Pup weight (on Days 0 and 4 of lactation) Females were killed on Day 4 of lactation together with their pups.
Postmortem examinations (parental animals):
Gross pathologyHistopathology
Postmortem examinations (offspring):
Gross pathologyHistopathology
Body weight, food and water consumption, mortality, clinical signs, dosing, reproduction data,and fetal weight data were recorded and stored in the PROVANTIS™ system.
Reproductive indices:
-Mating index: percent ratio between the animals with positive smear + females found to bepregnant but without positive smear and the animals mated.- Fertility index: percent ratio of females having evident signs of pregnancy with respect to thefemales that had positive vaginal smear and females found to be pregnant but without positivesmear.- Pregnancy index: the percent ratio of females with live births with respect to the pregnantfemales.- Pregnancy period: the duration of pregnancy was determined for all those dams that reachedpregnancy term as being the time that elapsed between the positive vaginal smear and the start ofparturition.- Mean pre-coital interval: was calculated on the dams proved pregnant and was expressed foreach group as the mean time lapse (in days) between the beginning of the mating period and theascertainment that copulation occurred. The fem
Offspring viability indices:
- The mean F1 body weight was obtained by averaging the mean weight of each litter at thevarious times.- The F1 probability of survival was analyzed per group. Animals that died owing to accidentalcauses or were sacrificed at the end of lactation were statistically censored.- The mean value per litter of live pups (number of males and females and total) was calculatedat different times (Days 0, 4 of lactation).- The following indices of each litter were calculated:Birth index:No. of live newborns at birth----------------------------------- x 100No. of implantationsViability index on Day 4:No. live pups on Day 4 after birth----------------------------------------- x 100No. of live birthsGroup mean values were calculated from individual data in two ways:Mean A: calculated on all the surviving females having evident signs of pregnancy, includingthose that presented 100% post-implantation losses.Mean B: calculated only on those females with viable fetuses at term.The external, visceral and skeletal malformations and variations found were described for eachlitter.All data were recorded and stored in PROVANTIS TM System.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
expect piloerection in males receiving 75 mg/kg for a few days during treatment
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: lower mean daily food consumption in females in 1st week of gestation @75 and 225 mg/kg

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

All animals survived the treatment period.No clinical signs were observed in males and females at any time point except in one male of the75 mg/kg/day group that showed piloerection for a few days during the treatment period.No compound-related changes were seen in body weight, or food and water consumption of maleanimals during the 28-day treatment period.At 75 and 225 mg/kg a slightly lower mean daily food consumption was observed in femaleanimals during the first weeks of gestation that resulted in minimal changes (about 5% less thancontrols) in mean body weight of these two groups at the end of gestation and up to end of thestudy (Day 4 of lactation). No changes were seen in the mean daily water consumption of treatedfemales except for a slightly higher consumption at 225 mg/kg at the end of gestation.No changes were seen in body weight, or food and water consumption of female animals at 25mg/kg.At necropsy of the adult animals no macroscopic changes were seen and no differences werenoted in the mean weight of testes and epididymides of treated males in comparison with thecontrols.Histological examination performed on the ovaries, testes and epididymes (with special emphasison stages of spermatogenesis and histopathology of interstitial testicular cell structure) of theanimals of the 225 mg/kg/group and the control group did not evidence any toxicologicalpathomorphological lesions.The reproductive performance was unaffected, in fact, no differences were seen in the matingand fertility indices (ratio between mated and paired animals and ratio between pregnant andmated animals, respectively) among the various experimental groups, no compound-relatedeffects were seen in the mean pre-coital interval was similar in all groups.No differences were seen in the pregnancy length and in the pregnancy index (ratio betweenfemales with live births and pregnant females) between treated and control groups.No compound-related effects were seen on pre-implantation losses (namely corpora lutea minusimplantations) and on post-implantation losses (namely implantations minus live birth) at anydoses, and no compound-related differences were observed in the viability index calculated onDay 4.Changes seen at 75 mg/kg as regards pre and post natal losses were considered not compound relatedsince not confirmed at the highest dose and since the values fell within the normal rangeof variability of the historical control data for the laboratory.

Effect levels (P0)

Key result
Dose descriptor:
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

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:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

The mean weight of male and female pups at birth and on Day 4 post partum did not show anystatistically significant differences between the treated groups and the control group.The ano-genital distance of pups (males and females) belonging to the treated group was similarto those of male and female control pups respectively, either when recorded at birth or on Day 4at necropsy and the external sex corresponded to the internal one, checked at sacrifice, in allpups.Necropsy of pups on Day 4 did not evidence any compound-related changes.

Effect levels (F1)

Dose descriptor:
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

In conclusion, the test material given orally to male rats for 28 consecutive days and to females during the pre-mating, mating and gestation periods and up to day 3 of lactation at the doses of 0, 25, 75 and 225 mg/kg/day only induced minimal changes, not dose-related, in food consumption and body weight of the 75 and 225 mg/kg/day females.Reproductive performance was unaffected and no changes were evidenced in the reproductive organs at any dose. Offspring development and survival were considered not to be affected by the test-item, since changes seen at 75 mg/kg in the pre and post natal losses and in the postnatal survival were not confirmed at the highest dose and since the values fell within the normal range of variability of the historical control data for the laboratory. On the basis of the above findings, 225 mg/kg was considered the NOAEL for reproduction and developmental toxicity.