Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: CP12
- Lot/batch No.: 0012
- Storage condition of test material: Room temperature in the dark.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy SpA
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 289-345 g; Females: 189-238 g
- Fasting period before study: No
- Housing: Polysulphone solid bottom cages, Group caged except for in pairs (1 male/1 female) for mating and females individually caged during gestation
- Diet (e.g. ad libitum): Mucedola 4RF21 pelleted diet available ad libitum
- Water (e.g. ad libitum): Municipal supply tap water available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-18 To: 2012-12-09

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared daily by dissolution/suspension in 0.5% (aq) carboxymethylcellulose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Proposed formulation procedure checked for concentration and homogeneity in the range from 10 to 100 mg/mL to confirm that the method was suitable. All levels were within 80-120% of nominal for concentration and the CV for homogeneity was < 20%. Stability after 24 hours at room temperature was verified in the range from 10 to 100 mg/mL and determined to be within acceptable limits (80-120% of nominal of concentration and CV for homogeneity < 20%).
Samples of the formulations, prepared on Weeks 1 and 6, were analysed to check for homogeneity and concentration. Results were within acceptable limits (90-110% of nominal for concentration and the CV for homogeneity was < 10%).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating observed (4 days/1 oestrus)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Males: from 14 days before pairing for a total of approximately 5 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)
Frequency of treatment:
Daily, 7 days/week
Duration of test:
Males: from 14 days before pairing for a total of approximately 5 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on data findings from 2-week preliminary study
- Rationale for animal assignment (if not random): Random, stratified body weight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health and moribund condition
- Cage side observations were included.: Yes - Daily, 2 or 3 times following dose administration

DETAILED CLINICAL OBSERVATIONS: Yes (functional observation battery)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: days 0, 7, 14, 20, gestation days 0, 7, 14 and 20, post partum Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable - Not a dietary study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No - Not applicable as animals dosed by gavage

SACRIFICE
- Male animals: All surviving animals following 42 days of treatment
- Maternal animals: All surviving animals day 4 of lactation

GROSS NECROPSY:
Gross necropsy consisted of external and internal examinations of all organs & including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The following tissues were weighed: adrenal glands, brain, epididymides, heart, kidney, liver, ovaries (with oviduct), spleen, testes, thymus.
The following tissues were prepared for microscopic examination:(5 males/5 females examined from high dose and control groups) - adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
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

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: Examined for external abnormalities and sex confirmation by gonadal inspection
- External examinations: Yes - all per litter
- Soft tissue examinations: Yes: - early decedents
- Skeletal examinations: No
- Head examinations: No
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.
Indices:
Copulation Index: No. of pairs with successful copulation/no. of pairs mated X 100
Fertility Index: No of pregnant females/no. of pairs with successful copulation X 100
Implantation index: No. of implantation sites/no. of corporea lutea X 100
Delivery index: No. of pups born/no. of implantation sites X 100
Gestation index: No. of females with live pups delivered/no. of pregnant females X 100
Nursing index: No. of females nursing live pups/no. of females with normal delivery X 100
Live birth index: No. of live pups at birth/no. of pups at birth X 100
Viability index: No. of live pups on d4/no.of live pups at birth

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the study. All females were pregnant and with live pups on Day 4 post partum.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences were found in body weight and body weight gain between groups throughout the study
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable between groups
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No mortality occurred in the study. Hair loss and/or salivation were observed in some males treated at 1000 mg/kg/day and in single females treated at 300 or 1000 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): No effects on bodyweight or food consumption.

ORGAN WEIGHTS (PARENTAL ANIMALS): No differences were found in organ weights between groups.

GROSS PATHOLOGY (PARENTAL ANIMALS): No treatment-related changes were noted. Those changes that were observed had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age.

HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment-related findings were noted. Changes that were observed had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age. Examples of such findings included liver inflammatory cell foci and thymus atrophy. A single case of moderate mucosa hyperplasia associated with inflammation was seen in the forestomach of a single female treated at 1000 mg/kg/day. This was considered to be an incidental finding unrelated to treatment.

A detailed qualitative evaluation of testes performed on 5 randomly selected control and high dose males took into account the tubular stages of the spermatogenic cycle, in order to identify potential treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day

Any other information on results incl. tables

Reproductive performance

Dose level (mg/kg body weight/day

0

100

300

1000

Number of mated pairs

10

10

10

10

Number of pregnant animals

10

10

10

10

Copulation index – males (%)

100

100

100

100

Fertility index – males (%)

100

100

100

100

Copulation index – females (%)

100

100

100

100

Fertility index – females (%)

90

100

100

100

Developmental toxicity parameters

Dose level (mg/kg body weight/day

0

100

300

1000

Number of pregnant animals

10

10

10

10

Number of pregnant animals with live young

10

10

10

10

Gestation length (days)

22.00 ± 0.47

21.90 ± 0.32

22.10 ± 0.32

22.00 ± 0.00

Number of corpora lutea

15.40 ± 1.71

15.20 ± 1.81

16.10 ± 2.13

15.70 ± 3.13

Number of implantation sites

15.40 ± 1.71

15.20 ± 1.81

16.00 ± 2.05

15.70 ± 3.13

Pre-implantation loss (%)

0.00 ± 0.00

0.00 ± 0.00

0.56 ± 1.76

0.00 ± 0.00

Pre-birth loss (%)

7.99 ± 6.90

8.10 ± 11.45

3.59 ± 5.36

15.43 ± 13.58

 

 

 

 

 

Lactation day 0:

 

 

 

 

Number of pups born

14.10 ± 1.10

14.00 ± 2.58

15.40 ± 1.90

13.60 ± 4.17

Number of pups alive

14.10 ± 1.10

13.90 ± 2.69

15.30 ± 1.89

13.50 ± 4.12

Pup weight (g)

6.89 ± 0.60

7.03 ± 0.92

6.85 ± 0.57

7.00 ± 0.29

Sex ratio (% males)

43.03 ± 18.51

57.39 ± 15.08

51.43 ± 12.99

56.05 ± 16.41

 

 

 

 

 

Lactation day 4:

 

 

 

 

Number of live pups

13.90 ± 1.20

13.50 ± 2.46

15.20 ± 1.75

13.50 ± 4.12

Pup weight (g)

9.42 ± 1.24

9.73 ± 1.34

9.29 ± 0.71

9.62 ± 1.07

Sex ratio (% males)

42.88 ± 17.95

57.88 ± 15.64

51.97 ± 12.70

55.63 ± 16.98

Applicant's summary and conclusion

Conclusions:
In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, no adverse effects on oestrous cycle, copulation, fertility, delivery or lactation and no changes related to gestation index, gestation length, numbers of corpora lutea, implantation sites or implantation index were observed. There were no changes in sex ratio, body weight, viability or morphology of pups. The No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day, this being the highest dose level investigated.
Executive summary:

In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, no adverse effects on oestrous cycle, copulation, fertility, delivery or lactation and no changes related to gestation index, gestation length, numbers of corpora lutea, implantation sites or implantation index. There were no changes in sex ratio, body weight, viability or morphology of pups. The No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day, this being the highest dose level investigated.