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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods. For read across justification see Section 13.

Data source

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

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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : CP12
- Physical state: Liquid
- Lot/batch No.: 0012
- Expiration date of the lot/batch: 2015-08-01
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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% aqueous carboxymethylcellulose.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance is poorly soluble in water
- Concentration in vehicle: As required to achieve nominal dose, up to 25% w/v
- Amount of vehicle (if gavage): 2mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days (until sperm detected in vagina).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): singly
- Any other deviations from standard protocol: None reported
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%).
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 during treatment period
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
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

Positive control:
No

Examinations

Parental animals: Observations and 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:

Oestrous cyclicity (parental animals):
Examined pre-dose and during mating period
Sperm parameters (parental animals):
Parameters examined in P males/group : yes
- testis weight and microscopic pathology, epididymis weight and microscopic pathology

Litter observations:
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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following approximately 6 weeks of treatment
- Maternal animals: All surviving animals day 4 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all organs & including the cervical, thoracic, and abdominal viscera. Pups: dead pups examined internally and externally. Live pups examined externally and sex confirmed by gonadal inspection..

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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination): Yes

GROSS NECROPSY
- Gross necropsy of dead pups consisted of external and internal examinations. Examination of live pups was limited to external examination.
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
Reproductive indices:
Copulation Index: No. of pairs with successful copulation/no. of pairs mated x 100
Fertility Index: No of males inducing pregnancy/No. of males paired x 100 and No. pregnant females/no. of pairs with successful copulation x 100
Offspring viability indices:
Pre-birth loss: (No. visible implantations - total litter size at birth)/No. visible implantations x 100
Pup loss at birth: (total litter size - live litter size)/total litter size x 100
Cumulative pup loss: (total litter size at birth - live litter size Day 4)/total litter size at 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:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No mortality occurred in the study. Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the tested substance. 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): Body weight and body weight gain were unaffected by treatment. Food consumption was unaffected by treatment.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Measurements of oestrous cycle did not show differences between treated and control groups

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): See histopathology, below.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Measurements of oestrous cycle, copulation plugs, copulatory index and fertility index did not show differences between treated and control groups. No significant differences were observed in the number of implantation, corpora lutea, pre-implantation loss, total litter size, pre-birth loss and gestation length between control and treated groups.

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 (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of effects on reproductive performance or sex organs

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)

VIABILITY (OFFSPRING): No effects

CLINICAL SIGNS (OFFSPRING): Apparently small pups were observed in litters from all treatment groups except for control. Some pups from all groups, including controls, appeared cold to the touch and with no apparent food intake.

BODY WEIGHT (OFFSPRING): No effects

GROSS PATHOLOGY (OFFSPRING): No milk in stomach and autolysed organs of the abdomen were observed at necropsy of decedent pups of control and treated groups. No abnormalities were found in pups sacrificed on Day 4 post partum.


Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of effects on pup weight; sex ratio; survival index; viability index

Overall reproductive toxicity

Reproductive effects observed:
not specified

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:
A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 for both parent animals and offspring, this being the highest dose level investigated.
Executive summary:

A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 for both parent animals and offspring, this being the highest dose level investigated.