Registration Dossier

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:
16 July 2007 to 31 October 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. These data have been read-across from a structurally similar compound.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
Details on test material:
dicerium tricarbonate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks old at the beginning of the treatment period
- Weight at study initiation: at the beginning of the treatment period, the animals had a mean body weight of 419g (range: 392g to 442g) for the males and 263g (range: 239g to 283g) for the females
- Fasting period before study: no
- Housing: The males were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). The females were individually housed, except during pairing, until late gestation in wire-mesh cages (43.0 x 21.5 x 18.0 cm). The females were individually housed from late gestation, and with their litter during lactation, in polycarbonate cages (43.0 x 21.5 x 20.0 cm).
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet
- Water: free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 50 +/- 20%
- Air changes: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 14-AUG-2007 to 10-OCT-2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 30, 90 and 200 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were prepared at a frequency based on stability data (up to 9 days) and were stored at +4°C, protected from light, prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqueous suspension for a poorly hydrosoluble test substance, as recommended in the guideline
- Concentration in vehicle: 30, 90 and 200 mg/mL
- Amount of vehicle (if gavage): a constant volume of 5 mL/kg/day was used
- Lot/batch no. (if required): methylcellulose batch No. 017K0052, supplied by Sigma
- Purity: data not available
Details on mating procedure:
- M/F ratio per cage: one female was placed with one male
- Length of cohabitation: Each female was placed with the same male until mating occurs or 14 days had elapsed. Any pairs with no evidence of mating after 14 days were separated and the female was placed for 7 days with a different male from the same dose level group who had already mated.
- Proof of pregnancy: Confirmation of mating was made in the morning, every day up to proof of mating, by checking for the presence of a vaginal plug or of sperm in a vaginal lavage.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During a pre-study period, the homogeneity, stability and concentration of two dosage forms prepared at the lowest and highest concentrations of the present study (30 and 200 mg/mL) were checked using ICP-OES (Inductively Coupled Plasma/Optical Emission Spectrometry) after validation of the analytical method. The results showed acceptable homogeneity and stability of both concentrations over 9 days at +4°C.

During the study, the concentration of the test item and homogeneity of the dosage forms was determined in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 6. The results showed acceptable homogeneity and concentration of all dosage forms analyzed. Precision (RSD =< 10%) and accuracy (100 +/- 10%) of the method were found to be satisfactory.
Duration of treatment / exposure:
- In males: 15 days before mating, during the mating period (up to 3 weeks), until sacrifice (i.e. at least 4 weeks in total)
- In females: 15 days before mating, during the mating period (up to 3 weeks), during pregnancy, during lactation until day 5 post-partum inclusive

Frequency of treatment:
Each animal was given the appropriate dosage form once a day, at approximately the same time each day, 7 days a week.

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 450 and 1000 mg/kg
Basis:
nominal conc.
corrected ffrom the water content)
No. of animals per sex per dose:
10/sex/group
Details on study design:
Dose selection rationale: on the basis of a previous 14-day toxicity study in the rat in which the dose-levels of 150, 450 and 1000 mg/kg/day elicited no significant treatment-related effects.
- Rationale for animal assignment: during the pre-treatment period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition and allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.
Positive control:
not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From arrival, the animals were observed once a day as part of routine examinations. From the start of treatment period, each animal was observed once a day, at approximately the same time for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each male was recorded once a week, over a 7 day period, from the first day of treatment until sacrifice. The quantity of food consumed by each female was recorded once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice. During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION: No

OTHER:
MORTALITY AND MORBIDITY: each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period.

Functional Observation Battery (FOB): on the first five males and the first five females, once at the end of the treatment period. This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.

HEMATOLOGY: peripheral blood
The following parameters were determined for the last five males and females of each group on the day of sacrifice: Erythrocytes, Hemoglobin, Mean cell volume, Packed cell volum, Mean cell hemoglobin concentration, Mean cell hemoglobin, Thrombocytes, Leucocytes, Differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and Large Unstained Cells), Monocytes, Reticulocytes, Prothrombin time, Activated partial thromboplastin time, Fibrinogen.

BLOOD BIOCHEMISTRY:
The following parameters were determined for the last five males and females of each group on the day of sacrifice: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total proteins, Albumin, Albumin/globulin ratio, Total cholesterol, Triglycerides, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Bile acids.
Oestrous cyclicity (parental animals):
The pre-coital time was calculated for each female. The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females are mated.

Sperm parameters (parental animals):
The microscopic examination made special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- Stillbirths, live births
- The total litter size and numbers of pups of each sex (sex ratio) was recorded as soon as possible after birth.
- Postnatal mortality: The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- The pups were observed daily for clinical signs.
- Weight gain: The weight of each pup was recorded on days 1 and 5 post-partum.

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE
After overnight fasting, at least 14 hours, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
- males: after the end of the mating period (after at least 4 weeks of treatment),
- females: on day 6 post-partum,

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on:
- all the tissues listed in the Table 1 for the first five males and females of the control and high-dose groups (groups 1 and 4) sacrificed as scheduled
- all macroscopic lesions
The microscopic examination made special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (offspring):
Pups found dead, prematurely sacrificed and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities and a macroscopic examination was performed. There was no preservation of tissues.

Statistics:
Mean values were compared by one-way variance analysis and Dunnett test, (mean values being considered as normally distributed, variances being considered as homogeneous). Percentage values were compared by Fisher exact probability test.
Reproductive indices:
The following parameters were evaluated:
- Pre-implantation loss,
- Post-implantation loss,
- Mating index,
- Fertility index,
- Gestation index,
- Number of corpora lutea,
- Number of implantations,
- Number of pups delivered

Offspring viability indices:
The following parameters were evaluated:
- Live birth index,
- Viability index on day 4 post-partum,
- Mean litter size,
- Pup sex ratios

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
Other effects:
not examined
Description (incidence and severity):
Test substance intake: substance administered by gavage

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): there were no treatment-related mortalities or clinical signs.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Females had lower mean body weight gains during pregnancy and lactation and lower food consumption during lactation but this may be related to the higher number of fetuses/pups in the control group. There were no effects during the pre-mating period and no effects on mean male body weight or food consumption.


HISTOPATHOLOGY (PARENTAL ANIMALS):
No microscopic treatment related changes were observed in the testes and ovaries.

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
for reproductive performance (mating and fertility)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

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)

There were no effects on pup survival after birth or on pup sex. Mean pup body weight was higher at all dose-levels but the mean body weight gain was comparable to the controls. There were no relevant macroscopic findings at pup necropsy.

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
(for toxic effects on progeny)
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 2: Summary of reproductive data

Dose level (mg/kg/d)

0

150

450

1000

Paired males + females

n

10 + 10

10 + 10

10 + 10

10 + 10

Pairs mated

n

10

10

9 (10)

10

Mating index

%

100.0

100.0

90.0 (100.0)

100.0

Mean number of days of pairing before mating

n

3.4

2.4

4.3

2.3

Pregnant female partners

n

10

8

10

10

Fertility index

%

100.0

80.0

100.0

100.0

Females with live concepti

n

10

8

10

10

Gestation index

%

100.0

100.0

100.0

100.0

( ): female with no evidence of mating

Table 3 : Summary of delivery data

Dose level (mg/kg/day)

0

150

450

1000

Number of pregnant females

Mean duration of gestation (days)

Mean number of corpora lutea

Mean number of implantations

Mean pre-implantation loss (%)

Mean number of pups delivered

Mean post-implantation loss (%)

10

21.8

18.6

16.5

11.3

15.6

5.7

8

21.9

17.6

16.5

5.6

14.6

11.6

10

22.1

18.5

15.1

17.0

12.0

22.2

10

22.0

18.0

14.7

17.4

12.9

12.3

Table 4: Reproductive and litter data

Dose (mg/kg/day)

0

150

450

1000

Females on Study

N

10

10

10

10

Females Mated

       Mating Index

N

%

10 f

100.0

10

100.0

10

100.0

10

100.0

Females Pregnant

        Female Fertility Index

N

%

10 f

100.0

8

80.0

10

100.0

10

100.0

Females with Liveborn

        Gestation Index

N

%

10 f

100.0

8

100.0

10

100.0

10

100.0

Females Surviving Delivery

 

       Duration of gestation

 

 

       With stillborn pups

 

 

       With all stillborn

 

 

        With entire liveborn litter dying and/or missing, cannibalized, culled

 

                               Days 0-4

 

 

                               Days 0-5

N

 

Mean

S.D.

 

N

%

 

N

%

 

 

 

 

N

%

 

N

%

10 f

 

21.8 d

0.4

 

0 f

0.0

 

0 f

0.0

 

 

 

 

0 f

0.0

 

0 f

0.0

8

 

21.9

0.4

 

0

0.0

 

0

0.0

 

 

 

 

0

0.0

 

0

0.0

10

 

22.1

0.6

 

0

0.0

 

0

0.0

 

 

 

 

1

10.0

 

1

10.0

10

 

22.0

0.0

 

0

0.0

 

0

0.0

 

 

 

 

0

0.0

 

0

0.0

Litters with liveborn pups

N

10

8

10

10

Pups Delivered (total)

 

 

       

        Liveborn

        Live birth index

 

        Stillborn

 

        

        Culled (total)

        Cannibalized

        Missing

        Died

 

        Liveborn, not culled

        Prior to day 5

N

Mean

S.D.

 

N

%

 

N

%

 

N

N

N

N

 

N

156

15.6 d

2.7

 

156 f

100.0

 

0 f

0.0

 

0

2

0

4

 

156

117

14.6

2.3

 

117

100.0

 

0

0.0

 

0

4

0

1

 

117

120

12.0

5.2

 

120

100.0

 

0

0.0

 

0

2

0

1

 

120

129

12.9

4.4

 

129

100.0

 

0

0.0

 

0

0

0

1

 

129

Pups dying, missing, and/or cannibalized

        Day 0

 

       

        Days 1-4

 

 

        Days 5-7

 

N

%

N

%

N

%

0 f

0.0

 

6 f

3.8

 

0 f

0.0

0

0.0

 

5

4.3

 

0

0.0

 

0

0.0

 

3

2.5

 

0

0.0

 

0

0.0

 

1

0.8

 

0

0.0

Pups surviving 4 days

        Viability index

N

       %

150 f

96.2

112

95.7

117

97.5

128

99.2

Pups surviving 5 days

        Lactation index

N

       %

150 f

100.0

112

100.0

117

100.0

128

100.0

Implantation sites per litter

N

Mean

S.D.

165

16.5 d

2.5

132

16.5

1.1

151

15.1

4.1

147

14.7

4.1

Corpora lutea

Total

Mean

S.D.

186

18.6 d

1.0

141

17.6

2.2

185

18.5

3.3

180

18.0

3.3

Preimplantation loss

Mean%

S.D.

11.3 d

12.2

5.6

8.1

17.0

21.7

17.4

21.9

Live pups / litter

         Day 1

 

        

Day 4

 

       

       Day 5

Mean

S.D.

 

Mean

S.D.

 

Mean

S.D.

 

15.3 d

2.5

 

15.0 d

2.3

 

15.0 d

2.3

 

14.5

2.2

 

14.0

1.9

 

14.0

1.9

 

11.8

5.1

 

13.0

3.6

 

13.0

3.6

 

12.8

4.3

 

12.8

4.3

 

12.8

4.3

Pup weight / Litter (grams)

       Day 1

 

 

       Day 5

Mean

S.D.

 

Mean

S.D.

 

7.3 d

0.5

 

12.0 d

1.5

 

7.5

0.4

 

12.2

1.2

 

7.6

0.9

 

12.6

2.1

 

7.9

0.5

 

12.8

1.8

Sex ratio – male pups : total pups

       Day 0

 

       

       Day 5

N

%

 

N

%

 

85 f

54.5

 

83 f

55.3

 

54

46.2

 

52

46.4

 

61

51.3

 

60

51.3

 

61

47.3

 

60

46.9

Statistical key: d=ANOVA + Dunnett-test                f =Fishers exact test N= number of animals

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study, the NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day and the NOEL for toxic effects on progeny was 1000 mg/kg/day.
Executive summary:

 The test item, Cerium carbonate, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 150, 450 or 1000 mg/kg/day. There were no adverse effects of treatment at any dose-level on mortality, clinical signs, body weight or food consumption. Animals treated at 1000 mg/kg/day had lower white blood cell counts and females in this group had lower chloride concentration and higher glucose, potassium, inorganic phosphorus and urea levels. There were no effects in any group on mating, fertility or delivery and no treatment-related effects on the mean numbers of corpora lutea, implantations or pups. There were no effects on mean pup body weight, survival or sex. There were no treatment-related organ weights and macroscopic changes in rats treated at 1000 mg/kg/day. At 450 or 150 mg/kg/day, there were no treatment-related organ weights and macroscopic changes. There were microscopic treatment related findings in the stomach that consisted of increased incidence and/or severity of submucosal eosinophil infiltration and pyloric gland hyperplasia in rats from both sexes treated at 1000 mg/kg/day and males at 450 mg/kg/kg. No microscopic treatment related changes were observed in the testes and ovaries. Based on the experimental conditions of this study, the No Observed Effect Level (NOEL) for parental toxicity was considered to be 150 mg/kg/day in female and male rats. The NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day and the NOEL for toxic effects on progeny was 1000 mg/kg/day.

These data have been read-across from a structurally similar compound.