Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive 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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. Read across was performed with methyl methacrylate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (= GD 0)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
Duration of treatment / exposure:
until one day before sacrifice
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 450 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
F0 generation parental animals: 25
F1 generation parental aniumals: 25
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).



OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical symptoms, sexual maturation


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.
Reproductive indices:
- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.
Offspring viability indices:
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Test group 03 (400 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY
- Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
- Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
- Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
- Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
- Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
- Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
- Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
- Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 17%)

Test group 02 (150 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
- Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
P PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

F1 PARENTAL ANIMALS:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY PATHOLOGY
- no test substance-related adverse effects/findings

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: P and F1 parental animals (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Generation: F1 and F2 progeny (migrated information)

Results: F1 generation

Details on results (F1)

Test group 03 (400 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 02 (150 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d):
F1 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

F2 PUPS:
CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ PUP ORGAN WEIGHTS/ GROSS FINDINGS
- no test substance-related adverse effects/findings

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

- Analytics:

The various analyses:

  • demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
  • confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
  • showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group

Nominal Dose
(mg/kg bw/d)

Analytical Dose
(mg/kg bw/d)
[minimum]

Analytical Dose (mg/kg bw/d)
[maximum]

% Nominal Dose
[minimum]

% Nominal Dose
[maximum]

00 / 10

0

0

0

 

   

01 / 11

50

43.40

46.61

86.8

93.2

02 / 12

150

132.90

169.80

88.6

113.2

03 / 13

400

359.20

379.90

89.8

95.0

 

Applicant's summary and conclusion