Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
historical data are not given, food consumption data male animals third week are missing, missing details on mating procedure: how many attempts, replacement of male after unsuccesful mating and how females were caged after mating, read-across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 002
EC Number:
603-331-4
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material:
Synthesia, a.s. and E0986

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
in a supplied container in dry room at the temperature < 30°C
- Stability under test conditions:
15 years
- Solubility and stability of the test substance in the solvent/vehicle:
based on the sponsor information, main component is insoluble in the used vehicle, suspension; stability can not be verified analytically
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
suspended in the vehicle daily

FORM AS APPLIED IN THE TEST: suspension, yellow non-transparent, undissolved particles easily visible in the application form and homogeneity could be checked by eye

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Kolec u Kladna, Czech Republic, RCH CZ 21760152
- Age at study initiation: 10 weeks
- Weight at study initiation: males 307.89 - 309.22g, females: 224.33 - 226.55g
- Housing: exept from mating period, two rats of the same sex per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: males from: 19.01.2011 To: 22.02.2011 females: 19.01.2011 - 13.03.2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
suspension
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily just before administration
- Storage temperature of food: test item was administered by gavage

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: 7d
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
According to the information from Sponsor the main component of the test substance is insoluble in the used vehicle (water for injection).
Mixture of sample and deionised water results in yellow non-transparent suspension but undissolved particles of the test substance were easily visible in the application form and homogeneity could be checked by eye. Stability of the test substance in the application form cannot be verified analytically. The application form was prepared just before the application and there was no indication that test substance would have been unstable in the suspension in deionised water for that short time period.
Duration of treatment / exposure:
males and females - 2 weeks prior to the mating period and during the mating period,
pregnant females - during pregnancy and till the 3rd day oflactation,
males - after mating period - totally for 28 days,
nonpregnant females (mated females without parturition) - for 25 days after the confirmed mating
Frequency of treatment:
daily, 7d/week
Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on data of 14d range finder
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
males - weekly
females - weekly in premating and mating period,
during pregnancy: day 0, 7th , 14th, 20th day, during lactation: 0. or 1st and 4th day;
pups (litters) - 0 or 1st and 4th day;
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, prostate gland weight, pituitary glands weight, sperm motility, sperm morphology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, physical or behavioural abnormalities

Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, males were killed at the end of the administration period - after 28 days of administration
- Maternal animals: All surviving animals, parental females were killed on the 4th day of lactation. Mated females without delivery were killed 26th day after confirmed mating

GROSS NECROPSY
- macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:
relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson's suspension), cervix of uterus, ovaries, uterus and vagina
Postmortem examinations (offspring):
SACRIFICE
- sacrificed on day 4 of lactation

GROSS NECROPSY
- examination of thoracic and abdominal tissues and organs
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight, biometry of organs and number of pups. Control group with vehicle was compared with three treated groups. The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.
Reproductive indices:
Post-implantation loss, Pre-implantation loss, Post-natal loss, Male mating index, Female mating index, Male fertility index, Female fertility index
Offspring viability indices:
Gestation index, Survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
yellow stained feces at mid and high dose groups
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
atrophic changes in prostate gland of high dose males

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)

- males: decrease of absolute and relative weight of prostate gland (dose-independent) was recorded in males of all dose levels. Changes did not exceed range of historical control. The histological examination demonstrated the increased occurrence of atrophic changes in prostate gland of males at the dose level I000 mg/kg/day. This finding was not dose dependend and is not considered to be an adverse effect.

- females: Dose dependent decreased relative weight of uterus was recorded in females at the dose level 1000 mg/kg/day (connection with oestrous cycle)

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: the value is based on the incidence of atrophic changes in prostate gland
Remarks on result:
other: the value is based on the incidence of atrophic changes in prostate gland
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: the value is based on the incidence of atrophic changes in prostate gland
Remarks on result:
other: the value is based on the incidence of atrophic changes in prostate gland
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: the value is based on the incidence of atrophic changes in prostate gland
Remarks on result:
other: the value is based on the incidence of atrophic changes in prostate gland

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:
other: not applicable
Based on:
not specified
Sex:
not specified
Basis for effect level:
other: not applicable
Remarks on result:
other: not applicable
Remarks:
not applicable

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects observed.
Remarks on result:
other: no effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

Effect levels (F2)

Dose descriptor:
other: not applicable
Generation:
other: not applicable
Effect level:
other: not applicable
Based on:
not specified
Sex:
not specified
Basis for effect level:
other: not applicable
Remarks on result:
other: not applicable

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean weight of organs

Organs Dose level
0 160 400 1000
Absolute weight (g) Relative weight Absolute weight (g) Relative weight Absolute weight (g) Relative weight Absolute weight (g) Relative weight
MALE
Testes 3.4895 0.8932 3.3634 0.8710 3.3428 0.8642 3.4594 0.9154
Epididymis 0.6705 0.1714 0.6417 0.1660 0.6386 0.1652 0.6508 0.1719
Prostate gland 0.8450 0.2171 0.7581 0.1948 0.7629 0.1974 0.6923 0.1814
Pituitary gland 0.0112 0.0029 0.0097 0.0025 0.0097 0.0025 0.0103 0.0027
FEMALE
Ovaries 0.1300 0.0418 0.1249 0.0419 0.1266 0.0414 0.1243 0.0428
Uterus 1.1200 0.3611 1.0072 0.3390 1.0236 0.3360 0.9550 0.3298
Pituitary gland 0.0158 0.0051 0.0157 0.0053 0.0170 0.0056 0.0146 0.0050

Table 2: Histopathological (microscopical) findings in females

Microscopical findings (number of affected animals)
Pathological findings Dose level
0 160 400 1000
Number of examined animals 12 12 12 12
Without microscopical changes 4 4 8 3
Pituitary gland: cysts 0 0 0 1
Ovaries: follicular or luteal cysts 0 1 0 1
Uterus: signs of previous gravidity 5 6 4 4
Uterus: hydrometra 3 0 0 4
Vagina: blood and/or cell detritus in lumen 2 2 0 0

Table 3: Fertility paramters determined in the study

Calculated parameters Dose level
0 160 400 1000
Male mating index 100.00 100.00 100.00 100.00
Female mating index 100.00 100.00 100.00 100.00
Male fertility index 66.67 83.33 75.00 66.67
Female fertility index 66.67 83.33 75.00 66.67
Gestation index 87.50 90.00 77.77 75.00
Survival index 96.30 100.00 98.85 100.00
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implants)
Pregnant females with 0 (N) 4 7 6 3
Pregnant females with 1 (N) 2 1 1 2
Pregnant females with 2 (N) 2 1 2 1
Pregnant females with ≥ 3 (N) 0 1 0 2
Post-implantation (implants minus live births)
Pregnant females with 0 (N) 6 3 5 1
Pregnant females with 1 (N) 1 2 1 5
Pregnant females with 2 (N) 0 1 2 1
Pregnant females with ≥ 3 (N) 1 4 1 1
Post-natal (live births minus alive at post-natal day 4)
Pregnant females with 0 (N) 6 9 6 6
Pregnant females with 1 (N) 0 0 1 0
Pregnant females with 2 (N) 0 0 0 0
Pregnant females with ≥ 3 (N) 1 0 0 0

Applicant's summary and conclusion

Conclusions:
Reproductive performance - ability of male and female animals to successfully mate and produce viable offspring was unaffected by the test substance treatment.
The NOEL (No Observed Effect Level) for the toxic effect on reprodnction organs of males was established as 400 mg/kg body weight/day (the value is based on the incidence of atrophie changes in prostate gland).
The NOEL (No Observed Effect Level) for the toxic effect on reproduction organs of females was established as 1000 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION was established as 1000 mg/kg body weight/day.