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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011-01-05 until 2011-06-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
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:
other: OECD 421 Reproduction/Developmental Toxicity Screening Test
Version / remarks:
27 July 1995
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
Details on test animals 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 - 309.22 g, females: 224.33 - 226.55 g
- 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 SUSPENSIONS:

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

SUSPENSION
- Amount of suspension: 10 ml/kg bw

VEHICLE
- Lot/batch no.: Ardeapharma a.s., Sevetin, Batch No.: 0102110510, 0102121110, 0206160910, 0203071210
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
- homogeneity could be checked by eye
- stability of the test substance in the application form cannot be verified analytically
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 to 1
- Length of cohabitation: one week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
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
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 finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly in premating and mating period, during pregnancy: day 0, 7th , 14th, 20th day, during lactation: 0. or 1st and 4th day

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 4
- Examination: macroscopic, all organs

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- weight of ovaries and pituitary gland
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No data

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
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.
Indices:
Male mating index
number of males with confirmed mating x 100 / number ofmales cohabited

Female mating index
number of sperm-positive females x l00 / number of females cohabited

Male fertility index
number of males impregnating a females x 100 / number ofmales cohabited

Female fertility index
number ofpregnant females x 100 / number of sperm-positive females

Gestation index
number of females with live born pups x l00 / number of pregnant females

Survival index
number of live pups on day 4 post partum* x l00 / number of pups born alive+

Note: * WIthout shll born pups (dead pups WIth anaenallungs)
+ with dead pups with aerial lungs

Pre-implantation loss: Number of corpora lutea - number of implantations
Post-implantation loss: Number of implantations - number of live births
Post-natal loss: Number of live births - number of alive at postuatal day 4
Historical control data:
yes, 13 control groups from Reproduction / Developmental Toxicity Screening tests

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- yellowish colored excrements at dose levels of 400 (since the 3rd week of application) and 1000 (since the 1st week of application) mg/kg bw/day
- in females the colouring was not discovered in all females in the last week of substance application
Mortality:
no mortality observed
Description (incidence):
- no unscheduled deaths observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- no statistically significant differences compared to the control groups
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- absolute and relative weights of testes, epididymis and pituitary gland similar in treated and control males
- in females at 1000 mg/kg bw/day: absolute weights of evaluated organs were slightly lower whilst relative weights of ovaries and pituitary gland were balanced
- relative weight of uterus of the high dose group was decreased against the control
No statistically significant differences were detected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- colouring of chymus in caecum and stomach recorded in all males of the dose level 1000 mg/kg/day
- no effects observed in male animals of the mid- and low-dose groups
- macroscopic changes of genital organs recorded in control and treated females dilatation of uterine horns, atrophy of one uterine horn and absence of one oviduct in one high-dose female
- stomach and content of intestine substance coloured in all females of the middle and the highest dose level and one female of the lowest dose level

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The number of corpora lutea and implantations was insignificantly decreased at the dose level 1000 mg/kg/day compared to control.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation losses were slightly increased at the dose level 1000 mg/kg/day. Slightly increased post-implantation losses were described at the dose level 160 mg/kg/day. No significant differences were detected in post-natal losses.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The number of duration of pregnancy was comparable between the control and treated groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of females achieving pregnancy was comparable between the control and treated groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: no effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
- mean weights of the litters and pups were balanced in the control and treated groups period
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
- total number of live pups (on the day of parturition / 1st day after parturition and the 4th day after parturition) at the dose level 1000 mg/kg/day was lower than at the control
- mean number of pups per litter at the dose level 400 mg/kg/day was increased compared to control

The statistical evaluation of the number of live pups did not find any significant changes.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio of pups was balanced in treated and control groups.
External malformations:
no effects observed
Description (incidence and severity):
- no statistically significant pathological findings were recorded in pups of contral group and treated groups
Details on embryotoxic / teratogenic effects:
Pups with macroscopical findings were recorded only sporadically at the dose levels 400 and 1000 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No statistically significant effects observed.
Remarks on result:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: 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 2: Total number of live pups and sex distribution/ratio

Number of pups and sex (mean per litter)
Dose level 0 160 400 1000
Day of study Total number (mean) Number of males and females Total number (mean) Number of males and females Total number (mean) Number of males and females Total number (mean) Number of males and females
0/1 day first check of litter after parturition 81 (11.57) 45 M, 36 F 99 (11.00) 51 M, 48 F 87 (12.43) 44 M, 43 F 68 (11.33) 37 M, 31 F
4th day of lactation 78 (11.14) 43 M, 35 F 99 (11.00) 51 M, 48 F 86 (12.29) 43 M, 43 F 67 (11.17) 37 M, 30 F

M: male pups, F: female pups

Table 3: Overview of the reproduction parameters of the study

Reproduction data
Observed parameters Dose level
0 160 400 1000
Paris started (N) 12 12 12 12
Females showing evidence of copulation (N) 12 12 12 12
Females achieving pregnancy (N) 8 10 9 8
Females with abortion (N) 1 1 2 2
Conceiving days (duration of mating) 1-5 (N) 12 11 12 12
Conceiving days (duration of mating) 6-10 (N) 0 1 0 0
Mean duration of mating (days) 2.42 2.25 1.83 1.92
Pregnancy ≤ 21 days (N) 1 2 2 1
Pregnancy = 22 days (N) 4 5 4 4
Pregnancy ≥ 23 days (N) 2 2 1 1
Mean duration of pregnancy (days) 22.14 22.00 21.86 22.00
Females with live pups born (N) 7 9 7 6
Females with live pups at day 4 after parturition (N) 6 9 7 6
Corpora lutea/pregnant females (mean) 11.63 12.40 11.11 10.88
Implantations/pregnant females (mean) 10.88 11.80 10.56 9.63
Live pups/mother at birth (mean) 11.57 11.00 12.43 11.17
Live pups/mother at day 4 after parturition (mean) 11.14 11.00 12.29 11.17
Sex ratio (M/F) at birth (mean) 6.43/5.14 5.67/5.33 6.29/6.14 6.17/5.17
Sex ratio (M/F) at day 4 after parturition (mean) 6.14/5.00 5.67/5.33 6.14/6.14 6.17/5.00
Litter weight at birth (mean) 76.31 71.91 80.29 69.55
Litter weight at day 4 after parturition (mean) 128.53 112.28 121.97 118.15
Pup weight at birth (mean) 6.67 6.58 6.51 6.24
ABNORMAL PUPS 10.02 10.32 9.96 10.72
Mothers with 0 (N) 0 0 0 0
Mothers with 1 (N) 0 0 1 1
Mothers with ≥ 2 (N) 0 0 0 0

Applicant's summary and conclusion

Conclusions:
The influence of the test substance treatment expressed mainly at the highest dose level (limit dose) - decrease of absolute and relative weight of prostate gland and occurrence of atrophie changes in prostate gland of parental males.
The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of pups was established as 1000 mg/kg body weight/day.