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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 414 (adopted 2001)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
(Harlan Laboratories Ltd.)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biofert Plusz
Details are presented in "Confidential details on test material"

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: HanRcc: WIST(SPF)
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: 181 to 234 g
- Housing: individually
- certified diet (analysis available) ad libitum
- Tap water ad libitum
- Acclimation period: 7 days; under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
according to Guideline

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Milli-Q-water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared every third day, based upon the dry mass (28.1%) of the main component in the provided test item. Therefore, a correction factor of 3.56 was used. Prior to dose formulation, the contents of the test item container were homogenized to avoid uneven distribution of the dry mass. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dosing volume: 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were stored in the refrigerator (2 - 8 °C) in glass beakers. On the first treatment day samples from the control group as well as three samples (top, middle, and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (3 days). The test item content in all samples was found to be within the accepted range of ±20% of the nominal content (ranged from 111.4% to 119.8% of the nominal concentration). In addition, the homogeneous distribution of Biofert Plusz in Milli-Q-Water was demonstrated. The application formulations were considered to be stable for at least 3 days when kept at 2-8 °C.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1; same source and strain; synchronized timing to initiate the nightly mating), until evidence of copulation was observed. The day of mating was designated day 0 post coitum when a) the daily vaginal smear was sperm positive, or b) a copulation plug was observed. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
day 6-20 post coitum
Frequency of treatment:
once daily
Duration of test:
scheduled necropsy on day 21 post coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: nominal, based to dry mass
No. of animals per sex per dose:
22 dams per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: results of a preliminary dose-range-finding study (see further study record in Section 7.8.2)

Examinations

Maternal examinations:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
Body Weights: recorded daily from day 0 until day 21 post coitum.
Termination day 21 post coitum: females sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
Necropsy: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea; uteri (and contents) of all females with live fetuses was weighed during necropsy to enable the calculation of the corrected body weight gain.
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
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:
- Number of live and dead fetuses and abnormal fetuses
- fetal sex ratios
- fetal body weights
- External examinations: Yes: [all per litter]
- Soft tissue and head examinations: Yes: [half per litter]; microdissection technique; combination of serial sections of the head and microdissection of the thorax and abdomen.
- Skeletal examinations: Yes: [half per litter]; fetuses were eviscerated; Alizarin red S staining.
Statistics:
- Means and standard deviations of various data calculated.
- Dunnett-test (many to one t-test) based on a pooled variance estimate (normal distribution)
- Steel-test applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test
Historical control data:
yes, available in Appendix V

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- non-pregnant rats only 2/22 in control and 1/22 in the low dose group
- no clinical signs
- mean food consumption significantly reduced from day 6 to 9 post coitum in the high dose group, however, this transient reduction was considered to be test item-related but not adverse (no effect on body weight; reduction less than 10%)
- mean body weight and body weight gain was similar in all groups
- no effects on corrected body weight gain
- no effects on post-implantation loss and mean number of fetuses
- No test item-related findings at necropsy

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: dry mass of the liquid test item
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: dry mass
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No test item-related effects on the sex ratio, fetal body weights, implantations, resorptions, dead fetuses, abnormal fetuses.
No treatment related increase in external, visceral, or skeletal malformations or variations. Significantly higher incidence of incompletely ossified sternebra 5 was noted at the high dose level. Since this incidence was in the range of the historical control data it was considered to reflect the range of natural biological variability.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Food consumption in pregnant rats

Means +- standard deviation

Observation period

Control group

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Day 3-6 pc

21.7+-1.8

20.7+-1.9

20.9+-1.6

20.8+-2.3

Day 6-9 pc

22.9+-1.7

22.1+-1.8

21.6+-2.0

21.0+-2.2*

Day 9-12 pc

23.0+-2.1

22.5+-2.0

21.7+-2.2

21.7+-2.5

pc: post coitum; *: p<0.01 (Dunetts test);

 

Body weight gain in dams (in % of absolute body weight

Means +- standard deviation

Day pc

Control group

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

6

0

0

0

0

7

2+-1.1

1+-1.2

1+-1.1

2+-1.1

8

3+-1.3

3+-1.4

3+-1.1

3+-1.1

9

5+-1.4

5+-1.6

6+-1.6

5+-1.7

pc: post coitum

Applicant's summary and conclusion

Conclusions:
In Wistar rats no maternal and developmental toxicity was detected even at the high dose level of 1000 mg/kg bw/day (related to dry weight) which corresponds to the limit dose recommended in OECD Guideline 414.