Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.3.2006 - 15.3.2007
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
other: Body responsible for the test
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EC 92/69/EEC, B.35 Two generation reproduction study OECD 416 Guideline for Testing of Chemicals
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test batch: No.: 003/2006

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Intact female and male, initial mass: 180-220 g , Species: rat, Strain: Wistar, number: 180 (120 female and 60 male), number of female depends of request of test (1,2) that in tested group shoul be minimally 20 gravid female.
Utilization in test: 180 rats (parent P-generation) + 180 (F1-generation).

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: By the rat mass ( in kg) and tested substance dose the amount of Dusantox L was calculated for the specific group, the substance wos diluted in olive oil so that administrated volume was 0,5 ml/100g of animal body mass.


DIET PREPARATION
- Rate of preparation of diet (frequency):aplication doses were prepared in frequency 1x per week by actual rats body mass
- Mixing appropriate amounts with (Type of food): Peletizet laboratory diet - standard food mixing for rats, waterfeed: ad libitum
- Storage temperature of food: 22 +/-2°C


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage): 0,5ml/100g body mass
- Lot/batch no. (if required): L602 022
- Purity:Olivae oleum raffinatum
Details on mating procedure:
- M/F ratio per cage: 1male for 2 female, 15males + 30 females per group
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: daily check of sperm in vaginal plug by microscope
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. no
- Further matings after two unsuccessful attempts: [no / yes (explain)] no
- After successful mating each pregnant female was caged (how): each pregnat female was stabled in individual cage in 0. day of gravidity( the day when the presence of sperm in vagina was positive )
- Any other deviations from standard protocol:-
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
P 0-parents generation:
Duration of exposure: 70 days
Duration of mating: 21 days
Males euthansia: after mating
Births: 22 days
Weaning of FI generation: 28 days after birth
F1 first generation:
Duration of exposure: 70 days
Duration of mating: 21 days
Males euthansia: after mating
Births: 22 days
Weaning of F2 generation: 21 days after birth -euthanasia

Frequency of treatment:
Dosing regime P0: 7 days/week (males)
Dosing regime P0: 7 days/week (females)
Dosing regime F1: 7 days/week (males)
Dosing regime F1: 7 days/week (females)
Dosing regime F2: 7 days/week (males)
Dosing regime F2: 7 days/week (females)
Details on study schedule:
Number of litters per F1 generation (in relation to female parent group size):
Group 1: 8; Group 2: 8; Group 3: 7; Group 4: 6; Group 5: ;

Number of litters per F2 generation (in relation to female parent group size)dose/conc.:
Group 1: ; Group 2: 9; Group 3: ; Group 4: 7; Group 5: ;Group 1: ; Group 2: ; Group 3: ; Group 4: ; Group 5:
Doses / concentrations
Remarks:
Doses / Concentrations:
0 mg/kg ; 5 mg/kg ; 25 mg/kg ; 50 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
P0 Generation Males: 0 mg/kg or mg/l; No. of animals: 15
P0 Generation Males: 25 mg/kg or mg/l; No. of animals: 15
P0 Generation Males: 5 mg/kg or mg/l; No. of animals: 15
P0 Generation Males: 50 mg/kg or mg/l; No. of animals: 15
P0 Generation Females: 0 mg/kg or mg/l; No. of animals: 30
P0 Generation Females: 5 mg/kg or mg/l; No. of animals: 30
P0 Generation Females: 25 mg/kg or mg/l; No. of animals: 30
P0 Generation Females: 50 mg/kg or mg/l; No. of animals: 30
F1 Generation Males: 0 mg/kg or mg/l; No. of animals: 15
F1 Generation Males: 5 mg/kg or mg/l; No. of animals: 15
F1 Generation Males: 25 mg/kg or mg/l; No. of animals: 15
F1 Generation Males: 50 mg/kg or mg/l; No. of animals: 15
F1 Generation Females: 0 mg/kg or mg/l; No. of animals: 30
F1 Generation Females: 5 mg/kg or mg/l; No. of animals: 30
F1 Generation Females: 25 mg/kg or mg/l; No. of animals: 30
F1 Generation Females: 50 mg/kg or mg/l; No. of animals: 30
Control animals:
yes

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

FIRST GENERATION STAGE: Effects on parental animals
The substance was applied to males after quarantine ending
within 70 days. Application was ended in last day before
coupling. The substance was applied to females 14 days
before coupling so that two ovulatory periods were captured.
The daily female application kept on non-stop during
coupling, pregnancy, lactation, weaning, 28 days after
birth.

At males from 50 mg/kg doses group P-generation direct
negative effect of Dusantox L was found to spermatogenesis
development and maturing of the wealthy fertilizable
spermatozoa.

The fact why the strongest degenerative changes in follicle
are at P-generation males however can be caused by
application of Dusantox L high doses 75 mg/kg within first
month of this study.

The effect of Dusantox L was most considerable in P-
generation; its negative effect worsened the animal
condition, increased salivation and decreased food intake
and followed to decrease of male weight of P- generation.
Evaluation of reproduction indicators in the highest dosed
group of P- generation expressly decreased the volume of
females as well as young animals. The litter weight of young
animals born in P- generation considerable decreased in the
highest dosen group on 14thdays post partum. Fertility
index decreased in dose relation of Dusantox L and the
lowest value got in application of dose 50 mg/kg. Index
values of viability and lactation were in all doses
comparable; the evaluation of pathology results in P-
generation found the increase of liver weight of males and
females with application of Dusantox L. The increase of
relative weight of follicles was in males of all dose
groups.

SECOND GENERATION STAGE: Effects on parental animals (F1 generation)
From the total number of 30 females in every group the
gravidity in the highest number was identically proved in
control and first dosen group where 27 females were gravid,
in second dosen group the number of gravid females decreased
to 26 and in the highest dosen group the number of gravid
females considerable decreased and gravidity was proved in
17 females. The number of females with life young animals in
first and second dosen group was identical. In the control
group the number of females with life young animals
decreased by 1 female and in third dosen group every gravid
female had life young animals. Fertility index was in the
control and first dosen group same. (90%). In second group
it slightly decreased (86.67%) and in third group was the
lowest value (56.67%). Index of gestation, viability and
lactation were in all groups equal, the differences against
the control were minimal.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 5 mg/kg bw/day
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
>= 25 mg/kg bw/day
Sex:
male/female

Results: F1 generation

Details on results (F1)

FIRST GENERATION STAGE: Effects on F1 generation
Dusantox L was applied on the animals of F1- generation
during all prenatal and postnatal life. The males were free
from test substance before mate, but females were in contact
with substance during the gravidity, birth, lactation and
till wean of young animals at the age of 21 days. It was
shown in clinical observation mostly in highest dosen group.
The weight of males considerable decreased on 42nd day of
application. The weight of females was higher than in
control, but in the gravidity it considerable decreased and
this situation was until the birth. In the reproduction
parameters the females from the group with dose 50 mg/kg of
Dusantox L are near to values P - generation of their
mothers, mostly in term of fertility. Reproduction values
of F1- generation females of two lower dosen groups are
better comparable against the P- generation of their
mothers and do not differ from values of females -
generation mates - from the control group. It is related to
favourable index values of fertility, gestation, viability
and lactation of females with application of 5 and 25 mg/kg
of Dusantox L. The females with dose of 50 mg/kg remain in
the fertility index, which is decreased against control and
both lower dosen groups. The other index values were not
adverse affected by tested substance.

SECOND GENERATION STAGE: Effects on F2 generation
Adverse reaction of Dusantox L was only in progeny of F2 -
generation in the highest dosen group. F1 and F2 generations
were in contact with Dusantox L from the moment of
conception constantly via mother organism and after birth in
lactation term. During observation of average number of
young animals the F2- generation litter, the average number
of young animals in control and second dosen group was
comparable to the number 10.At descendants of F2 generation
improvement of some reproductive indicators was observed
against to F1-generation. The total number of young was
increased evidently in F2-generation against to F1-
generation in all groups and control. An only negligible
difference was found between I. and II. dose group and
control. The lower number was recorded in III. highest dose
group only, even thought against the same data at F1-
generation the number was increased gently. The average
number of young per throwing was in the highest dose
decreased against the other doses and control but was higher
to F1-generation again.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 25 mg/kg bw/day
Sex:
male/female
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
> 25 mg/kg bw/day
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion