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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline studie

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Uvinul MC 80 N
- Physical state: Liquid
- Analytical purity: 99.9 area %
- Lot/batch No.: uv2-01.019
- Stability under test conditions: Proven by reanalysis after the inlife phase of
the study
- Storage condition of test material: Room temperature



Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany.
- Age at study initiation: (P) 37 +/- 1 days ;

- Weight at study initiation:
(P) Males: 123.4 (113.1-135.2)g; Females: 108.0 (96.4- 119.0)g;

- Housing:
individually in type DK lll stainless steel wire mesh cages supplied by BECKER& CO., Castrop-Rauxel, Germany (floor area of
about 8 00 cm2)
overnight matings: male and female mating partners housed together in type DK lll cage
gestation day 18- lactation day21: pregnant animals and their litters housed in Makrolon type M lll cages.
nesting material (cellulose wadding) toward the end of gestation
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours( 12h light from 6 .00 a .m. to 6.00 p .m. and
12 h darkness from 6 .00 p .m. to 6.00 a .m.)


IN-LIFE DATES: From: 28.Feb.2002 To: 03.Dec.2002

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: unchanged, mixed with diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat "GLP" meal
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight matings for 4d (F0), 6d (F1)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
Mating indices were 100%
- Further matings after two unsuccessful attempts:
no, mating indices were 100%
- After successful mating each pregnant female was caged (how):
From gestation day 18 to lactation day 21: pregnant animals and their litters were housed in
Makrolon type Mlll cages and nesting material (cellulose wadding) was provided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Feed samples were extracted with acetonitrile. Aliquots of the extracts were used for HPLC-analysis.
Duration of treatment / exposure:
F0 generation: continuous administration of the test substance until or up to
about 16 hours before they were sacrificed (Feb.28- Jul.17.2002)
F1 generation: After weaning, continuous administration of the test substance until or up to
about 16 hours before they were sacrificed. (Jul.3.-Nov.11.2002)
F2 generation: After weaning, continuous administration of the test substance until or up to
about 16 hours before they were sacrificed (Nov.7.-Dec.3.2002)
Frequency of treatment:
continuous administration via diet
Details on study schedule:
- F1 parental animals not mated until at least 74 days after selected from the F1 litters.

for further details, see appendix 1
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 450 ,1000 mg/kg body weight/day
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: parental animals daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Exeptions for female animals:
During gestation period parental females were weighed on day 0,7,14,20 post coitum.
Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
Females with litter were weighed on the day after parturition day 1, 4, 7, 14 and 21 post partum.
Females without litter were not weighed during the lactation phase.
After weaning female F0 parental animals were weighed again once weekly (in parallel to male) until scheduled sacrifice.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculation:
ITx=(FCx*C)/BWy
ITx= TS intake on day x(mg/kg bw/day)
FCx= daily food consumption day x (g)
C= TS concentration (ppm)
BWy= body weight day y (last weighing before day x)

Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluate daily (F0 and F1 parental animals)
for a minimum of 3 weeks prior to mating and were continued throughout the mating
period until the female exhibited evidence of mating.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
testis weight,
epididymis weight,
sperm count in testes,
sperm count in epididymides,
sperm motility,
sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter as nearly as possible. Standardisation was not performed <= 8 pups /litter
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups
stillbirths,
live births
postnatal mortality
presence of gross anomalies
weight gain
physical or behavioural abnormalities



GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrifieced after weaning period of respective offspring
- Maternal animals: All surviving animals were sacrifieced after weaning period of respective offspring


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Appendix 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after standardization or weaning (14-28d), except for the
group of reared F2 pups, which were sacrificed shortly after achieving sexual maturity (43-56d)

- These animals were subjected to postmortem examinations as follows:
Pups were examined externally and eviscerated their organs were assessed macroscopically

ORGAN WEIGTHS
brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed.
Statistics:
For statistics of clinical examinations see appendix 3
For statistics of pathology see appendix 4
Reproductive indices:
Male mating Index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
Male fertility Index (%) = (number of fertile males**/number of males placed with females) x100
Female mating index (%) = (number of females mated*/number of females placed with males) x100
Female fertility index (%) = (number of females pregnant***/ number of females mated*) x 100
* animals with vaginal sperm or that gave birth to a litter or with pups/implantations in utero
**defined by a female giving birth to a litter or with pups/implantation in utero
*** defined as the number of females that gave birth to a litter or with pups/implantations in utero

Offspring viability indices:
Gestation Index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females that gave birth to a litter or with pups/implantations in utero
Live birth Index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Post lmplantation loss (%) = ((number of implantations - number of pups delivered)/number of implantations) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

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)

Test substance related findings:

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
1000 mg/kg bw /day:
Males F0: significantly decreased mean bodyweights (16% below control) and body weight gain (average 22 % below control)
Males F1: significantly decreased mean body weights(14% below control) and body weight gain (average 14% below control)
Females F0: decreased mean bodyweights during premating (4 %), gestation (8 %) and early lactation (6 % below control)
Females F1: decreased mean body weights during premating(6 %), gestation(10%) and lactation( 6% below control)
Females F0: decreased body weight gain during premating (9 %) and gestation (18% below control)
Females F1: decreased body weight gain during gestation(1 7% below control)
Males F1: Significantly decreased mean food consumption(4 % below control)
Females F1: significantly decreased mean food consumption during gestation(9%) and mid lactation(11 %) below control.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Test substance related findings with histological correlate:
1000 mg/kg bw/day
F0: Significantly increased mean absolute liver weight in female rats (17% above control)
F0: Significantly increased mean relative liver weight in males/fermales ( 17 and 23% above control, respectively)

HISTOPATHOLOGY (PARENTAL ANIMALS)
1000 mg/kg bw/day
F0/F1: eosinophilic homogeneous appearance of the liver cell cytoplasm indicative of
enzyme induction in males/females
F0/F1: increased amount of hemosiderin in the spleen of males

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0: One low dose female (150 mg/kg body weight) was found dead: not considered as substance related.
F1: Mortality in two mid dose females (found dead/ sacrificed for to humane reasons): not considered as substance related.
F0: Urine-smeared fur around the anogenital region in 2 animals (1000 mg/kg bw/day)
F1: Urine-smeared fur around the anogenital region in 3 animals (1000 mg/kg bw/day)
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0: mean number of implantation sites was statistically significantly lower for the high dose group (12.0; 12.3; 11.3; 10.0 implants/dam for test group 0;150; 450; 1000 mg/kg bw/day, respectively)
F1: mean number of implantation sites was statistically significantly lower for mid/ high dose group (12.4; 11.5; 10.7; 10.3 implants/dam for test
group 0;150; 450; 1000 mg/kg bw/day, respectively)
F0: mean number of F1 pups delivered/dam was statistically significantly lower for the high dose group (11 .0 ; 11.9; 10.9; 9.2 for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)


GROSS PATHOLOGY (PARENTAL ANIMALS)
F0:Erosion/ulcer in the mucosa of the glandular stomach ( 0/1/3/4 females for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)
F1:Erosion/ulcer in the mucosa of the glandular stomach ( 0/0/0/4 males and 3/3/2/4 females for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic parental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: body weight; gross pathology; organ weights; histopathology;
Remarks on result:
other: Generation: P/F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Fertility and reproduction parameters
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: secondary number of implantation sites; secondary delayed sexual maturation
Remarks on result:
other: Generation: P/F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: pup weights
Remarks on result:
other: Generation: F1/F2 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

Test substance related findings:

BODY WEIGHT (OFFSPRING)
1000 mg/kg bw/day
F1: significantly decreased mean body weights( 13% below control at weaning) and body
weight gain( 15% below control) males/females during lactation
F2: significantly decreased mean body weights( 16% below control at weaning) and body
weight gain(20% below control) males/females during lactation.
F2 reared: significantly decreased mean body weights (final weight 12% below control) and body
weight gain (average 10% below control) in males
F2 reared: significantly decreased mean body weights (final weight 6% below control) in females

SEXUAL MATURATION (OFFSPRING)
1000 mg/kg bw/day
F1: slight delay of preputial separation in males (average age 45.4d vs.43.6 d in control) exceeds historical control (42.5- 45.0d), secondary to delayedbody weight development
F2 reared: slight delay of preputial separation in males (average age 45.1d vs.42.8 d in control) exceeds historical control (42.5- 45.0d), secondary to delayed body weight development
F1: apparent slight delay of vaginal patency in females (average age 33.8d vs. 31.3d in control) within historical control (30.8d- 33.8d), secondary to
delayed body weight development
F2 reared: apparent slight delay of vaginal patency in females (average age 35.5d vs. 32.9d in control) within historical control (30.8d- 33.8d),
secondary to delayed body weight development.

OTHER FINDINGS (OFFSPRING)
F2 reared: Significantly decreased mean food consumption(10 % below control) in males





Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion