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:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Analogue substance tested; GLP-study using standard method

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Annex V (2-generation fertility study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Portage, Michigan, USA
- Age at study initiation: no data
- Weight at study initiation: (P) 52 -78 g
- Fasting period before study: no
- Housing: individual, suspended stainless steel, wire-mesh cages; during mating, parturition & lactation: lpastic cages with wood chip bedding
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18,9 -24,4 (66-76 F)
- Humidity: 34 - 77 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% carboxymethylcellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved directly in appropriate volumes of 0,05 % CMC in water

VEHICLE
- Concentration in vehicle: 0 -10 -30 -100 mg/mL depending on desired dose level
- Amount of vehicle (if gavage): 10 mL/ kg bw / day

Details on mating procedure:
- Start after at least 10 weeks of treatment
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of mating was found (vaginal smears)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing mating ended.
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no second mating of parental animals
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test article suspensions were analysed for stability, homgeneity, concentration.
Homogeneity was determined before treatment initiation at 10 and 100 mg/mL.
Stability was determined over the range of doses in this study in a separate study after storage of samples for 14 days in a refrigerator.
Concentrations were determined in samples of vehicle and all tes article concentrations weekly during the first month and monthly thereafter.
Duration of treatment / exposure:
F0 Parents: 70 days before mating until euthanasia
F1 pubs: indirect: in utero and during lactation
F1 parents: direct (via gavage):starting when the youngest selected animal had reached an age of 28 days until 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 (lactation only, males) Dosing regime F2: 7 days/week (lactation only, females)
Details on study schedule:
- F1 parents selection: 1 male and 1 female from each litter were selected on day 21 of lactation (weaning) as parents for F2;
- Sipling matings were avoided
- F1 parental animals not mated until 10 weeks of treatment after selected from the F1 litters.

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw / day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw /day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
P0 Generation Males: 0 mg/kg or mg/l; No. of animals: 26
P0 Generation Males: 100 mg/kg or mg/l; No. of animals: 26
P0 Generation Males: 1000 mg/kg or mg/l; No. of animals: 26
P0 Generation Males: 300 mg/kg or mg/l; No. of animals: 26
P0 Generation Females: 0 mg/kg or mg/l; No. of animals: 26
P0 Generation Females: 100 mg/kg or mg/l; No. of animals: 26
P0 Generation Females: 300 mg/kg or mg/l; No. of animals: 26
P0 Generation Females: 1000 mg/kg or mg/l; No. of animals: 26
F1 Generation Males: 0 mg/kg or mg/l; No. of animals: 26
F1 Generation Males: 100 mg/kg or mg/l; No. of animals: 26
F1 Generation Males: 300 mg/kg or mg/l; No. of animals: 26
F1 Generation Males: 1000 mg/kg or mg/l; No. of animals: 26
F1 Generation Females: 0 mg/kg or mg/l; No. of animals: 26
F1 Generation Females: 100 mg/kg or mg/l; No. of animals: 26
F1 Generation Females: 300 mg/kg or mg/l; No. of animals: 26
F1 Generation Females: 1000 mg/kg or mg/l; No. of animals: 26
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose range finding study
- Rationale for animal assignment (if not random): random
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2 x per day
- parameters: mortality, morbidity, signs of injury

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1 x per week
Parameters: general condition, skin, fur (body surface), eyes, ears, nose, oral cavity, respiration, genitals regarding size, shape, color, other changes

BODY WEIGHT: Yes
- Time schedule for examinations: day of arrival, first day of dosing, then weekly
mated females during gestation: days 0, 7, 14, 20 of gestation
parental females during lactation: days 0, 4, 7, 14, 21 of lactation

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 calculated as time-weighted averages from the consumption and body weight gain data: No (gavage)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (gavage)
Estrous cyclicity (parental animals):
Estrous cyclicity was determined in all parental females beginning 3 weeks prior to initiation of mating
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, litter size, sex distribution,
- reduction of litter site to 8 /sex/litter (as nearly as possible); excess pups were killed and necropsied.

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;
possible cause of death was determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

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

ORGAN WEIGHTS (absolute & relative): Tissues weighed:
Males: Adrenal, brain, epididymides, liver, kidney, pituitary gland, prostate, seminal vesicle, spleen, testes
Females: Adrenal, brain, liver, kidney, ovaries, oviduct , pituitary gland, spleen, uterus,

HISTOPATHOLOGY : Tissues examined:
Males: Adrenal, epididymides, pituitary, seminal vesicle, testis (left), gross lesions
Females: Adrenal, intestine (large & small), lung, lymph nodes (mesenteric), nasal cavity, ovary, oviduct, pituitary, uterus (incl. cervix), vagina, gross lesions
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGHTS (absolute & relative): Tissues weighed:

F 1 & 2 pups (1 random pup/sex/litter)
Males & females: Brain, spleen, thymus

HISTOPATHOLOGY : Tissues examined:

F 1 & 2 pups (1 random pup/sex/litter)
Males & females: Brain, spleen, thymus
Statistics:
The following table defines the set(s) of comparisons used in the statistical analyses. If more than 1 set of comparisons was required, all analyses were conducted separately on each set, unless stated otherwise. Data for each sex within a set were analyzed separately, where appropriate.

Group Pair-wise Comparisons: Parental In-life Data; Premating Body Weights; Premating Body Weight Changes; Premating Food Consumption; Gestation Body Weights; Gestation Body Weight changes; Gestation Food Consumption; Lactation Body Weights; Lactation Food Consumption;

Fertility Indices
Group Pair-wise Comparisons; Gestation Length Copulatory Interval; Estrous cycle (mean cycle time); Estrous cycle (# cycles/period)
Fisher's Exact Test; Male Fertility Index; Female Fertility Index; Male Mating Index; Female Mating Index; Male Fecundity Index; Female Fecundity Index; Gestation Index

Pathology
Group Pair-wise Comparisons: Male reproduction organ weights; Female reproduction organ weights
Spermatogenesis
Arcsin-Square-Root Transformation: % Motility; % progressive Motile; % abnormal
Group Pair-wise Comparisons: Total sperm per cauda epididymidis (*10E-8); Concentration per gram cauda epididymidis (*10E-8); Total sperm per testis (*10E-8); Concentration per gram testis (*10E-8); Daily sperm production; Spermatogenic efficiency;

Primordial follicle count (F1) Group Pair-wise Comparisons
Uterine Examination
Implantations / dam Group Pair-wise Comparisons

Litter
Group Pair-wise Comparisons: Litter Size; Viable Pups
Arcsin-Square-Root Transformation: Pup Sex Ratio (% males); Live Birth Index; Pup Survival Indices (Days 0-4 pre-cull, 4 post-cull-21)
Stillborn Index Arcsin-Square-Root Transformation
Covariate Analysis: Mean Pup weights

Developmental Indices
Group Pair-wise Comparisons: Preputial Separation; Vaginal Opening

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
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
Description (incidence and severity):
A test article-related increase in absolute and relative kidney weight was noted in females at 1000 mgkglday. There were statistically significant increases in absolute and relative (to brain and body weight) kidney weight in females at 1000 mgkglday when
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Test substance intake: gavage

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

FIRST GENERATION STAGE: Effects on parental animals
One female from the 1000 mg/kg/day group was found dead in week 17 of the study, this death was considered to be due to intubation error. One female each from the 300 and 1000 mg/kg/day groups were terminated in extremis in week 13; it was considered that these deaths were not treatment related. The general condition of the remaining males and females was satisfactory throughout the study, no clinical signs were observed that were considered to be treatment-related. No treatment-related effects were seen on bodyweight or food consumption in animals of either sex. A treatment-related, statistically significant increase in relative (to bodyweight) kidney weight (~9%) and pititary weight (~20%) was observed in females from the 1000 mg/kg/day group only, compared with control values. No treatment-related microscopic changes in these or any other organs were evident in females. microscopic changes were noted in two males from the 1000 mg/kg/day group: one had small testes bilateral and aspermia of the epididymides; this male did not mate. Another male from this group had small epididymides containing spermatid giant cells and a flacid left testis; however this animal did mate and sire a litter, successfully. One male from the 300 mg/kg/day group had small testes with mild hypospermia in the left epididymidis. Sperm analysis of this individual revealed reduced sperm count and motility with 94% abnormal sperm; this male did mate but not sire a litter. No treatment-related changes were observed in oestrus or gestation length, gestation or female mating indices. Slight, but not statistically significant decreases were seen in the fertility and fecundity indices of both sexes of all test substance-treated animals and in male mating index, however these were well within the range of historical control data for this strain in the performing laboratory and were therefore not considered to be toxicologically significant. The copulatory interval was statistically significantly increased for animals in the 1000 mg/kg/day group; this was due to two females who did not become pregnant until 8 and 13 days post-pairing. These females were cycling normally and the males had normal sperm parameters, therefore this effect may not be toxicologically significant. No overall treatment-related or toxicologically significant effects on sperm analyses were observed in F0. SECOND GENERATION STAGE: Effects on parental animals (F1 generation) No treatment-related changes in bodyweight, bodyweight gain or food consumption were noted during the premating, post- mating, gestation or lactation intervals. No toxicologically significant macroscopic or microscopic findings were noted in F1 parents. One control male had epididymal aspermia amd one 1000 mg/kg/day male had trace degeneration of the seminiferous tubules; neither sired a litter and these findings were not considered to be treatment-related. A treatment-related, statistically significant increase in relative (to bodyweight) kidney weight was observed in males (~8%) and females (~14%) from the 1000 mg/kg/day group and in females of the 300 mg/kg/day (~6%) compared with the control values. A statistically significant increase in relative adrenal weight was also observed in females at 1000 mg/kg/day (~19%) although no associated microscopic changes were seen at this dose. No treatment-related changes were observed in F1 parental oestrous cycle length and number, follicle count, gestation length, gestation index, copulatory interval or male and female fecundity or female mating indices. Slight, non dose- dependent or non-statistically significant decreases were seen in fertility indices of both sexes or in the male mating index of all test-treated animals, however these were well within the range of historical control data for this strain in the performing laboratory and were therefore not considered to be toxicologically significant. No overall treatment-related effects on sperm analyses or morphology were observed in F1 males

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes in kidney weight were noted at 300 and l000 mgkglday. There were statistically significant increases in absolute and relative kidney weight in both males and females at 1000 mgikglday as well as relative kidney (to body weight
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

FIRST GENERATION STAGE: Effects on F1 generation
The general condition of the offspring was similar in all groups and showed no adverse responses to treatment of the F0 parents.
The number of uterine implantation sites, litter size at birth, survival of offspring and sex ratios did not indicate any adverse effects of treatment.
A statistically significant increase (~6%) in male bodyweight and consequently combined male and female offspring bodyweight (~6%) was observed at birth only in the 1000 mg/kg/day group.
The timing of vaginal opening and preputial separation for the F1 animals showed no adverse treatment-related response and the bodyweights of both males and females was similar between groups at the time of sexual maturation.
There were no treatment-related effects on organ weights of offspring and macroscopic examination of offspring dying before weaning. The offspring killed at weaning after F1 selection did not reveal any treatment-related findings.
SECOND GENERATION STAGE: Effects on F2 generation
The general condition of the offspring was similar in all groups and showed no adverse responses to treatment of the F1 parents.
The number of uterine implantation sites, litter size at birth, live birth, stillborn index, survival of offspring and sex ratios did not indicate any adverse effects of treatment.
Dose-dependent and treatment-related increases in pup bodyweights were observed at birth and at the time of culling (day 21). At some time points these increases reached statistical significance, however these changes were not considered to be adverse.
There were no treatment-related effects in organ weights of the brain, spleen or thymus of the F2 offspring. No treatment-related macroscopic changes were noted at necropsy.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this study, in which 2 generations of rats were exposed to the Analogue substance 2 (refer to IUCLID chapter 13), at doses of 0, 100, 300 and 1000 mg/kg/day during an approximate 10-week premating period, and continuing during mating, gestation and lactation to evaluate the effects on reproductive performance as well as growth and development of offspring, there was a test article-related increase in kidney weight that was evident in P females and in F1 males and females at 1000 mg/kg/day. No other evidence of adverse, test article-related effects was noted in either parental generation. Furthermore, no test article-related effects on reproductive performance were noted for either parental generation.
No adverse, test article-related changes in growth or development of offspring were noted in either the F1 or F2 generations. Body weight was higher in F2 pups from all treatment groups when compared with controls, but although this was considered to be test article-related, the effect was not considered to be adverse or biologically significant.
Based on the results of this study, the No-Observed-Adverse-Effect Level (NOAEL) for parental toxicity was 300 mg/kg/day and for parental reproductive performance, the NOAEL was 1000 mg/kg/day. For offspring growth and development, the NOAEL was also 1000 mg/kg/day.