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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Dec 2012 - 29 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Wistar Hannover RccHan
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Models, S.L., Barcelona, Spain
- Age at study initiation: 11-12 weeks
- Mean weight at study initiation: males: 312 to 364 g; females: 198 to 219 g
- Fasting period before study: no
- Housing: cages with standard, granulated, softwood Lignocel S8/15 bedding
Pretreatment period: 5 per cage
Mating period: 1 male and 1 female per cage
Postmating: single housing
- Diet: pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet; ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose+1% Tween 80 in aqueous solution
Details on exposure:
VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 18 h
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 postcoitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment by GC-FID analysis.
Duration of treatment / exposure:
Males: two weeks prior to mating and at least up to and including the day before sacrifice (day 49 of treatment).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).
Frequency of treatment:
Once daily; 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S40412 14-day Oral (gavage) DRF using dose levels of 100, 500 and 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 hours after dosing
- Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: twice weekly during the pre-pairing and pairing period and daily during postpairing period.
F0 females: twice weekly during the pre-pairing and pairing period and daily until day 4-5 postpartum.

FOOD CONSUMPTION: Yes
F0 males: once weekly during the pre-pairing period and two weeks of postpairing period.
F0 females: once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 postcoitum and days 1-4 postpartum.
No food consumption was recorded during the mating period.

WATER CONSUMPTION: Yes
F0 males: one week during the pre-pairing and postpairing period.
F0 females: one week during the pre-pairing, postcoitum period and days 1-4 postpartum.
No water consumption was recorded during the remaining periods.:
Oestrous cyclicity (parental animals):
Smearing of individual females was evaluated during pairing period and was discontinued when sperm are found.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control
and high-dose groups
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 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):
GROSS PATHOLOGY: Yes (males on day 50, females on day 5 postpartum)
- Organ weights: epididymes and testicles, adrenal gland, brain, heart, kidney, liver, ovaries, pituitary, spleen, thymus, thyroid and parathyroids, uterus and oviducts
- Fixation: Adrenals, Aorta (thoracic), Bone with bone marrow- (Sternum), Peyer’s patches, Pharynx, Pituitary, Bone marrow smear (femur) (air-dried smear), Brain (cerebrum, cerebellum, medulla/pons), Epididymides (Bouin's solution), Esophagus, Eyes with optic nerve (Davidson’s fixative), Femur (with articular surface), Heart (with papillary muscle), Intestine, large (cecum, colon and rectum), Intestine, small (duodenum, jejunum and
ileum), Kidneys, Larynx, Liver, Lungs, with main bronchi and bronchioles, Lymph nodes (mandibular and mesenteric), Nose (the entire head will be collected), Mammary gland area, Ovaries, Pancreas, Prostate, coagulating gland and seminal vesicles, Salivary glands (mandibular, sublingual and parotid), Sciatic nerve, Skeletal muscle, Skin (abdominal), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach (glandular and nonglandular), Testes (Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder, Uterus (cervix, corpus and oviducts), Vagina, All gross lesions
Postmortem examinations (offspring):
On day 4 postpartum, all live pups were sacrificed by an intraperitoneal injection of sodium pentobarbital and examined macroscopically.
Any F1 offspring that died during the study was examined externally and necropsied, except those excessively cannibalized. Samples of the organs and tissues with macroscopic alterations were taken and preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.
Statistics:
The following statistical methods were used to analyse food consumption during postpairing period, body weight, clinical laboratory data, organ weights, postnatal development and reproduction data, as well as macroscopic findings:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
Fisher's exact-test was applied to the macroscopic findings.
Armitage/Cochran Trend Test [5] for non-neoplastic lesions, if appropriate.
Bonferroni-test was applied to some reproduction parameters.
Reproductive indices:
- Percentage Mating [%] = Number of females mated/Number of females paired x 100
- Fertility Index female [%] = Females achieving pregnancy/Number of females paired x 100
- Gestation Index [%] = Number of litters with live pups/Number of pregnant rats x 100
- Concenption rate [%] = Females achieving pregnancy/Number of females mated x 100
- Pre-implantation loss [%] = Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100
- Post-implantation loss [%] = Number of implantation sites - Total number of offspring born/Number of implantation sites x 100
Offspring viability indices:
- Post-natal loss [%] = Number of offspring/Number of offspring alive on day 4 x 100
- Viability Index [%] = Number of offspring alive on day 4/Number of offspring alive on day 1 x 100

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):
females: lower left ovary weight at 1000 mg/kg bw/day (non adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality and no relevant clinical signs were observed. One female from the control group, one female and three males at 100 mg/kg bw showed occasionally up of the product. In addition, one male at 300 mg/kg bw had missing upper incisors in week 4 of treatment. No clinical sings were observed at 1000 mg/kg bw in either males or females.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test-item-related differences from the control group were recorded for body weight. Some significant differences were observed in group 3 but these were not considered of toxicological relevance.
Food consumption was similar in all groups.

REPRODUCTIVE FUNCTION:

SPERM MEASURES (PARENTAL ANIMALS)
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There were only single cases with isolated tubules that showed minor changes. They consisted of single sperm resorption in stage VII. In epididymides, there was only one animal in group 1 with unilateral monounclear cell foci of a minimal serverity.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating Performance: Median precoital time and mean precoital time (approx. 3 days) were similar in animals treated with the test item at 100 and 1000 mg/kg bw with respect to the control group. However, mean and median precoital time was longer at 300 mg/kg bw (5.7 and 4 days, respectively). In addition, female no. 68 from this group did not mate with the first male (no. 28) after a pairing period of 14 days and was mated a second time with male no. 23. This female showed an anestrus cycle of 11 days. Moreover, some animals from all groups took more than 2-4 days to mate. Females nos. 43 (control group), 53 (100 mg/kg), 61 and 70 (300 mg/kg) and 73 (1000 mg/kg) took 12, 12, 13, 14 and 13 days, respectively, to mate and showed an anestrus cycle.

Fertility: No treatment-related differences were recorded in the percentage of mating or the gestation index in females at the three doses with respect to the control group. Although fertility index and conception rate was lower in test-item-treated groups (100% vs. 90%), it was considered to be within the range of normal background findings which may be seen in rats.
Females no. 53 at 100 mg/kg bw, no. 70 at 300 mg/kg bw and no. 77 at 1000 mg/kg bw, which mated with males nos. 13, 30 and 37, respectively, were not pregnant despite the presence of sperm in the vaginal smear and vaginal plug in the last two females. Duration of the mating period of those couples was 12, 14 and 4 days, respectively. Females no. 69 at 300 mg/kg bw and no. 74 at 1000 mg/kg bw, which mated with males nos. 29 and 34, did not show signs of pregnancy and were mated again to ensure eight pregnant females per group. These animals were identified as 81 and 82 to record the data and their second mating led to pregnancy. To check the fertility of the male whose first pairing did not result in mating, male no. 28 from the 300-mg/kg bw group was mated a second time with one female from a reserve group and mating led to pregnancy.

Reproduction data:
No treatment-related differences from the control group were recorded in the mean of implantation sites per litter or corpora lutea.
No treatment-related differences from the control group were recorded in the percentage of pre- or postimplantation losses. Female no. 45 from control group showed 100% implantation site loss. No differences in sex ratio were recorded.

Breeding data:
The length of pregnancy was similar in all groups with a mean of 21-22 days.
Female no. 78 at 1000 mg/kg bw had not enough milk and did not nurse correctly its pups; consequently, the pups died or were devoured between days 2 and 3 of lactation. Despite the fact that the pups had milk in the stomach when alive, they did not have any at necropsy and showed hypothermia and pallor. Female no. 78 showed slightly enlarged mammary glands. Even the percentage of pups showing milk in the stomach was similar in all groups. One pup in the litter of females no. 43 from control group, no. 63 at 300 mg/kg bw, no. 78 and two pups from no. 73 at 1000 mg/kg bw showed no milk in stomach on day 1 postpartum and died.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 1000 mg/kg bw, kidney weights tended to be slightly higher in males. These differences were not statistically significant. Moreover, lower pituitary and adrenal weights were recorded; the differences were statistically significant for pituitary in males. A trend to higher liver weight was recorded in females at 1000 mg/kg bw, but the differences were not statistically significant.
Concerning reproductive organs, lower left ovary weights were recorded at 1000 mg/kg bw. This difference was statistically significant in relation to body weight ratio.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At 1000 mg/kg bw, large right kidney was observed in one male and reddish thymus in another one. A reddish area on cecum wall was observed in one male and reddish foci in lungs in another one. Likewise, thickened gastric mucosa was observed in one female and small spleen in another one.
At 300 mg/kg bw, reddish foci were observed in the thymus of one male. No findings were observed in females.
At 100 mg/kg bw, pale kidneys were observed in one male and reddish foci in lungs in another one. Likewise, left seminal vesicle reduced in size was observed in one male and reddish thymus in three males. Thickened gastric mucosa was observed in one female.
In the control group, one male had left seminal vesicle reduced in size. One male and one female had pale kidneys. In addition, one male had pancreas reduced in size and thymus with reddish foci.
Yellowish-whitish gastric mucosa was recorded in few females from all groups (including control group).

HISTOPATHOLOGY (PARENTAL ANIMALS)
All microscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
No treatment-related differences from the control group were recorded in the mean of dead pups at the first litter check and living pups. During the first 4 days postpartum, the percentage and mean of postnatal losses was slightly higher at 1000 mg/kg bw; however the number of litters affected was similar to that of the control group (2 vs 1 litters) due to the fact that female no. 78 (1000 mg/kg bw) lost all its litter. The resulting viability index was lower than in the control group (84.7% versus 99.1%, respectively).
No differences were recorded at 100 and 300 mg/kg bw.


BODY WEIGHT (OFFSPRING)
No differences were recorded between groups and sexes. At 1000 mg/kg bw, one runt pup *no.7 from litter no.54) was observed.

GROSS PATHOLOGY (OFFSPRING)
No noteworthy findings were recorded in the control, 100, 300 or 1000 mg/kg bw pups.

MORPHOLOGICAL EXAMINATION
No treatment-related alterations were recorded in the morphological examination of the pups.
One pup from control group had a hematoma on toes and one pup from 300 and 1000 mg/kg bw group had a wound on scapula or limbs.

OTHER FINDINGS (OFFSPRING)
Motor development: Regarding postural reflexes in the pups, a significantly lower percentage of fetuses with positive response in the surface-righting reflex (righting reflex) at 1000 mg/kg bw was recorded compared to the control group. There were no treatment-related differences compared to the control animals at 100 or 300 mg/kg bw.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion