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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Nov 2012 - 25 Oct 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 422 and GLP. During the study period the automatic light cycle of 12 h on/12 h off was not functional. Therefore, all animals were subjected to 24 h light.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 Mar 1996
Deviations:
yes
Remarks:
animals were subjected to 24 h light
GLP compliance:
yes (incl. certificate)
Remarks:
Agence française de sécurité sanitaire des produits de santé, France
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: RccHan : WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 weeks
- Weight at study initiation: 311-368 g (males), 209-262 g (females)
- Housing: in groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops, during the pairing period, females were housed with sexually mature males (1:1), sterilized standard softwood was used as bedding with paper enrichment
- Diet: pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: community tap-water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 0/24

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test substance as supplied and weekly if stability of the test substance for 8 days was confirmed. Separate formulations were prepared for each concentration. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 2, 8 and 40 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: 332190038, 382191749 and 492194511
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: presence of sperm in the daily vaginal smear or presence of a copulation plug
- After 14 days of unsuccessful pairing replacement of first male by another male from the same group with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 1 g of each concentration were taken from the middle of each aliquot used on Day 7 of the treatment. On two further time points, samples were taken from the middle to confirm concentration. The samples were analyzed by GC/FID following an analytical procedure. The test substance was used as the analytical standard.
The test substance concentrations in the dose formulations ranged from 81.4% to 115.2% with reference to the nominal and were within the accepted range of ±20%. The homogeneous distribution of the test substance in the preparations was approved because single results found did not deviate more than 4.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.
In addition, the test substance was found to be stable in application formulations when kept 8 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.
Duration of treatment / exposure:
males: for at least 28 days, starting 2 weeks before mating and a maximum of 14 days during mating
females: for 2 weeks prior to mating, throughout gestation until the F1 generation reached Day 3 post partum (one day before necropsy)
Frequency of treatment:
once daily, 7 days/week
Details on study schedule:
not applicable for an OECD 422 study
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 40 and 200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main groups: initially 11
Additional: 6 at 0 and 40 mg/kg bw/day; 8 at 10 mg/kg bw/day

Because of the low percentage of mated females, additional animals were assigned to the control, low and mid dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats using dose levels of 0, 50, 150 and 500 mg/kg bw/day. The study was performed in the same laboratory and by the same study director in 2012. Four groups of 5 males and 5 females were treated by gavage with the test substance once daily over a 14-day period. At 500 mg/kg bw/day, two females were found dead on Day 4 and one male and one female were found dead on Day 5. The remaining males and females at this dose level were sacrificed on Day 5 of the treatment period for ethical reasons. At 50 and 150 mg/kg bw/day, all males and females survived until the scheduled necropsy. At 500 mg/kg bw/day, mean food consumption and body weight gain were statistically significantly reduced in the males and females up to necropsy. At 150 mg/kg bw/day, mean food consumption in males was slightly but not statistically significantly reduced during the treatment period. No test substance-related macroscopical findings were observed in the males and females at any dose level. Based on these observations, dose levels of 10, 40 and 200 mg/kg bw/day were considered to be suitable for the main study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test substance and weekly thereafter. In females, it was performed once prior to the first administration of the test substance, weekly during the pre-pairing and pairing periods and on Days 0, 6, 13 and 20 of the gestation period. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Food consumptions of males were recorded on pre-pairing period days 1-4, 4-8, 8-11 and 11-13 and after the pairing period weekly. Food consumptions of females were recorded on pre-pairing period days 1-4, 4-8, 8-11 and 11-13, on gestation days 0-7, 7-14 and 14-21 and on days 1-4 of the lactation. Food consumption was not recorded during the pairing period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: Haematology, clinical chemistry, neurobehavioural examination (for details refer to IUCLID section 7.5.1)
Sperm parameters (parental animals):
qualitative examination of the stages of spermatogenesis in the testis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: litter size, live births, still births and any gross anomalies, sex ratio, body weights on Day 0, 1 and 4 post partum
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All males were sacrificed after treatment for at least 28 days, when no longer needed for the assessment of reproductive effects. The additional males were terminated following mating of the respective females.
- Maternal animals: All dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (Day 21 post coitum), the dam was sacrificed on Day 25 post coitum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the scheduled sacrifice, the following organs were weighted (*) and preserved in neutral phosphate buffered 4% formaldehyde solution: epididymides* (fixed in Bouin's solution), ovaries, prostate gland and seminal vesicles incl. coagulating glands, testes* (fixed in Bouin's solution), adrenal glands*, bone marrow (femur), brain - including section of medulla/pons*, cerebral and cerebellar cortex, cecum, colon, duodenum, heart including auricles*, ileum (with Peyer's patches), jejunum (with Peyer's patches), kidneys*, liver*, lungs (preserved by inflation with fixative and then immersion), lymph nodes – mesenteric and mandibular, rectum, sciatic nerve, spinal cord - cervical, midthoracic, lumbar, spleen*, stomach, thymus*, thyroid (incl. parathyroid gland, if possible), trachea, urinary bladder (preserved by inflation with fixative and then immersion), uterus (incl. oviducts, cervix and vagina), all gross lesions

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 µm and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Reproductive indices:
Percentage mating (%) = (females mated / females paired) x 100
Fertility index (%) = (females achieving a pregnancy / females paired) x 100
Conception rate (%) = (females achieving a pregnancy / females mated) x 100
Gestation index (%) = (number of females with living pups / females pregnant) x 100
Offspring viability indices:
Birth index (%) = (pups born alive / number of implantations) x 100
Viability index (%) = (pups alive before culling on Day 4 post partum / pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
increased incidence and severity of hyaline droplets in the renal tubular epithelium in males at 200 mg/kg bw/day, increased incidence of decreased lymphocytes in the thymus in females at 200 mg/kg bw/day
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
increased pre-coital time in females at 200 mg/kg bw/day

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One female at 200 mg/kg bw/day was found dead on Day 3 of the lactation period. This female had ruffled fur, a hunched posture and decreased activity. At macroscopical examination, the thymus and mandibular lymph nodes in this female were reduced in size and there was red discoloration in the lungs. Microscopical examination revealed the cause of death to be attributed to bacterial contamination of the pregnant uterus/vagina with secondary systemic changes induced by thrombo-embolic bacterial dissemination and associated poor health status, unrelated to the test substance. All other males and females survived until the scheduled necropsy.

Findings at daily clinical observation:
On the first day of treatment reduced activity was observed in 5 females and 1 male at 200 mg/kg bw/day. Two females and one male had ptosis in both eyes. In the lactation period, one female had ruffled fur at 200 mg/kg bw/day. No clinical signs were observed at 40 mg/kg bw/day. At 10 mg/kg bw/day, one male had a wound on the tail apex in the pre-pairing and pairing period. No further clinical signs were observed.

Findings at detailed weekly clinical observations:
At all dose levels, there were incidences in males and females of delayed pupil reflex/absent iridic reflex, especially in the acclimatization period. This was considered to be a result of the lighting problem, to which the animals acclimatized. Incidences of salivation were observed in males and females at 200 mg/kg bw/day. Isolated incidences of swaying gait, decreased activity, increased activity, ruffled fur and Straub phenomenom were observed, which were considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
Males:
At 200 mg/kg bw/day, mean body weight gain was statistically significantly reduced from Day 2 until the end of the pre-pairing period. Over the whole pre-pairing period, the difference in mean body weight gain was 4% compared to 8% in the control group. Mean body weight was statistically significantly reduced from Day 7 of the pre-pairing period and remained so until the end of the study. The difference to the control group was 5% at 200 mg/kg bw/day at the end of the study. Mean body weight and body weight gain were not affected by treatment with the test substance at 10 and 40 mg/kg bw/day.
Females:
During the pre-pairing period mean body weight and body weight gain were statistically significantly reduced on Day 9 at 200 mg/kg bw/day. However, the difference to the control group was only slight (2%). During the gestation period, mean body weight and body weight gain were reduced without statistical significance (+44% compared to +53% in the control group). During the lactation period, although body weight gain was increased compared to the control group, mean body weight remained reduced. Mean body weight and body weight gain were not affected by treatment with the test substance at 10 and 40 mg/kg bw/day.

FOOD CONSUMPTION
Males:
At 200 mg/kg bw/day, mean food consumption was statistically significantly reduced during the pre-pairing period up to Days 8 - 11. Over the whole pre-pairing period, mean food consumption was -11.3% compared to the control group. At 10 and 40 mg/kg bw/day, mean food consumption was not affected by treatment with the test substance in the pre-pairing or after pairing periods.
Females:
At 200 mg/kg bw/day, mean food consumption was statistically significantly reduced up to Days 8 - 11 of the pre-pairing period (-14.5% over the whole of the pre-pairing period). It remained reduced during the gestation period and was statistically significantly reduced over the last 2 weeks. Over the whole of the gestation period, mean food consumption was -11.7% compared to the control group. During the lactation period, mean food consumption was similar to the control group. At 40 mg/kg bw/day, mean food consumption was similar to the control group during the pre-pairing period. During the gestation period, it was reduced without statistical significance over the last week (-9.4%). In the lactation period, it was increased compared to the control group. At 10 mg/kg bw/day, mean food consumption was not affected by treatment with the test substance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Of the original females, 3 were not pregnant in the control group, 7 at 10 mg/kg bw/day, 3 at 40 mg/kg bw/day and 2 at 200 mg/kg bw/day. Further animals were added to the study to make up for the low numbers. Of these females, 4 at 10 mg/kg bw/day and 3 at 40 mg/kg bw/day were not pregnant. The unexpectedly low fertility was considered to have been directly influenced by a disturbance of the light/dark cycle, which resulted in the animals being subjected to 24 h light. This is known to have an impact on the estrous cycle. Since there was no dose-dependency, a test substance-related effect could be excluded.
At 200 mg/kg bw/day, the mean pre-coital time was increased (5.6 days compared to 2.7 days in the control group). This was outside the range of the historical control data.
At 40 mg/kg bw/day, pre-coital time was also dose-dependently (but not statistically significantly) increased in the first pairing period, also when the second pairing period was included. The increase in pre-coital time might also be related to the disturbance of the light/dark cycle.

No test substance-related effects on the corpora lutea were observed.

No test substance-related effects were observed on the duration of gestation. The duration was similar at all dose levels.

The mean number of implantations per dam was not affected by treatment with the test substance. Post-implantation loss was generally high at all dose levels. However, at 200 mg/kg bw/day mean post-implantation loss was increased compared to the control group (mean 5.7 per litter compared to 2.5 in the control group). This was partly due to one female (16 losses), which died during the lactation period due to a bacterial infection. Therefore, this increase was not considered to be test substance-related. Post-implantation loss was similar to the control group at 10 and 40 mg/kg bw/day.

ORGAN WEIGHTS
At 200 mg/kg bw/day, in the males the absolute liver weight and body weight ratio were statistically significantly increased (12 and 20%, respectively). In addition, in the females the absolute weight, body weight ratios and brain weight ratios of the liver (22, 28 and 21%, respectively) and kidneys (22, 26 and 20%, respectively) were statistically significantly increased. However, no microscopical findings were found that correlated to changes in the liver and kidneys. In the females at 200 mg/kg bw/day, the absolute weight, body weight ratios and brain weight ratios of the thymus were statistically significantly decreased (45, 43 and 46%, respectively). This correlated with the reduced lymphocytes in the thymus, observed at microscopical examination. No test substance-related findings were observed in males and females at 10 and 40 mg/kg bw/day.

GROSS PATHOLOGY
No test substance-related findings were observed at any dose level in males. The thymus in one female was reduced in size at 200 mg/kg bw/day. This correlated with histopathological findings. No other test substance-related findings were observed at any dose level.

HISTOPATHOLOGY
In the males, there were no test substance-related findings in any examined reproductive organs. In particular, the qualitative examination of the stages of spermatogenesis in the testis did not reveal any test substance-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. The only potentially test substance-related finding was an increased incidence and severity of hyaline droplets in the renal tubular epithelium at 200 mg/kg bw/day. In the females, there were no test substance-related findings in the ovaries of non-pregnant females. In the pregnant females, there were no test substance-related microscopic findings in the reproductive organs, including following evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. The only potentially test substance-related finding was an increased incidence of decreased lymphocytes in the thymus at 200 mg/kg bw/day. This was the histological correlate of the decreased thymus weight at 200 mg/kg bw/day when compared to control group. According to the authors, this was considered to be induced by the stress of pregnancy rather than a test substance-related finding.

OTHER:
For results on heamatology, clinical chemistry, neurobehavioural examination refer to IUCLID section 7.5.1.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 200 mg/kg bw/day: reduced food consumption and increased liver weights (m, f)
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased pre-coital time was not considered to be adverse.

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
only external examinations
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING) / EARLY POSTNATAL DEVELOPMENT
The litter size at the first litter check did not reveal any dose-dependent effects. Five dead pups were found in one litter in the control group and one dead pup at 40 mg/kg bw/day. Two female at 200 mg/kg bw/day were seen giving birth, however, at the first litter check, no pups were found.
One pup was lost in each of groups at 0 and 10 mg/kg bw/day, and 2 pups were lost at 200 mg/kg bw/day. These losses were considered to be incidental
No findings were observed in the pups at any dose level.
The sex ratios at the first litter check were close to 50% at all dose levels.

The birth index was statistically significant reduced at 200 mg/kg bw/day (59.5% compared to 77.6% in the control group). This was due to the higher post-implantation loss at this dose level, which was not considered to be a test substance-related effect.

BODY WEIGHT (OFFSPRING)
Although the mean pups weights at 40 and 200 mg/kg bw/day were slightly lower than in the control group, all weights were within the range of the historical control data. No test substance-related effect was observed in the increase in weight over days 1 - 4 of the lactation period.

GROSS PATHOLOGY (OFFSPRING)
No test substance-related macroscopical findings were observed in the pups at any dose level.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
development/teratogenicity
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was set at 40 mg/kg bw/day due to effects on food consumption and increased liver weights at 200 mg/kg bw/day. No adverse effects on development of offspring were observed up to and including 200 mg/kg bw/day, the highest dose tested.