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Developmental toxicity / teratogenicity

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developmental toxicity
combined repeated dose and reproduction / developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. certificate)
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:

Test material

Test material form:

Test animals

Details on test animals and environmental conditions:
- Source:
Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 14 - 15 weeks old
- Weight at study initiation: Males: 343 - 389 g; Females: 203 - 240 g
- Fasting period before study: no
- Housing: Housed in groups of 5 animals/sex/cage during the premating period for both males and females and during post-mating period for males depending on the mating status. Housed in ratio 1:1 (male to female) during the mating period. After the confirmation of mating, females were individually housed during gestation/lactation period and males were returned to their original cage. Animals of the recovery group were housed in groups of 3 animals/sex/cage.
- Diet: Altromin 1324 maintenance diet provided ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8 provided ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air.

- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
- Amount of vehicle: 4 mL/kg body weight
- Lot/batch no.: MKCD1021
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%.
Details on mating procedure:
- M/F ratio per cage: 1:1 ratio
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was individually caged during gestation/lactation period in type III H, polysulphone cages.
Duration of treatment / exposure:
28 days in males and up to 63 days in females
Frequency of treatment:
The test item was administered daily.
Duration of test:
28 days in males and up to 63 days in females
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
360 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
The 4 groups consisted of 10 male and 10 female rats.
Two recovery groups (control and high dose group) consisted of 12 male and 12 female rats.
Control animals:
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a 14-day oral dose range finding study, in which doses of 1000 mg/kg bw/day were found to be in the lethal toxic range, while at 100 and 300 mg/kg bw/day no signs of toxicity were noted.
- Rationale for animal assignment: Randomisation was performed with validated IDBS Workbook 10.1.2 software.


Maternal examinations:
- Time schedule: At least once a day, preferably at the same time each day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded. Prolonged parturition of pregnant females and maternal behaviour during lactation period were also monitored.

- Time schedule: Once before the first exposure, and at least once a week thereafter.

- Time schedule for examinations: Once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OTHER: Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as supported rearing and non- supported rearing, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded. In the case of recovery groups, urinalysis was performed for all males and females at the end of the recovery period.

Haematology and clinical chemistry parameters were also evaluated.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded.
A statistical assessment of the results of behavioural parameters, body temperature, body weight, food consumption, clinical pathology parameters, organ weights and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test.
Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100
Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At the high and mid dose, frequently observed clinical sings included moving the bedding, salivation, piloerection and alopecia. In the low-dose group females, few incidences of moving the bedding, salivation, piloerection and alopecia. Salivation and moving the bedding were mainly observed immediately after dose administration and, therefore, were considered to be a sign of discomfort due to a local reaction to the test item or adverse taste.
Dermal irritation (if dermal study):
not examined
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In females, no statistically significant effect on body weight was observed during the premating and mating period. At the end of the gestation and start of the lactation period, however, body weights were statistically significantly lower in high-dose dams when compared to the controls (between 6 and 11% below controls). Statistically significantly lower body weight gain was observed in those animals on gestation day 14-20. During the recovery period, a slight but statistically significantly higher body weight was seen in high-dose females when compared to the recovery control group (approx. 6% higher). A tendency towards higher body weights, however, was seen in these animals already during the treatment period showing a statistical significance on day 42.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly lower food consumption was also found in high-dose females, i.e. between gestation days 7 and 20 (approx. 16% below controls).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A slight decrease or increase in the mean % neutrophils in mid-dose females at the end of the treatm ent period and % reticulocytes in high-dose males at the end of the recovery period, were not consi dered biologically relevant as these values were within the historical range of this strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In females of the high-dose group slightly lower alanine aminotransferase level and slightly higher potassium level are not considered toxicologically relevant as values were in the range of historical control data.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period statistically significantly higher relative kidneys weights in high-dose females were observed when compared to the control group (22% above controls). In the presence of histopathological findings in these organs the effect on organ weights at the end of treatment period was considered to be adverse. No considerable difference in kidney weight was observed at the end of the recovery period.

Females sacrificed at the end of treatment period, showed statistically significantly lower absolute and relative thymus weights in high dose (47% and 42% in females below controls, respectively). This was associated with thymic atrophy found histopathologically in this dose group.

A tendency towards increased liver weight (approx. 10% above controls) was observed in high- and mid-dose females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In females, thickened wall of urinary bladder was observed in the high-dose group (6/10).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A backflow nephrosis was recorded in four females at 360 mg/kg bw/day. In the kidneys, an increased incidence of minimal tubular basophilia also was observed in females at 360 mg/kg/day. This finding was associated with inflammatory lesions, i.e., pyelitis and interstitial inflammation, interstitial fibrosis and papillary necrosis. The papillary necrosis appeared focally at the urothelium. Urothelial hyperplasia also was observed in four females at 360 mg/kg bw/day.
The ureters from three females at 360 mg/kg bw/day were dilated. This finding was associated in some cases with mucosal and/or muscularis hyperplasia, and/or inflammation. Mucosal hyperplasia was occasionally observed, but the highest severity degrees were noted in ureter segments adjacent to the vagina.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Maternal abnormalities

Key result
effects observed, treatment-related
other: urinary system

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: There were no effects on litter data, litter weight data, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in pups sacrificed on PND 13.

Fetal abnormalities

Key result
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:

Applicant's summary and conclusion

A well reported oral combined repeated dose/reproductive and developmental screening study, conducted according to the current guideline and in accordance with GLP, reported a NOAEL for developmental effects at the highest tested dose of 360 mg/kg bw/day. General systemic maternal effects were reported at the high dose of 360 mg/kg bw/day.