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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

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:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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: not reported
- 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

ENVIRONMENTAL CONDITIONS
- 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
Vehicle:
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.

VEHICLE
- 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
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.
Analytical verification of doses or concentrations:
yes
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%.
Duration of treatment / exposure:
28 days for males and up to 63 days for females
Frequency of treatment:
The test item was administered daily.
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:
yes
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.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- 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.

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

BODY WEIGHT: Yes
- 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:
- 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.
Oestrous cyclicity (parental animals):
Estrous cycles were monitored before the start of the treatment to select for the study females with regular cyclicity (using vaginal smears). In addition, vaginal smears were also examined daily from the beginning of the mating period until evidence of mating of main study females.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight, tubular stages of the spermatogenic cycle
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, runts, presence of gross anomalies, weight gain, any abnormal behaviour, anogenital distance (AGD), and presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed by using anesthesia (e.g. ketamine/xylazine), followed by exsanguination. The males of the recovery groups were subjected to necropsy 14 days after the last administration (end of recovery period).
- Maternal animals: All surviving animals were sacrificed on the respective PND 13 by using anesthesia (e.g. ketamine/xylazine), followed by exsanguination. Non-pregnant females were sacrificed on day 26 from the day of mating or from the last day of mating period. The females of the recovery groups were subjected to necropsy 14 days after the first scheduled necropsy of dams of any main group.

GROSS NECROPSY
- Gross necropsy consisted of careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved.

All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in tables below were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
-On PND 13 dead pups and all surviving pups.

GROSS NECROPSY
- Blood samples were collected from the defined site in serum separator tubes from 2 female pups/litter on day 4 after birth from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations.

- Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination, if required.
Statistics:
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.
Reproductive indices:
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

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, the number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 LD 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
Mortality:
mortality observed, treatment-related
Description (incidence):
One high-dose recovery male rat (no. 92) was euthanised on study day 15. Clinical signs included markedly reduced spontaneous activity, prone position, slight salivation, wasp waist, dehydration, moderate piloerection, hypothermia, abnormal breathing. At necropsy, both kidneys were found enlarged, one kidney was white and of viscous consistency and ureters were dilated. Histopathologic al examination results show that the cause of morbidity is assumed to be a consequence of backflow nephrosis and it is considered to be treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were statistically significantly lower in high-dose males throughout the study period when compared with the control group (up to 10 % below controls). In recovery males, no statistically significant changes were observed during the recovery period when compared to the respective control group.

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):
During the premating period food consumption of high-dose males was slightly lower when compared to controls (30% and 28% below controls in premating week 1 and 2, respectively), while food consumption of low- and mid-dose males was comparable to the controls.

Slightly lower food consumption was also found in high-dose females, i.e. between gestation days 7 and 20 (approx. 16% below controls).

Food consumption during the recovery period in males and females were found to be comparable to the 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 treatment period and % reticulocytes in high-dose males at the end of the recovery period, were not considered biologically relevant as these values were within the historical range of this strain.

Blood coagulation was not affected by the test item. Minimal but statistically significant differences in activated partial thromboplastin time in high-dose recovery males are not considered to be biologically relevant, as the individual values were within the normal range of variation for this strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In male animals a slight but statistically significant decrease in creatinine level in the mid group and in aspartate-aminotransferase level in the mid- and high-dose groups are not of biological relevance as they were slight decreases and not associated with pathological conditions. Slight statistically significant increases in albumin in the males of the low-dose group and total cholesterol in the males of the mid-dose group are not considered toxicologically relevant as values were in the historical control ranges and without dose-dependency.

At the end of the recovery period the slight but statistically significant increase in glucose in male animals of the high-dose group and the decrease in aspartate-aminotransferase are not considered toxicologically relevant as they were in the range of historical control data. 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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A backflow nephrosis was recorded in two males at 120 mg/kg bw/day, and in all males and four females at 360 mg/kg bw/day.

In the kidneys of males at 120 mg/kg bw/day, the incidence of minimal tubular basophilia exceeded the background incidence levels. At 360 mg/kg bw/day, both sexes were affected, whereby the mean severity increased in both sexes, and males were much more affected than females. This finding was associated with inflammatory lesions, i.e., pyelitis was noted in males at 120 mg/kg bw/day, and pyelitis and interstitial inflammation, interstitial fibrosis and papillary necrosis were noted in both sexes at 360 mg/kg bw/day. The papillary necrosis appeared focally at the urothelium. Furthermore, the incidence of pelvic dilatation increased in males with dose (all doses), and tubular dilatation was noted in almost all males at 360 mg/kg bw/day. The findings caused urothelial hyperplasia, in two males at 120 mg/kg bw/day, and in all males and four females at 360 mg/kg bw/day.

The ureters of one male at 120 mg/kg bw/day, and from all males and three females at 360 mg/kg bw/day were dilated. In animals at 360 mg/kg bw/day, the findings were 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 and prostate gland.

The findings in the urinary bladder were characterized mainly by diffuse urothelial hyperplasia in both sexes at 120 and 360 mg/kg bw/day. This finding was associated with an increased incidence and severity of mononuclear cell foci.

In one male, the urethra was found in its full length and diameter within the prostate tissue. The urothelium showed a moderate hyperplasia accompanied by a minimal subacute inflammation. At some locations, in mucosal folds, precipitation of an unknown material was seen.

After the recovery period, findings were still present but decreased in severity. All animals were affected. Pelvic dilatation was noted in the decedent male only. No tissue from the urethra could be evaluated from recovery animals.

Diffuse cortical hypertrophy was noted in females at 40 mg/kg bw/day, and in both sexes at 120 and 360 mg/kg bw/day. After the recovery period, there were no differences between controls and test item-treated animals.

Increased thymic atrophy was noted in females at 40 mg/kg bw/day, and in both sexes at 120 and 360 mg/kg bw/day. After the recovery period, there were no differences between controls and test item-treated animals.

There were no abnormalities in the male reproductive organs. In addition, during sperm staging of PAS stained testicular sections, there were no indicators for induced lesions.

There were no abnormalities in the female reproductive organs. Female no. 65 from the mid dose group was non-pregnant. The reproductive organs of this female and its mating partner, male no. 25, did not reveal any specific abnormality in reproductive organs.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly reduced copulation index of 90% in the high-dose group was observed compared to 100% in the control group. This is considered as biological variation. Fertility index was slightly lower in the high-dose group (80%), when compared to controls. As this included the non-mated female only a single high-dose females was affected. Moreover, as this value was within the range of historical control data, this is not considered to test item related.

Details on results (P0)

See background material.

Effect levels (P0)

open allclose all
Key result
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:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects on estrous cyclicity, precoital interval, gestation duration. No adverse effect were observed with regards to sperm. There were no specific lesions noted in the reproductive system organs from males and females.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test item related effect was observed on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in low- and mid-dose groups. However, a slightly higher mean mortality of pups on PND 1 was observed in the mid-dose group (3.7 %) and high-dose group (1.4 %). As this effect was only slight and not dose-dependent, it is not considered to be an adverse effect of the test item.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
In male pups, slightly but statistically significantly lower absolute and relative anogenital distance (AGD) was observed in the low-dose group when compared to the controls. At the time of measurement male pup weight was slightly but statistically significant lower in the high dose group when compared to controls (approx. 6% below controls). This is likely to be related to slightly lower body weight of the high-dose group dams at the end of the gestation period. In female pups, slightly but statistically significantly higher absolute and relative anogenital distance was observed in the low-dose group when compared with the controls. As no considerable difference was seen in the mid- and high-dose groups and values were within the historical control data, this is not assumed to be test item related.

No statistically significant effect or toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effect of toxicological relevance or statistical significance was observed on pup thyroid weight in males and PND 13 pups of the dose groups when compared to the controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Few pups were found dead/still born on PND 0. Few specific findings like absent tail tip were observed. The external findings like absent hair coat on back, black, absent tail tip were considered to be spontaneous and not related to test item treatment.

No test item-related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.

Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Serum thyroxine hormone (T4) levels were in the normal range of variation in all groups. No differences between the dose groups and control group were seen that can be considered biologically relevant. A minimal tendency towards lower values observable in the dose groups is related to control values that were in the upper range of historical control data.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

See background material.

Effect levels (F1)

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

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

Conclusions:
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 reproductive effects at the highest tested dose of 360 mg/kg bw/day. General systemic parental effects were reported at the mid dose of 120 mg/kg bw/day.