Registration Dossier

Administrative data

Endpoint:
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 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Crl:WI(Han) (outbred, SPF-quality), time-mated females arrived at Day 0 or 1 post coitum at the laboratory.
- Age at study initiation: Approximately 10-14 weeks.
- body weight at start of dosing: 178-271 g
- Housing:
Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material was supplied; paper was provided as environmental enrichment and nesting material
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
The actual daily mean temperature ranged from 21 to 22°C, and the relative humidity from 45 to 55%. At least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle were maintained.

IN-LIFE DATES
From: November - December 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 16, 50, 150 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations and homogeneity of test substance in water were controlled analytically. A validated method using UPLC with UV-detection and a C18 column was used.
The concentrations analysed in groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).
The concentrations were homogeneous (i.e. coefficient of variation < 10%).
Details on mating procedure:
Time-mated females were provided by the animal breeding facility.
Duration of treatment / exposure:
Dams were dosed from Day 6 to Day 20 post coitum.
Frequency of treatment:
The test substance was administered once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: results from OECD 422 study
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: dams were weighed on Days 2, 6, 9, 12, 15, 18, and 21 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post coitum

GROSS NECROPSY
- Gross necropsy consisted of external, thoracic, and abdominal examinations.

HISTOPATHOLOGY
- thyroid glands of all animals of the control and high dose groups were examined histopathologically; as no changes were observed the thyroid glands of low dose and mid dose animals were not examined

CLINICAL BIOCHEMISTRY
- Thyroid hormone parameters were determined at the end of dosing in all dams; triiodothyronine (T3) and thyroxine (T4) as well as thyroid stimulating hormone (TSH)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- number and distribution of live and dead fetuses
- number and distribution of embryo-fetal deaths
- the sex of each fetus based on ano-genital distance
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.


Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, piloerection was noted for 15/22 dams, generally during the second week of treatment. Piloerection was also observed for one female at 80 mg/kg/day and for two females at 250 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight remained in the same range as controls up to 750 mg/kg/day over the treatment period.
Body weights before and after correction for gravid uterus weight at 80 and 250 mg/kg/day were considered not affected by the test item.
At 750 mg/kg/day, mean body weight gain corrected for gravid uterus was statistically significantly lower (49%) compared with concurrent control mean (6.3% weight gain vs 11.7% in the control group). The mean at 750 mg/kg/day was below the range considered normal for rats of this age and strain. This was essentially due to corrected body weight of four animals at 750 mg/kg/day with weight loss ranging from 0.5 to 19%, compared with Day 6 post-coitum.
(see Tables 1.6 and 2.5 in attached background material)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 250 and 750 mg/kg/day, food consumption was 20 and 25% lower than the control mean respectively over post-coitum Days 6-9, achieving a level of statistical significance. Mean food intake at both dose levels was slightly lower than control means on several occasions during treatment, achieving a level of statistical significance. Food intake at 250 and 750 mg/kg/day remained 9% and 13% lower than controls respectively at the end of treatment, and mean of mean values were 14% lower than the control mean at the end of the treatment period. During the last week of the treatment period at 750 mg/kg/day, a notable reduction in food intake was recorded for several animals, where food intake between post-coitum Days 18-21 was notably lower than over post-coitum Days 15-18. At 250 mg/kg/day, only a single animal showed a comparable reduction in food intake.
(see Table 1.8 in attached background material)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone parameters were determined at the end of dosing in all dams; triiodothyronine (T3) and thyroxine (T4) as well as thyroid stimulating hormone (TSH)

At 250 mg/kg/day, mean Total T3 and T4 was reduced (18% and 16%, respectively) compared to concurrent controls. At 750 mg/kg/day, mean Total T3 and mean Total T4 levels were decreased (16% and 28%, respectively) compared to concurrent controls. Besides that, mean TSH was increased (75%) at 750 mg/kg/day. These thyroid hormone changes were not accompanied by test item-related changes in thyroid weights or thyroid gland histopathology. At 80 mg/kg/day, mean TSH, and total T3 and T4 remained similar to the control mean. These changes in TSH, and Total T3 and T4 were considered to be test item-related. However, possible adversity of these effects could not be assessed within this type of study and was therefore not taken into account when determining the maternal NOAEL.
(see Table 1.10 in attached background material)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Only thyroid weights were determined in dams; no effects were observed.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No effects were observed in thyroid, the only tissue examined microscopically.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
one high dose female delivered on the day of necropsy. One high dose female was not pregnant (see Table 1.13 in attached background material).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
(see Table 1.14 in attached background material).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 250 - < 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, mean male, female and combined fetal weights were statistically significantly lower compared with concurrent controls (6% lower for combined weights). Mean combined fetal body weights were 5.3, 5.2, 5.2 and 5.0 gram for the control, 80, 250 and 750 mg/kg/day groups, respectively. Mean body weight of female fetuses remained within the historical control range, while mean body weight of male fetuses was marginally below this range. At 80 and 250 mg/kg/day, fetal body weights were considered not affected by the test item.
Litters with the lowest mean body weights were in general from litters of dams with the lowest weight gain or even weight loss. Accordingly, the lower fetal body weights may have occurred secondarily to the lower maternal body weights. Overall, these fetal body weight changes were of a slight magnitude and occurred in the absence of associated fetal morphological abnormalities.
(see Table 1.15 in attached background material).
The test substance has strong antimicrobial activity leading to disruption of the gut microbiome of orally dosed rats. Subsequently, animals of high dose groups suffered from disruption of nutritional homoeostasis and imbalanced nutrition leading secondarily to adverse effects on reproduction and developmental parameters (see the review and evaluation of effects observed in reproduction toxicity studies attached to the endpoint summary).
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:
not examined
External malformations:
no effects observed
Description (incidence and severity):
(see Table 1.16 and 1.18 in attached background material)
Skeletal malformations:
no effects observed
Description (incidence and severity):
(see Table 1.16 and 1.18 in attached background material)
Visceral malformations:
no effects observed
Description (incidence and severity):
(see Table 1.16 and 1.18 in attached background material)
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects were observed at the highest dose level

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study maternal toxicity was observed at the highest dose level. No adverse effects were noted in fetuses in any of the developmental parameters up to and including the highest dose level.