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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
liquid
Details on test material:
homogenous, slightly yellowish liquid
storage at room temperature
batch 0017160490

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI[Han]
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: average 165 g (range 141.3 – 191.5 g) (gestation day 0), average 200g (GD6)
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-02-28 To: 2018-03-22

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations
were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer
until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight
Concentration of test item: 0, 2, 6, 10 g/100ml (taking into account a solid content (active ingredient) of the test item of 50.1% in aqueous solution.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature.
The homogeneous distribution of the test substance in the vehicle (drinking water) was confirmed.
The correctness of the prepared concentrations was shown.

Given that the test substance was completely miscible with deionized water, solutions were considered to be homogenous without further analysis.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory.
Duration of treatment / exposure:
gestation days 6- 19
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of existing subacute data.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.


POST-MORTEM EXAMINATIONS: Gross pathology
- Sacrifice on gestation day: GD 20

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: Site of implantations in the uterus
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
Statistics:
DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses

FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test
Proportion of fetuses with findings per litter
Indices:
sex ratio
conception rate (in %)
preimplantation loss (in %)
postimplantation loss (in %)
Historical control data:
Historical control data is included in the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nearly all (23 out of 25) females of the high-dose group (2000 mg/kg bw/d) showed occasional salivation during the treatment period. Salivation occurred in the respective animals only
shortly, i.e. within 0-2h, after treatment and was observed during the whole administration period (GD 6-19). No clinical signs or changes of general behavior were detected in any female
of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic
toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high-dose dam (No. 90 - 2000 mg/kg bw/d) died beyond 2 hours after treatment on GD 9. Gross pathological examination revealed findings indicating a gavage error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean food consumption of the high-dose dams (2000 mg/kg bw/d) was statistically significantly reduced during GD 6-8 (7% below control) but recovered afterwards and was comparable
to the control values again throughout the remaining study period (GD 7-20). If calculated for the entire treatment period (GD 6-19), the mean food consumption of the high-dose dams
was comparable to the concurrent control group. Therefore, it was assessed as treatment related but not adverse.
The mean food consumption of the mid- and low-dose dams (600 and 200 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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):
The mean gravid uterus weights of the animals of test groups 1-3 (200, 600 and 2000 mg/kg bw/d) were not influenced by the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One finding was noted associated with an unscheduled maternal death in one individual female of the test group 3 (No. 90, died after gavage error on GD 9), i.e. thoracic cavity filled with bloody fluid.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The conception rate was 92% in the control group (0 mg/kg bw/d), 96% in the low-dose group (200 mg/kg bw/d) and 100% in the mid- and high-dose groups (600 and 2000 mg/kg bw/d).
With these rates, a sufficient number of pregnant females were available for the purpose of this study.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at the limit dose

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
One fetus in test group 3 (2000 mg/kg bw/d) had a skeletal malformation (specifically a malpositioned and bipartite sternebra). The isolated finding occurred in one single fetus. The mean value of affected fetuses/litter (0.7 %) was within the historical control range (HCD; affected fetuses/litter, range of 0.0 – 1.1 %). No ontogenetic pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus each of test groups 1 and 3 (200 and 2000 mg/kg bw/d) had a soft tissue malformation. These findings were not related to dose and single events in individual fetuses.
The mean values of affected fetuses per litter of both findings (situs inversus und abnormal lung lobation) were within the range of historical control data. Thus, they are not considered as treatment-related and adverse.
The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Other effects:
no effects observed
Description (incidence and severity):
Three soft tissue variations were detected, i.e. short innominate in the control group, dilated renal pelvis and dilated ureter in all treated and the control groups. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects at the limit dose.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1_ Total soft tissue malformations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 23
112
24
115
25
130
24
124
Fetal incidence N (%) 0.0 1 (0.9) 0.0 1 (0.8)
Litter incidence N (%) 0.0 1 (4.2) 0.0 1 (4.2)
Affected fetuses/litter Mean% 0.0 0.8 0.0 0.8
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 2: Total skeletal malformations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 23
125
24
128
25
142
24
134
Fetal incidence N (%) 0.0 0.0 0.0 1 (0.7)
Litter incidence N (%) 0.0 0.0 0.0 1 (4.2)
Affected fetuses/litter Mean% 0.0 0.0 0.0 0.7
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 3: Total soft tissue variations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 23
112
24
115
25
130
24
124
Fetal incidence N (%) 7 (6.3) 3 (2.6) 6 (4.6) 6 (4.8)
Litter incidence N (%) 7 (30) 3 (13) 5 (20) 3 (13)
Affected fetuses/litter Mean% 5.8 2.4 4.3 5.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 4: Total fetal skeletal variations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 23
125
24
128
25
142
24
134
Fetal incidence N (%) 123 (98) 125 (98) 137 (96) 132 (99)
Litter incidence N (%) 23 (100) 24 (100) 25 (100) 24 (100)
Affected fetuses/litter Mean% 98.4 96.2 96.6 98.6
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 5: Total fetal malformations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 22
237
24
243
25
272
24
258
Fetal incidence N (%) 0.0 1 (0.4) 0.0 2 (0.8)
Litter incidence N (%) 0.0 1 (4.2) 0.0 2 (8.3)
Affected fetuses/litter Mean% 0.0 0.4 0.0 0.8
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 6: Total fetal variations

    Test group 0
0 mg/kg bw/d
Test group 1
200 mg/kg bw/d
Test group 2
600 mg/kg bw/d
Test group 3
2000 mg/kg bw/d
Litter Fetuses N N 23
237
24
243
25
272
24
258
Fetal incidence N (%) 130 (55) 128 (53) 143 (53) 138 (53)
Litter incidence N (%) 23 (100) 24 (100) 25 (100) 24 (100)
Affected fetuses/litter Mean% 54.8 51.8 52.5 53.6
mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Applicant's summary and conclusion

Conclusions:
Oral dosing of rats with 0, 200, 600 or 2000 mg/kg bw did not cause developmental toxicity or teratogenicity (OECD 414, GLP).
Executive summary:

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 200, 600 or 2000 mg/kg bw/d test material and controls. Nearly all (23 out of 25) females of the high-dose group (2000 mg/kg bw/d) showed transient salivation during the treatment period. Salivation persisted in the respective animals only for some time after daily gavage dosing (maximum up to 2 hours) and was initially observed on GD 6. Transient salivation shortly after dosing most likely reflects a reaction of the animals to the taste and smell of the test substance. It is not considered to be a sign of systemic toxicity and was, therefore, not assessed as treatment-related and adverse.

No differences of toxicological relevance between the control and the treated groups (200, 600 or 2000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.