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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep 2013 - 28 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 11-13 weeks
- Weight at study initiation:
- Housing: Makrolon cages type M III (1 animal per cage)
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): yes
- Acclimation period: from GD 0 (day of supply) to the beginning of administration (GD 6), the animals were accustomed to the environmental conditions and to the diet.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % suspension in drinking water
Details on exposure:
Gavage exposure from gestation day (GD) 6 through GD 19.

PREPARATION OF DOSING SOLUTIONS:
10 ml/kg body weight were administered. Test substance preparations were suspended in 1 % Carboxymethylcellulose in drinking water.
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. The preparations were kept in a refrigerator.
For the test substance preparations, the specific amount of test substance were weighed, topped up with 1% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.
During administration the preparations were kept homogeneous with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations of the test substance preparations, and verification of stability of the test substance in 1 % Carboxymethylcellulose suspension in drinking water over a period of a maximum of 7 days prior to the start of the study (Project No.: 01Y0066/05Y009).
Details on mating procedure:
The animals were paired by the breeder (time-mated animals) and were supplied at noon on the day of evidence of mating. This day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
Gavage exposure from gestation day (GD) 6 through GD 19.
On GD 20, blood samples were obtained from dams by retrobulbar venous puncture.
Following blood sampling on GD 20, all dams were sacrificed and examined. Fetuses were removed from the opened uterus.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, for GD 0-1 , 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17,
17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes, on GD 20, following anesthetisia with isoflurane, sacrificion by decapitation.
Fetuses were be removed from the uterus.
- Organs examined: Adrenal glands, Kidneys, Liver, Spleen. Organ / Tissue fixation for all gross lesions and organs examined.

HEMATOLOGY /CLINICAL CHEMISTRY: Yes
Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
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
- Site of implantations in the uterus
Fetal examinations:
- Weight of each fetus
- Sex
- Weight of the placentas
- Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord);
- fetuses wer sacrificed by subcutaneous injection of pentobarbital, and about half of the fetuses of each dam were skinned, fixed in ethyl alcohol and, after fixation, stained according to a modified method (KIMMEL and TRAMMELL) to show the skeleton and the cartilage. The other half of the fetuses of each dam were fixed in Harrison's fluid.
- examination of skeletons/cartilage and soft tissues.
Statistics:
Food consumption, body weight, organ weights - DUNNETT's test.
Implantations, resorptions and live fetuses - DUNNETT's test.
Number of pregnant animals, mortality and number of litters with fetal findings - FISHER's exact test.
Proportion of fetuses with findings per litter - WILCOXON test.
Clinical pathology parameters - KRUSKAL-WALLIS and WILCOXON test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no test substance-related or spontaneous mortalities in any females of all test groups.
Seven (out of 25) high-dose females showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 20 minutes) and was initially observed on GD 10.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of the low- and the mid-dose groups during the entire study period.

The mean food consumption of the high-dose dams was statistically significantly reduced at the beginning of the treatment period (GD 6-13; up to 15% below control), but recovered afterwards.
If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the high-dose dams consumed 8% or 6%, respectively, less food in comparison to the concurrent control group.
The mean food consumption of the dams in test groups 1 and 2 (30 or 100 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. The only exception was a slightly, but statistically significantly lower mean food consumption of the low- and high-dose dams on GD 0-1, which was a solitary event and considered to be accidental.

The mean body weights of the high dose dams were in general comparable to the controls throughout the entire study period.
The body weight change of the high-dose dams was statistically significantly reduced on GD 8-10 (approx. 29% below control) and if calculated for the entire treatment period (GD 6-19; 8% below control).
The mean body weights and the average body weight gains of the low- and mid-dose dams were in general comparable to the controls throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of high dose was clearly lower than the concurrent control value (approx. 10% below control), but without attaining statistical significance.
The corrected body weight gain of low and mid dose groups revealed no difference of any biological relevance to the corresponding control group.
Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Mean gravid uterus weights of the animals of all test groups were not influenced by the test substance.

No necropsy findings which could be attributed to the test substance were seen in any dam.
In the high dose spontaneous findings were noted in individual females of the high dose group (a diaphragmatic hernia (this female was not pregnant), a dilated renal pelvis, and a hemometra).

The conception rate varied between 96% in the high dose group 3 and 100% in the other test groups. With these rates, a sufficient number of
pregnant females were available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The sex distribution of the fetuses in all dose groups was comparable to the control fetuses.
The mean placental weights of all dose groups were comparable to the corresponding control group.
The mean fetal weights of all dose groups were not influenced by the test substance and did not show any biologically relevant differences in
comparison to the control group.

No soft tissue malformations were recorded.
Some soft tissue variations were detected in all test groups including the control, i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant. Most of them can be found in the historical control data at comparable incidences.
No unclassified soft tissue observations were recorded.

One mid-dose fetus with multiple external malformations was seen (misshapen head and absent face). The overall incidences of external malformations were comparable to those found in the historical control data. An association of these findings to the treatment is not assumed.
No external variations were recorded.
One unclassified external observation, i.e. blood coagulum around placenta, was recorded in two fetuses of the high-dose group. This finding was not considered biologically relevant.

Skeletal malformations were detected in all test groups exept low dose group (0, 100 and 300 mg/kg bw/d) affecting the skull, sternum and forelimbs.
One fetus of the mid dose group (same animal as external malformation) was multiple-malformed (malformations affected the skull, vertebral column, ribs, pelvic girdle and forelimbs) and had associated external findings. The average rate of affected fetuses per litter showing skeletal malformations was statistically significantly increased in the high-dose group. However, each of the findings leading to that increased rate ist present in the historical control data and no abnormality pattern became obvious. The total incidences of skeletal malformations in the low- and mid-dose groups were comparable to the concurrent control group. These scattered observations in individual fetuses of these groups are are common for this rat strain.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data, and no dose dependency was observed.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no observed effects at highest dose level

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Terminal body weights, food consumption and relative organ weights [g].

dose [mg/kg bw/day]

0

30

100

300

food consumption (0-20)

19.3

19.1

19.3

18.2

absolute terminal bw

295.9

302.4

297.8

291

corrected bw gain

40.9

43.7

40

36.9

carcass weight

236.8

242.8

239.1

235.2

bw change 6 -19

85.2

89.8

84.3

78.6*

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of DHDPS to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 300 mg/kg bw/d caused evidence of maternal toxicity, such as reduced food consumption and (net) body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d.
There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d.
Executive summary:

In a prenatal developmental toxicity study the test substance DHDPS 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, the homogeneous distribution and the stability of the test substance in the vehicle.

The test substance caused no mortality nor clinical symptoms of systemic toxicity in any of the exposed groups receiving 30, 100 or 300 mg/kg bw/d DHDPS. Some females (7 out of 25) of the high-dose group (300 mg/kg bw/d) showed transient salivation after treatment. This salivation persisted in the respective females for a few minutes immediately after each administration. It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The high-dose of the test substance (300 mg/kg bw/d) caused a significant decrease in food consumption (mainly at the beginning of treatment) and body weight gain as well as a distinct decrease in the corrected (net) body weight gain. These effects are considered to be treatment-related and adverse. No toxicologically relevant effect on food consumption and body weight gain was noted for the animals exposed to 30 or 100 mg/kg bw/d DHDPS.

No differences of toxicological relevance between the control and the treated groups (30, 100 or 300 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.