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Toxicological information

Developmental toxicity / teratogenicity

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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:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany Gmb
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 148.2 - 191.1 g
- Housing: individually (Polycarbonate cages type III)
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: 6 days

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:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The oily test substance solutions 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 corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany.The stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); 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. The following day was designated as “GD 1”.
Duration of treatment / exposure:
From implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning
Frequency of treatment:
once daily
Duration of test:
On GD 20 all surviving dams were sacrificed and examined
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-19) all animals were checked daily for any abnormal clinically signs before administration as well as within 2 hours and within 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13- 15, 15-17, 17-19 and 19-20. Before start of the treatment period, some spilling of food in single dams on individual gestation days (GD 0-3) was observed in test groups 2 and 3 without relevance for the study.

POST-MORTEM EXAMINATIONS: Yes
Cesarean section:
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the
following data were recorded: weight of the unopened uterus, number of corpora lutea. Number and distribution of implantation sites classified as: live fetuses and dead implantations ( a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy); b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible; c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the dams (except of gross pathology including organ weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Pathology:
Organ weights
The following weights were determined in all animals sacrificed on schedule: adrenal glands, kidneys, liver, spleen. The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.
Organ / Tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution: all gross lesions, adrenal glands, kidneys, liver, spleen. No further examinations or procedures were performed in the study.
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
DUNNETT-test (twosided), FISHER'S EXACT test (onesided), WILCOXON-test (onesided)
Indices:
The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/number of fertilized animals) x100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations)/number of corpora lutea) x100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses)/number of implantations) x100
Historical control data:
Historical contraol data were available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Eight out of 25 high-dose females (120 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals shortly after treatment (i.e. within 2 hours post-dosing) and was observed from GD 12 to GD 19. After 2 hours and up to 5 hours post-dosing no clinical signs or changes of general behavior were detected in any female of all test groups (0, 10, 40 or 120 mg/kg bw/d), nor were any clinical signs observed before dosing in the morning. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 10, 40 or 120 mg/kg bw/d).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Corresponding to reduced food consumption, the mean body weight change of the high-dose dams (120 mg/kg bw/d) was statistically significantly reduced on GD 6-8 (approx. 41% below control), but recovered afterwards and was comparable to the concurrent control. This temporary decrease of body weight change in the high-dose group is considered as treatment-related and adverse. The mean body weights and average body weight change of the low- and mid-dose dams (10
or 40 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (120 mg/kg bw/d) was statistically significantly decreased during GD 6-8 (about 18% below control), but recovered afterwards and was comparable to the concurrent control group throughout the remaining study period (GD 8-20). If calculated for the entire treatment period (GD 6-19) or study period (GD 0-20), the mean food consumption of the high-dose dams was generally comparable to the concurrent control. This temporary reduction of food consumption during GD 6-8 was considered as treatment-related and adverse. The mean food consumption of the dams in test groups 1 and 2 (10 and 40 mg/kg bw/d) was comparable to the concurrent control throughout the entire study period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of gestation in dams of test group 3 (120 mg/kg bw/d), red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) were significantly decreased and absolute reticulocyte counts, mean corpuscular hemoglobin content (MCH) and mean corpuscular hemoglobin volume (MCV) were significantly increased. RBC counts were significantly decreased in dams of test groups 1 and 2 (10 and 40 mg/kg bw/d). MCV in test group 2 (40 mg/kg bw/d) was higher compared to controls. However, MCV in test group 2 was within, and RBC counts in test groups 1 and 2 were only marginally below the historical control range (dams, MCV 50.4-54.3 fL, RBC 5.58-7.86 tera/L). RBC count means in test groups 1 and 2 were not dose-dependently changed, and RBC was the only measured red blood cell parameter which was altered in the mentioned test groups. Therefore, the RBC decrease in dams of test groups 1 and 2 as well as the MCV increase in dams of test group 2 were regarded as incidental and not treatment-related (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. In dams of test group 3 (120 mg/kg bw/d) total bilirubin and creatinine levels were significantly decreased. Creatinine was already significantly lower in rats of test group 2 (40 mg/kg bw/d). However, all values were within historical control ranges (dams, creatinine 22.8-33.6 μmol/L; total bilirubin 0.74-1.37 μmol/L). Therefore, the creatinine and total bilirubin alterations were regarded as incidental and not treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased in one or more test groups: spleen. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased or decreased in one or more test groups: adrenal glandy, kidneys, liver and spleen.
The significantly decreased absolute and relative spleen weight as well as the significantly increased relative weight of adrenal glands in test group 2 animals was regarded as incidental, since there was no dose-response relationship. The significantly increased absolute and relative spleen weights in animals of test group 3 were regarded as treatment related.
The slightly, but significantly increased relative liver weights of animals in test groups 2 and 3 lay above the range of historical control values. Therefore, a relation to treatment cannot be excluded.
The significantly increased relative weight of kidneys in test groups 1 to 3 was regarded as incidental, since there was no clear dose-response relationship and the weight parameters lay within the range of historical control values.
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The postimplantation loss was statistically significantly lower in test groups 1 and 2 (mean: 11.9%/1.6%**/3.2%*/8.6% in test groups 0-3). Statistical significance was caused by the rather low values in the low- and mid-dose groups which showed no relation to dose and were assessed as incidental.
The mean gravid uterus weights of the animals of test groups 1-3 (10, 40 and 120 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of resorptions was statistically significantly lower in the low- and mid-dose groups (1.5/0.2**[p<=0.01]/0.4**[p<=0.01]/0.8 in test groups 0-3). Consequently, the mean number of live fetuses was also higher in those dams (mean: 88.1%/98.4**%/96.8*%/91.4% in test groups 0-3). As specified above, these statistically significantly greater litters came from a comparatively low resorption rate at the low- and mid-dose levels and are neither treatmentrelated nor adverse.
Other effects:
no effects observed
Description (incidence and severity):
The mean placental weights of test groups 1-3 were comparable to the concurrent control group.
Details on maternal toxic effects:
The conception rate reached 96% in the control and the low-dose group (0 and 10 mg/kg bw/d) and 100% in the mid- and high-dose groups (40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study (according to the test guidelines). There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 10, 40 and 120 mg/kg bw/d) in conception rate, in the mean number
of corpora lutea and implantation sites or in the values calculated for the preimplantation loss.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
haematology
organ weights and organ / body weight ratios

Maternal abnormalities

open allclose all
Abnormalities:
effects observed, treatment-related
Localisation:
other: spleen
Abnormalities:
effects observed, treatment-related
Localisation:
other: food consumption and compound intake
Abnormalities:
effects observed, treatment-related
Localisation:
other: haematology

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (10, 40 and 120 mg/kg bw/d) was comparable to the control fetuses.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal external malformations:
External malformations were seen in one control litter (0 mg/kg bw/d) and one high-dose litter (120 mg/kg bw/d). For the one affected fetus of test group 3 with skeletal examination, these external findings were associated with skeletal malformations. The finding anasarca was observed in one litter of control and one litter of test group 3 (two affected fetuses). In the control, it was associated with gastroschisis. In test group 3, it was associated with microstomia. On a litter basis, litter incidence of the test group 3 was within the historical control data (HCD of anasarca, litters, range: 0.0 – 4.2°%). Furthermore, no ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of the high-dose group. The findings were assessed as incidental and not-treatment related.
Fetal external variations:
One external variation in one fetus each of test groups 0 and 1 was recorded (0 and 10 mg/kg bw/d), i.e. limb hyperextension. The incidence of this single finding was not statistically significantly different from control. The finding was not related to dose and it can be found in the historical control data at comparable or higher incidences (HCD of affected fetuses per litter: mean 0.0 % [0.0-0.7 %]). Thus, it is not considered as treatment-related and adverse.
Fetal external unclassified observations:
One unclassified external observation, i.e. pointed lower jaw, was recorded in one fetus of test group 3 (No. 93-01; 120 mg/kg bw/d). Since this finding was not confirmed during skeletal observation and occurred only in one single fetus, it is not considered biologically relevant.

Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Fetal skeletal malformations:
Severely malformed vertebral column and/or ribs, cleft sternum, and bent ribs were observed in one fetus each of test group 2 (40 mg/kg bw/d). In case of malformed vertebral column and/or ribs and cleft sternum, there was no dose-dependency, and the incidences were within the historical control range of the rat strain (HCD). The finding bent ribs was observed in each one fetus of test groups 2 and 3 without relation to dose. Furthermore, bent ribs have been shown to be reversible postnatally. Therefore, the findings in test group 2 fetuses were not assessed as treatment-related and adverse. Test group 3 (120 mg/kg bw/d) fetuses showed 3 findings in 4 fetuses of 4 litters. The finding misshapen tuberositas deltoidea of test group 3 was seen in one individual fetus and is present in the historical control data. Generalized disturbance of ossification was observed in two fetuses of two litters and is not present in the historical control data. Although there is no ontogenetic pattern recognizable for these individual malformations, a relation to treatment cannot be excluded. The number of these malformations adds up to a statistically significantly higher value for the total skeletal malformation rate in the mid- and high-dose groups. However, the incidence of total malformations of test group 2 was within the historical control range (HCD of affected fetuses per litter: mean total malformations: 0.8%, [0.0 – 3.1]) whereas the incidence of test group 3 was slightly above.
Fetal skeletal variations:
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 skeletons and were neither statistically significantly nor dose-dependently changed in comparison to the concurrent control group. The overall incidences of skeletal variations were comparable to the historical control data.
Fetal skeletal unclassified cartilage observations:
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 ribs and the sternum 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.

Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue malformations:
No soft tissue malformations were recorded.
Fetal soft tissue variations:
Two soft tissue variations were detected in all test groups including the control (0, 10, 40 or 120 mg/kg bw/d), i.e. 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. All of them can be found in the historical control data at comparable incidences.
Fetal soft tissue unclassified observations:
No soft tissue unclassified observations were recorded.


Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal: general disturbance of ossification
Description (incidence and severity):
two fetuses of two different litters

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-(2-ethoxyethoxy)-2-methylpropane to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused adverse findings at the highest dose level of 120 mg/kg bw/d. The effects in dams consisted of a temporary reduction in food consumption and decrease in body weight during GD 6-8, a macrocytic-hypochromic, regenerative anemia in clinical pathology and an increase in spleen weights in pathology. At this dose level, in presence of maternal toxicity, fetal findings affecting skeletal structures were observed in test group 3 fetuses (120 mg/kg bw/d). Therefore, no observed adverse effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity is 40 mg/kg bw/d.