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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
Study period:
2015/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Name of test material (as cited in study report): Sovermol 1102
Analytical purity: 100 %
Specific details on test material used for the study:
Batch identification: CE03250005
Content: UVCB
The test item is a complex mixture of isomers and homologues components, so no purity was stated.
Producton date: 21.11.2010
re-certification date: 21.11.2011
Expiry date: 26 May 2016
water content: 0%
Physical state/appearance: viscous, yellowish liquid

Test animals

Species:
rat
Strain:
Wistar
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:
- 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°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Due to technical reasons, the study was carried out in 2 cohorts. Each dose group was represented in every cohort:
1st cohort: in-life phase 08/09/2015 - 28/09/2015
2nd cohort: in-life phase 09/09/2015 - 29/09/2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Carboxymethylcellulose in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions 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 0.5% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): basel on trial formulations
- Concentration in vehicle: 0, 0.5, 2.0, 8.0 g/100 ml
- Amount of vehicle (if gavage): 10 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: referred to as day 0 of pregnancy
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Duration of test:
GD 6-19 / 14 days
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: range finding study, BASF 2016, 1 OR0565/11 R251

Examinations

Maternal examinations:
CAGE SIDE AND CLINICAL OBSERVATIONS:
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 (GO 0 to 20). If such signs occurred, the animals were examined several times daily (GD 0-20).

FOOD CONSUMPTION:
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.

BODY WEIGHT:
Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: 20.
- 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, in randomized order. 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.
- 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)
- Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
- 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 and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
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:
EXAMINATIONS OF THE FETUSES AFTER DISSECTION FROM THE UTERUS [all per litter ]:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

SOFT TISSUE EXAMINATION OF THE FETUSES [ half per litter ]:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

SKELETAL EXAMINATION OF THE FETUSES [ half per litter ]:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.

EVALUATION CRITERIA:
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. All fetal findings were listed in tables according to these classifications.
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
- Blood parameters
- Weight parameters

KRUSKAL-WALLIS test
- Blood parameters
- Weight parameters
Indices:
- sex ratio
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100
- 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) x 100
- 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) x 100
Historical control data:
yes, period over 5 years

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most of the females (22 out of 25) of the high-dose group (800 mg/kg bw/d) and one female of the mid-dose group (200 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval (i.e. <2h after treatment) and was initially observed on GD 7. During the 5-hour examination interval (i.e. >2h<5h after treatment), no clinical signs or changes of general behavior were detected in any female of all test groups. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 50, 200 or 800 mg/kg bw/d during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were comparable to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable to the control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes among haematological parameters were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20 in dams of test group 3 (800 mg/kg bw/d) total protein and globulin values were decreased whereas cholesterol values were increased. In dams of the mentioned test group, total bilirubin values were lower compared to controls. In absence of any sign of anemia, decreased bilirubin values were most probably due to an increased conjugation rate because of a liver enzyme induction, coupled with an accelerated excretion of bilirubin via the bile. This mechanism is regarded as adaptive rather than adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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 and relative weights of livers were significantly increased in test group 3 (both absolute (+26%) and relative (+22%) liver weights).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histopathological findings were observed in livers of females of test groups 2 and 3. The hypertrophy of hepatocytes in animals of test groups 2 and 3 and the increased glycogen storage in animals of test group 3 were considered to be treatment related. The mean gravid uterus weights of the animals of test groups 1-3 (50, 200 and 800 mg/kg bw/d) were not influenced by the test substance.
Minimal (grade 1) to mild (grade 2) centrilobular hepatocellular hypertrophy in17/25 animals of the high dose group.
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the control and high-dose groups (0 and 800 mg/kg bw/d) and 100% in the low- and mid-dose groups (50 and 200 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 200 and 800 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Details on maternal toxic effects:
- Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d): 1 female not pregnant
Test group 3 (800 mg/kg bw/d): 1 female not pregnant

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 (50, 200 and 800 mg/kg bw/d) 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 (50, 200 and 800 mg/kg bw/d) was comparable to the control fetuses.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations, variations or unclassified observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Three skeletal malformations were detected in one control fetus (male fetus No. 19-09 - 0 mg/kg bw/d), i.e. absent cervical arch, severely malformed sternum, fused rib. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data. 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 appeared without a relation to dosing. The overall incidences of skeletal variations were comparable between treated groups and concurrent control as well as with the historical control. 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 vertebral column, the ribs and the sternum and did not show any relation to dosing.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Two control fetuses and one high-dose fetus had soft tissue malformations. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Four soft tissue variations were detected in all test groups including the control (0, 50, 200 or 800 mg/kg bw/d), i.e. short innominate, small liver lobe, dilated renal pelvis and dilated ureter. The incidences of one of the above mentioned soft tissue variations (dilated renal pelvis) were statistically significantly increased (0.7/4.8*/6.3*/10.0**mean% affected fetuses/litter) in the substance-treated groups (50, 200 and 800 mg/kg bw/d). As the value of the control group is below and the values of the low and mid dose group are covered by the historical control range (2.6 - 6.3 mean% affected fetuses/litter) apparent increase in the low and mid dose group were assessed as incidental and biologically not relevant. The statistically significantly increased incidence in the high dose group was not covered by the historical control data and therefore assessed as possibly related to treatment. Consequently the total incidence of fetal soft tissue variations was also increased in the high dose group. One unclassified soft tissue observation, i.e. discolored kidney, was recorded in one low dose fetus (50 mg/kg bw/d). This finding was not considered biologically relevant.
Other effects:
no effects observed
Description (incidence and severity):
The mean placental weights of test groups 1-3 (50, 200 and 800 mg/kg bw/d) were comparable to the concurrent control group.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations:
External malformations did not occur in any of the fetuses in this study. There were noted soft tissue and skeletal malformations in three control and one high-dose fetuses (0 and 800 mg/kg bw/d). As these were scattered observations in individual fetuses which can be found in the historical control data they are considered as incidental events. External variations did not occur in any of the fetuses of this study. Some soft tissue variations and a range of skeletal variations occurred in all test groups including the controls. The majority of all variations were equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. The statistically significantly increased incidence of the soft tissue variation dilated renal pelvis in the high dose group was not covered by the historical control data and therefore assessed as possibly related to treatment. Consequently, the total incidence of fetal soft tissue variations was also increased in test group 3. The total number of fetal variations is not significantly affected by this single event. No unclassified external observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified soft tissue and the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 50, 200 and 800 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to a dose of 200 mg/kg bw/d.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: urinary

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Total soft tissue malformations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  117 25 120  25 113   24 127
 Fetal incidence  N (%)  2 (1.7)  0.0  0.0  1 (0.8)
 Litter incidence  N (%)  2 (8.3)  0.0  0.0  1 (4.2)
 Affectedfetuses/litter  Mean (%) 2.1   0.0  0.0  0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total soft tissue variations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  117 25 120  25 113   24 127
 Fetal incidence  N (%)  3 (2.6)  6 (5.0)  7 (6.2)  12 (9.4)
 Litter incidence  N (%)  2 (8.3)  5 (20)  7 (6.2)  9 (38)*Fi
 Affectedfetuses/litter  Mean (%) 2.4  4.8  6.3  10.8*Wi

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p ≤ 0.05 ** = p ≤ 0.01

Fi = Fisher’s exact test (one-sided) Wi = (Wilcoxon-test (one-sided)

Total fetal skeletal variations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  134 25 131 25 123   24 137
 Fetal incidence  N (%)  132 (99)  124 (95)  120 (98)  134 (98)
 Litter incidence  N (%)  24 (100)  25 (100)  25 (100)  24 (100)
 Affectedfetuses/litter  Mean (%) 98.5  94.1  97.6  98.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total unclassified cartilage observations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  134 25 131 25 123  24 137
 Fetal incidence  N (%) 101 (75)  84 (64) 83 (67) 98 (72)
 Litter incidence  N (%)  24 (100)  25 (100) 24 (96) 98 (72)
 Affectedfetuses/litter  Mean (%) 75.9  64.3  67.3  72.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total fetal malformations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  251 25 251 25 236 24 264
 Fetal incidence  N (%) 3 (1.2) 0.0 0.0 1 (0.4)
 Litter incidence  N (%) 3 (13)  0.0 0.0 1 (4.2)
 Affectedfetuses/litter  Mean (%) 1.4  0.0 0.0  0.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total fetal variations

   0 mg/kg bw/d  50 mg/kg bw/d  200 mg/kg bw/d  800 mg/kg bw/d 
 Litter Fetuses N N 24  251 25 251 25 236 24 264
 Fetal incidence  N (%) 135 (54) 130 (52) 127 (54) 146 (55)
 Litter incidence  N (%) 24 (100) 25 (100) 25 (100) 24 (100)
 Affectedfetuses/litter  Mean (%) 53.9 51.4 54.2 56.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Applicant's summary and conclusion