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Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information
NOAEL dev and maternal = 1000 mg/kg/d (BASF SE/Evonik, 2014, OECD 414)
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.04-16.12.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD and GLP guideline study
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
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: 10-12 weeks
- Body weight at beginning: 140.3-190.3 g
- Fasting period before study: no
- Housing: rats were housed individually in Makrolon type M III cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm); animals
were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

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

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The oily 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
- Diet preparation: the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed by shaking until it is dissolved.

VEHICLE
- Concentration in vehicle: 0, 2500, 7500, 25000 mg/100 ml
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in oil at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch.
Given that the test substance was completely miscible with corn oil, solutions were considered to be homogenous without further analysis.
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. After 6 days acclimatization, treatment started on day 6
Duration of treatment / exposure:
gd6-19
Frequency of treatment:
once daily
Duration of test:
animals were sacrificed on day 20
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

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

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: Yesa
- Number of late resorptions: Yes
- Number of dead fetuses: 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 (Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss,
proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight)
Siegel test (Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings)
Nikenhuis/Wilf and Hettmansperger (Proportions of fetuses with malformations, variations and/or unclassified observations in each litter)
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Two females of treatment group 2 (300 mg/kg/d) and one female assigned to treatment group 3 were not pregenant and hence not taken into account for further analyses.

Mortality: There were no test substance-related or spontaneous mortalities in female animals of any test group (0, 100, 300 or 1000 mg/kg bw/d).

Clinical Observations: Nearly all females (24 out of 25) of the high-dose group (1000 mg/kg bw/d) and some females (11 out of 25) of the mid-dose group (300 mg/kg bw/d) showed transient salivation during the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 9. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. 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 100, 300 or 1000 mg/kg bw/d during the entire study period.

Food concumption: The mean food consumption of the high-dose dams (1000 mg/kg bw/d) was significantly increased from GD 13 onwards until scheduled sacrifice on GD 20. However, if calculated for the entire treatment period or the entire study period, the high-dose dams did not consume significantly more food than the concurrent control group. Therefore, the changes were assessed to be incidental and not related to treatment.

Body weight: The mean body weights and the average body weight gains of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the significantly increased body weight change value in test group 1 (100 mg/kg bw/d) between GD 13-15. The corrected body weight gain of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

Uterus weight: The mean gravid uterus weights of the animals of test group 1-3 (100, 300 and 1000 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.

Necroscopy: No necropsy findings which could be attributed to the test substance were observed in any dam of the test groups 1, 2 or 3 (100, 300 or 1000 mg/kg bw/d). Two spontaneous findings occurred, i.e. dilated renal pelvis in two females of test group 2 and a diaphragmatic hernia in one female of the same test group. These findings were detected in single animals and were not assessed to be treatment-related.

Reproduction data: The conception rate was 92% in test group 2 (300 mg/kg bw/d), 96% in test group 3 (1000 mg/kg bw/d) and 100% in test groups 0 and 1 (0 and 100 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 listed in chapter 2.3.). No test substance-related and/or biologically relevant differences between the test groups 0, 1, 2 and 3 (0, 100, 300 and 1000 mg/kg bw/d) were observed with regard to conception rate, mean number of corpora lutea and implantation sites or the values calculated for the postimplantation loss, the number of resorptions and viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Sex distribution: The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Weight of plancentae: The mean placental weights of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were comparable to the corresponding control group.

Weight of fetuses: The mean fetal weights of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Fetal external malformations: External malformations were recorded for one fetus of the high-dose group (1000 mg/kg bw/d). Male fetus No. 83-03 had more than one malformation affecting the fetal head, i.e. domed head, anophthalmia, retarded development of right side of head (asymmetric face) and upturned nose. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Fetal external variations: One external variation, i.e. limb hyperextension, was detected in test group 1 (100 mg/kg bw/d). This single case was considered to be incidental and not related to treatment.

Fetal external unclassified observations: One unclassified external observation, i.e. placentae fused, was recorded in one fetus of the mid-dose group (300 mg/kg bw/d). This finding was not considered to be biologically relevant.

Fetal soft tissue malformations: Soft tissue malformations were recorded for one low-dose fetus. Female fetus No. 27-12 had more than one malformation affecting the urinary tract, i.e. hydronephrosis and hydroureter. The finding was assessed to be incidental and not related to treatment. There were no further soft tissue malformations in any of the other test groups

Fetal soft tissue variations: Three soft tissue variations were detected, i.e. short innominate, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the control nor dosedependently altered. Therefore, they were not considered to be biologically relevant

Fetal soft tissue unclassified observations: No unclassified soft tissue observations were recorded.

Fetal skeletal malformations: One high-dose male fetus (No. 83-03 – 1000 mg/kg bw/d) showed multiple malformations. In correlation to the external malformations, some skull bones were small on the right side (nasal, frontal and zygomatic bones, zygomatic process). Furthermore, this fetus had malformations affecting the cervical vertebrae and sternebrae. This single case of multiple malformations is considered to be an incidental finding. There were no further skeletal malformations recorded in any fetuses of the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d)

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 skeleton and their incidences were neither significantly different from control nor dose-dependent. Therefore, they were not considered to be biologically relevant. The overall incidences of skeletal variations were comparable to the historical control data. The increased incidences of skeletal variations were neither related to the dose nor were they outside the historical control range. They were in any case not considered as adverse events.

Fetal skeletal unclassified cartilage observations: Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (table 4.3.4.3.1.). The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing. However, the incidence of bipartite processus xiphoideus was significantly increased in test groups 1 and 3 (100 and 1000 mg/kg bw/d). As a consequence of this occasional increase, also the incidence of total skeletal unclassified cartilage observations was significantly increased in these test groups. However, this finding showed no dose-dependency and was therefore assessed to be without biological relevance.

Assessment of all fetal external, soft tissue and skeletal observations: There were noted external, soft tissue and skeletal malformations in test groups 1 and 3 (100 and 1000 mg/kg bw/d). The distribution of total malformations to the test groups was not related to the doses. Two fetuses were multiple malformed: low-dose female fetus No. 27-12 (100 mg/kg bw/d) had more than one visceral malformation affecting the urinary tract, i.e. hydronephrosis and hydroureter, while the findings in high-dose male fetus No. 83-03 (1000 mg/kg bw/d) consisted of multiple external malformations affecting the fetal head (domed head, anophthalmia, development of right side of head retarded [asymmetric face] and upturned nose) and multiple skeletal malformations affecting the skull, cervical vertebrae and sternebrae (small nasal, frontal and zygomatic bones, small zygomatic process, cervical hemivertebra, malpositioned and bipartite sternebrae). No ontogenetic pattern was recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. One external variation, three soft tissue variations and a broad range of skeletal variations occurred in all test groups including the controls. None of the incidences showed a relation to dosing. The majority of the skeletal variations were equally distributed among 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

Total fetal variations: No unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin was assumed for the unclassified external observation, which was observed in one fetus of test group 2 (300 mg/kg bw/d). This isolated finding did not suggest any relation to treatment. Several unclassified skeletal cartilage observations were observed in several fetuses of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d). Although the total incidences in test groups 1 and 3 were significantly increased, a relation to treatment was not assumed, because these findings were not related to the dose and can be found in the historical control data. Finally, fetal examinations revealed that there was no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Oxooel 9N to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 1000 mg/kg bw/d caused no evidence of maternal toxicity.
In addition, no toxicologically relevant adverse fetal findings were evident. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 1000 mg/kg bw/d, the highest dose tested. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity was also 1000 mg/kg bw/d.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

In a prenatal developmental toxicity study the test substance Oxooel 9N 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 maternal as well as its prenatal developmental toxic potential. Generally, clinical observations revealed no toxicologically relevant findings in the animals receiving 100, 300 or 1000 mg/kg bw/d. Salivation after treatment was observed for nearly all females (24 out of 25) of the high-dose group (1000 mg/kg bw/d) and some females (11 out of 25) of the mid-dose group (300 mg/kg bw/d). From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 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 influences of the test compound on fetal weight and sex distribution of the fetuses were noted at any dose level. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose level.

Thus, under the conditions of this prenatal developmental toxicity study, the oral administration of Oxooel 9N to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 1000 mg/kg bw/d caused no evidence of maternal toxicity. In addition, no toxicologically relevant adverse fetal findings were evident. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 1000 mg/kg bw/d, the highest dose tested. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity was also 1000 mg/kg bw/d.

Justification for classification or non-classification

Based on the available data, the test substance does not require classification with respect to development and fertility according to Regulation EC1272/2008 (CLP).