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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:
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 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
Details on test material:
Batch No.: Lot-Nr. 0007989197
Purity: 99.3 g/100g
Storage stability: Expiry date: 07 Nov 2014
ADDITIONAL TEST SUBSTANCE INFORMATION
Physical state/Appearance: Liquid/ light yellowish clear
Storage conditions: Room temperature, under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 10-12 weeks
- The body weight of the pregnant animals on gestation day 0 varied between 148.5 – 191.5 g
- Housing: singly
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- 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%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

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 preparation was stored in a freezer.
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 1% Carboxymethylcellulose in drinking water over a period of a maximum of 7 days at room temperature were carried out prior to the start of the study. Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
gestation days 6 to 19
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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 in pregnant rats

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uteri and the ovaries

OTHER:
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: live fetuses, dead fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
Dunnett Test, Fishers exact test, Wilcoxon test
Indices:
conception rate (in %), preimplantation loss (in %), postimplantation loss (in %)
Historical control data:
18 studies, from 2011 - 2013

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical examinations of the dams: 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 female was excluded from the above-mentioned calculations: Test group 3 (500 mg/kg bw/d): female No. 94 – not pregnant
Mortality: There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 50, 150 or 500 mg/kg bw/d).
Clinical symptoms: Most females (20 out of 25) of the high-dose group (500 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals for a few minutes after daily gavage dosing (i.e. about 10-15 minutes) and was initially observed on GD 9. Furthermore, high-dose female No. 95 (500 mg/kg bw/d) had an unsteady gait after treatment on GD 16. This finding persisted for approximately 2 hours. No 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 or 150 mg/kg bw/d during the entire study period.
Food consumption: The mean food consumption of the dams in test groups 1-3 (50, 150 or 500 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. This includes the statistically significantly increased food consumption value in the high-dose group on GD 15-19.
Body weight data: The mean body weights and the average body weight gains of the low-, mid- and high-dose rats (50, 150 or 500 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. The statistically significantly increased body weight gain value in test group 2 on GD 0-6 (before start of treatment) is considered to be an incidental finding.
Corrected (net) body weight gain: The corrected body weight gain of test groups 1-3 (50, 150 and 500 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 (50, 150 and 500 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.
Necropsy findings: No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred two spontaneous findings in test group 1, i.e. one diaphragmatic hernia and one dilated renal pelvis. These findings were detected in single animals and therefore were not assessed to be treatment-related.
Reproduction data: The conception rate varied between 96% in test group 3 (500 mg/kg bw/d) and 100% in test groups 0-2 (0, 50 and 150 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.). There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the value calculated for the postimplantation loss, the number of resorptions and viable fetuses. However, the value calculated for the preimplantation loss was statistically significantly increased in the mid-dose group (150 mg/kg bw/d). Due to the lack of dose-response relationship and a historical control range which covers this value (HCD: 6.6% [3.2-15.8%]), this finding is considered as incidental and not related to treatment. All other observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Sex distribution of the fetuses: The sex distribution of the fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Weight of the placentae: The mean placental weights in the high-dose group (500 mg/kg bw/d) were decreased (about 6-8% below control). Since the difference to the control was slight and as there were neither effects on intrauterine mortality nor on fetal weights, this apparent finding is not considered to be toxicologically relevant or adverse. The mean placental weights of the low- and mid-dose groups (50 and 150 mg/kg bw/d) were comparable to the corresponding control group.
Weight of the fetuses: The mean fetal weights of test groups 1, 2 and 3 (50, 150 and 500 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: No external malformations were recorded.
Fetal external variations: No external variations were recorded.
Fetal external unclassified observations: One unclassified external observation, i.e. blood coagulum around placenta, was recorded in two fetuses of the low-dose group (50 mg/kg bw/d). This finding was not considered biologically relevant.
Fetal soft tissue malformations: One soft tissue malformation was recorded for a control fetus. Male control fetus No. 13-08 had a situs inversus (abdominal and thoracic cavity). There were no soft tissue malformations in any of the treated groups.
Fetal soft tissue variations: Soft tissue variations were detected in all test groups including the control (0, 50, 150 or 500 mg/kg bw/d). The highest frequency was noted for dilated renal pelvis in all test groups including the control, and dilated ureter in the mid- and high-dose groups. The incidences of these findings were statistically significantly increased in the high-dose group. As a consequence, the total incidence of fetal soft tissue variations was also increased in this test group (Tab. 1.).
Fetal soft tissue unclassified observations: No unclassified soft tissue observations were recorded.
Fetal skeletal malformations: No skeletal malformations were recorded.
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 did not present a specific pattern. The overall incidences appeared without a relation to dosing and were comparable to the historical control data. For a better overview, all individual skeletal variations with statistically significant differences between the control and any treated group were compiled in the table below (Tab. 2). All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in bold types. As can be seen from Tab. 2. the rates of supernumerary thoracic vertebra and misshapen sternebra (unchanged cartilage) were statistically significantly increased in test group 3 (500 mg/kg bw/d) and slightly outside the historical control ranges. Concerning the other statistically significant findings, no dose dependency was observed and/or all values were clearly inside the historical control range, thus, an association to the test substance and a toxicological relevance is not assumed.
Fetal skeletal unclassified cartilage observations: Isolated cartilage findings without an impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Tab.3.). The observed unclassified cartilage findings were related to the skull, the sternum
and ribs and did not show any relation to dosing. However, the overall incidence of unclassified cartilage observations was statistically significantly increased in test group 3 (500 mg/kg bw/d), albeit only slightly above the historical control range (47.6 -80.3%).
Assessment of all fetal external, soft tissue and skeletal observations: External and skeletal malformations did not occur in any of the fetuses in this study. There was noted one soft tissue malformation in the control group, i.e. one situs inversus. External variations did not occur in any of the fetuses in this study. Some soft tissue variations and a range of skeletal variations were noted 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. At the top dose (500 mg/kg bw/d) four different variations - dilated renal pelvis and dilated ureter as well as supernumerary thoracic vertebra and misshapen sternebra (unchanged cartilage) were statistically significantly increased and slightly above their historical control ranges. These rather minor increases resulted in a statistically significantly increased high-dose incidence if all different types of variations were summarized which just exceeded the historical control range (mean% affected fetuses per litter 50.44 - 56.61). Since these variations did not present a specific pattern and their rate was only slightly increased above an inherently high background rate, their toxicological significance is considered to be rather low. No unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external observation and the unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. The total incidence of skeletal cartilage observations was slightly but statistically significantly higher in test group 3 (500 mg/kg bw/d) compared to control. However, since the control incidence was already at the upper limit of the historical control range (79.3 vs. 80.3 mean% affected fetuses per litter), this marginal increase is not considered to be biologically meaningful. Overall, fetal examinations revealed that there is no specific adverse effect of the test compound on fetal morphology at any of the tested dose levels. Particularly, there were no indications for a test substance-induced teratogenicity.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Stability analysis The stability of the test substance suspensions over a period of 7 days at room temperature was demonstrated

Homogeneity analysis of the test substance preparations The homogeneous distribution of the test substance in the vehicle (1% CMC) was demonstrated

Concentration control analyses of the test substance preparations The results of the analysis of the aqueous test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Tab. 1 Total soft tissue variations

    Test group 0, 0 mg/kg bw/d Test group 1, 50 mg/kg bw/d  Test group 2, 150 mg/kg bw/d   Test group 3, 500 mg/kg bw/d 
 Litter Fetuses     N  25  25  25  24
 N  118  119  112  105
 Fetal incidence  N%  3 (2.5)  2 (1.7)  2 (1.8)  10 (9.5)
 Litter incidence  N%  3 (12)  2 (8.0)  2 (8.0)  8 (33)
  Affected fetuses/litter  Mean %  2.3  1.8  1.8  10.9*

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

Tab. 2 Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

 Finding  Test group 0, 0 mg/kgbw/d Test group 1, 50 mg/kgbw/d  Test group 2, 150 mg/kgbw/d   Test group 3, 500 mg/kgbw/d  HCDMean %(range)
 Incomplete ossification ofnasal; unchanged cartilage  2.1 2.1  5.2  9.9*   1.4(0.0 – 10.1)
 Dumbbell ossification ofthoracic centrum;dumbbell-shaped cartilageof centrum 1.4  4.2  4.3  7.5*   4.9(0.0 – 19.4)
 Supernumerary thoracicvertebra  3.3 2.3  3.5  13.3**   3.9(0.8 – 8.0)
 Misshapen sternebra;unchanged cartilage  65.0 68.3  70.3  75.0*   52.7(29.5 – 66.8)
 Supernumerary rib (14th);cartilage present  3.5 9.2*  11.7*  6.8   5.9(1.4 – 11.7)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Tab. 3 Total unclassified cartilage observations

     Test group 0, 0 mg/kg bw/d  Test group 1, 50 mg/kg bw/d  Test group 2, 150 mg/kg bw/d  Test group 3, 500 mg/kg bw/d
 Litter  N  25  25  25  24
 Fetuses  N  130  129  128  121
 Fetal incidence  N (%)  103 (79)  97 (75)  107 (84)  107 (88)
 Litter incidence  N (%)  24 (96)  25 (100)  25 (100)  23 (96)
 Affectedfetuses/litter  Mean  79.3  76.2  85.1  87.1*

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

Tab. 4 Total fetal variations

     Test group 0, 0 mg/kg bw/d  Test group 1, 50 mg/kg bw/d  Test group 2, 150 mg/kg bw/d  Test group 3, 500 mg/kg bw/d
 Litter  N  25  25  25  24
 Fetuses  N  248  248 240 226
 Fetal incidence  N (%)  133 (54)  130 (52)  130 (54)  131 (58)
 Litter incidence  N (%)  25 (100)  25 (100)  25 (100)  24 (100)
 Affectedfetuses/litter  Mean  53.4  52.6  54.8  58.5*

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

Applicant's summary and conclusion

Executive summary:

In this guideline (OECD 414) study conducted with GLP certification, the test material (EC xxx-xxx-x) was determined to have a NOAEL of >= 500 mg/kg bw/day. The test substance doses (50-500 mg/kg bw/day) were administered via oral gavage to pregnant rats on the day of implantation until one day before the expected day of parturition. No differences of toxicological relevance between the control and the treated groups were observed. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).