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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
Remarks:
GLP guideline study. Adopted according to OECD SIDS (public available peer reviewed source).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
OECD Guidelines for Testing of Chemicals; Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (January 22, 2001)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tridecanol H
- Physical state: Liquid, oily/colorless
- Analytical purity: 99.7 corrected area-%
- Lot/batch No.: EDV 55712
- Test substance No.: 03/0197-1
- Stability under storage conditions: The stability under storage conditions was confirmed by reanalysis; The stability of the test substance solutions in olive oil Ph.Eur./DAB over a period of 7 days at room temperature could also be demonstrated;
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature, under N2

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: The animals were supplied at an age of about 70 - 84 days.
- Weight at study initiation: Based on the pregnant animals the body weight on day 0 varied between 146.2 - 188.1 g.
- Housing: single housing
- Diet: ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland; food was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy);
- Water: drinking water of tap water quality from water bottles; ad libitum
- Acclimation period: Between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
Ph. Eur./DAB
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 analytical results of the stability verification. For the preparation of the solutions, an appropriate amount of the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with olive oil Ph.Eur./DAB and subsequently thoroughly mixed.

Administered standard dose volume of 5 ml/kg body weight

VEHICLE
- Concentration in vehicle: 1.2, 5 or 15 g/100 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance solutions were analyzed by GC. The results of the analyses of test substance solutions in olive oil Ph.Eur./DAB 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.
Details on mating procedure:
The animals were mated by the breeder ("time- mated") and supplied on day 0 post coitum (p.c.). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1" p.c..

- Proof of pregnancy: vaginal plug / sperm referred to as day 0 p.c.
Duration of treatment / exposure:
day 6 through day 19 of gestation (from implantation to one day prior to the expected day of parturition)
Frequency of treatment:
1x/d
Duration of test:
on day 6 through day 19 post coitum (p.c.)
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 mated female Wistar rats/group
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p .c.).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8,10,13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p .c. minus weight of the unopened uterus minus body weight on day 6 p .c.).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (With the exception of day 0, the consumption of food was determined on the same days as was body weight.)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: liver, uterus, and ovaries

OTHER:

Clinical Pathology:
Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anaesthetized using isoflurane as anesthesia. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The following examinations were carried out in all female animals per test group:

Hematology:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (ADVIA 120 model; Bayer, Fernwald, Germany): leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes.

Furthermore, differential blood smears were prepared and stained without being evaluated.

The clotting analyses were carried out and the prothrombin time (Hepator Quick's test) was determined.

Clinical chemistry:
An automatic analyzer was used to examine the clinicochemical parameters. The following parameters were determined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-gIutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
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: dead fetuses
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: No data
Statistics:
STATISTICS
- Statistics of clinical, necropsy and fetal examinations: simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number 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.
- Organ weights (liver, kidneys, spleen): Non-parametric one way analysis using KRUSKAL-WALLIS- test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON-test (two-sided) for the equal medians.
- Pairwise comparison of each dose group with the control group using Fishers exact test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings;
- Pairwise comparison of each dose group with the control group using the Wilcoxon-test (one-sided) for the hypothesis of equal medians: Propotions of fetuses with malformations, variations and/or unclassified observations in each litter.

Statistics of clinical pathology:
Means and standard deviations of each test group were calculated for several parameters. Clinical pathology except reticulocytes and differential blood count. Non-parametric one way analysis using KRUSKAL-WALLIS test (two sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using Wilcoxon-te t (two-sided) for the equal medians.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high dose (750 mg/kg body weight/day) and the majority (17 out of 25) of the mid dose (250 mg/kg body weight/day) animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. This substance-induced symptom was observed first for high dose females Nos. 98, 99 and 100 on day 7 p.c. and for mid dose rat No. 75 on day 12 p.c.. After cessation of treatment on day 19 p.c., salivation did not occur any longer. The observed temporary salivation of the animals is considered to be substance-induced. It is very likely, that this finding was induced by bad taste of the test substance or local affection of the upper digestive tract. Salivation itself is not assessed as an adverse or toxic effect.

Additionally, 4 high dose dams (Nos. 77, 83, 98 and 100) showed urine-smeared fur on gestation days 10 - 13 or 17 - 20. This clinical finding is a sign of discomfort of the rats and is probably substance-induced.

No indications for disturbances of the general behavior, however, occurred in the dams of test groups 0 and 1 (0 and 60 mg/kg body weight/day).
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant or biologically relevant differences between the controls and the substance-treated dams in terms of body weights and body weight gain during the study. The observed slight and transient, but statistically significant reductions in food consumption at 750 mg/kg on the first treatment days induced no adverse effects on body weight development of these dams.

- Corrected body weight gain (net maternal body weight change): the corrected body weight gains (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) of the dams of test groups 1 - 3 (60; 250 and 750 mg/kg body weight/day) revealed no differences of any biological relevance to the corresponding control group. Moreover, a clear relation to dosing was not present.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the high dose dams (750 mg/kg body weight/day) was statistically significantly reduced (up to about 11 % below the concurrent control value) on treatment days 6 - 10 p.c.. Thereafter, food consumption of the top dose rats was similar to control values and was not influenced in a dose-related manner. The food consumption of the females of test groups 1 and 2 (60 and 250 mg/kg body weight/day) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reduction in food consumption of the high dose dams at initiation of dosing is considered to be substance-induced; however, the decreased food intake had no corroborative effects on body weight data and corrected body weight gain of these females.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment- related changes in the hematological parameters measured.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period serum enzyme examinations revealed slightly, but statistically significantly increased alanine aminotransferase activities in the high dose animals. The other serum enzymes were not affected by the test compound.
Blood chemistry investigations showed decreased total protein and globulin concentrations and increased triglyceride levels in the serum of the high dose females. No treatment- related changes were observed in the other blood chemistry parameters.
The slight changes seen in the clinical chemistry parameters alanine aminotransferase, total protein, globulins and triglycerides of the high dose animals are considered to be test substance-related and are indicative for enhanced metabolic activity or a mild adverse effect on the liver.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus weight:
The mean gravid uterus weights of the animals of test groups 1 - 3 (60, 250 or 750 mg/kg body weight/day) were not influenced by the administration of the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Liver weight:
Absolute and relative mean liver weights were statistically significantly increased at the high dose group (750 mg/kg body weight/day) and were about 14% (absolute) or 18% (relative) above control values. The increased liver weights at the top dose are in line with the slight changes seen in different clinical chemistry parameters (i.e. alanine aminotransferase, total protein, globulins and triglycerides); they are considered as substance-induced. Absolute and relative liver weights of the dams of test groups 1 and 2 (60 and 250 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant changes.

Kidney weights:
Absolute and relative mean kidney weights of the dams of test groups 1, 2, and 3 (60, 250 and 750 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant differences.

Spleen weight:
Absolute mean spleen weights were statistically significantly decreased at the high dose group (750 mg/kg body weight/day) and were about 9% lower than the control values. As the more important relative spleen weights of the top dose dams remained unaffected, this is considered to be spontaneous in nature and not as substance-induced. The absolute and relative spleen weights of the low and mid dose rats (60 and 250 mg/kg body weight/day) were similar to the respective control values and did not show any toxicologically significant changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related observations at necropsy in any of the dams..

Control dam (No. 10) had a hemorrhagic thymus. This gross finding is considered to be spontaneous in nature and is probably related to the method how the rats were killed.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There are additional statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the different test groups in the values calculated for the pre- and the postimplantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Low dose dam No. 34 had one dead fetus, but also 9 live fetuses in the uterus. Mid dose dam No. 62 was only pregnant by stain. It had only two very early resorptions, but no viable fetuses in the uterus. The isolated occurrence of one low dose rat with one dead fetus and of one mid dose female with total resorptions does not suggest any relation to treatment. Moreover, both findings occur also occasionally as spontaneous findings in the strain of rats used for this study.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 100% in test groups 0 and 1 (0 and 60 mg/kg body weight/day), 92% in test group 2 (250 mg/kg body weight/day) and 96% in test group 3 (750 mg/kg body weight/day). As all rats that became pregnant had implantation sites at necropsy, a sufficient number of females for the purpose of the study were available.
Other effects:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the different test groups concerning viable fetuses or in the mean number of corpora lutea and implantation sites.

Thus, all differences observed in respect to the gestational parameters in the various test groups were within the normal range of deviations for animals of this strain and age.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights in test groups 1, 2 and 3 (60, 250 and 750 mg/kg body weight/day) were not influenced by the test substance administration and were very similar to the corresponding control values.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1 - 3 (60, 250 and 750 mg/kg body weight/day) was comparable with that of the control fetuses. The observable differences in comparison to the concurrent control are without any biological relevance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The only skeletal malformations, which occurred, were observed for low dose fetus No. 10 from dam No. 34. This fetus was already dead and showed two external malformations, i.e. gastroschisis and scoliosis, when it was developed from the uterus of its mother. The observed additional skeletal malformations (i.e. misshapen thoracic arch (with changed cartilage) and fused rib (with present cartilage)) fit well to the above mentioned external findings.
In total, none of the 105 control fetuses (from 25 litters), one of 115 low dose fetuses [= 0.9%] in one of 25 litters [= 4.0%], none of the 99 mid dose fetuses (from 22 litters) and none of the 103 high dose fetuses (from 24 litters) showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 0.7, 0.0, and 0.0%.The occurrence of two skeletal malformations in one low dose fetus in the absence of any further skeletal malformations in the other examined fetuses from test groups 0 - 3 (0, 60, 250 and 750 mg/kg body weight/day) does not suggest a substance - induced background but is considered to be spontaneous in nature.

In all groups signs of skeletal variations with or without involvement of corresponding cartilaginous structures elicited. The observed variations were related to the skull (supraoccipital and/or basioccipital holes; incomplete ossification of the entire skull, basisphenoid, frontal, parietal, interparietal, supraoccipital and/or hyoid), the vertebral column (incomplete, dumbbell or bipartite ossification of cervical, thoracic, lumbar and/or sacral vertebrae ; supernumerary thoracic vertebra; fused sacral centrum and arch, misshapen sacral vertebra), the ribs (supernumerary 14 th, cervical or wavy ribs), the sternum (misshapen sternebra; unilateral, incomplete, bipartite or missing ossification of sternebra) and the pelvic girdle (incomplete ossification of pubis). The mean percentages of affected fetuses/litter with skeletal variations amounted to 95.1, 93.9, 93.0, and 96.3% at 0, 60, 250 or 750 mg/kg body weight/day. Most of the noted skeletal variations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data. This assessment includes the only skeletal variation, misshapen sacral vertebra, which occurred at a statistically significantly higher rate at 60 and 750 mg/kg body weight/day in comparison to the concurrent control group; 0.0%, 3.1 %* (* = p
Additionally, some isolated cartilage findings without any impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the controls. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum. The mean percentages of affected fetuses/litter with these findings amounted to 26.9, 32.3, 34.0, and 20.7% at 0, 60, 250 or 750 mg/kg body weight/day. A toxicological relevance for these findings, which did not show any relation to dosing, can be excluded with certainty.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations were recorded for one low dose and one high dose fetus (60 and 750 mg/kg body weight/day) each. Unilateral hydronephrosis in combination with hydroureter occurred in female fetus No. 8 from low dose dam No. 44 and a situs inversus was seen in male fetus No. 6 from high dose dam No. 76. The isolated occurrence of these soft malformations does not suggest any relation to dosing. In total, none out of the 92 examined control fetuses (from 25 litters), one out of 104 low dose fetuses [= 1.0%] (in one out of 25 litters [= 4.0%]), none out of the examined 87 mid dose fetuses (from 22 litters) and one out of 88 high dose fetuses (in one out of 24 litters [= 4.2%]) showed soft tissue malformations. The mean percentages of affected fetuses/litter with soft tissue malformations amounted to 0.0, 0.8, 0.0 and 1.4%, respectively without attaining statistical significance in any of the test groups (60, 250 or 750 mg/kg body weight/day).

Two soft tissue variations (uni- or bilateral dilation of the renal pelvis and/or ureter) were detected in each group including the controls in 8 - 12 fetuses from 4 - 9 litters without any dose-response relationship. The mean percentages of affected fetuses/litter with total soft tissue variations amounted to 11.8% (control), 13.1% (60 mg/kg body weight/day), 8.5% (250 mg/kg body weight/day) and 9.7% (750 mg/kg body weight/day), respectively. Thus, a relation to dosing is not present and a substance-induced effect concerning the occurrence of soft tissue variations can be excluded with certainty.

No so-called unclassified soft tissue observation (like blood imbibition of kidney(s)) was recorded in any of the fetuses.
Description (incidence and severity):
Weight of placentae: the mean placental weights in the substance-treated groups (60, 250 and 750 mg/kg body weight/day) were similar to the corresponding control values and did not show any dose dependency.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion