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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study (OECD).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Jan. 22, 2001)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Propylheptanol
- Physical state: liquid/colorless;
- Analytical purity: 99.8%
- Lot/batch No.: Tk 2012
- Test substance No.: 02/0356-1
- Stability: The stability under storage conditions was confirmed by reanalysis. The stability of Propylheptanol in doubly distilled water + Cremophor EL for a period of 7 days at room temperature was 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: 70 - 84 days at delivery
- Weight at study initiation: 145.5 - 187.9 g
- Housing: single (from day 0 - 20 p.c.)
- Diet: ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland; ad libitum;
- Water: drinking water of tap water quality from water bottles; ad libitum;
- Acclimation period: between the supply on day 0 and the first administration on gestational day 6

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: doubly distilled water with Cremophor EL
Details on exposure:
- Concentration of the emulsion (mg/100 ml): 500, 2000, and 6000
- Dose volume: 10 ml/kg bw

PREPARATION OF DOSING SOLUTIONS:
The test substance emulsions in doubly distilled water 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 emulsions, an appropriate amount of the test substance was weighed depending on the dose group in calibrated beakers, topped up with doubly distilled water and some drops Cremophor EL and subsequently thoroughly mixed using a high-speed homogenizer. A magnetic stirrer was used to keep the emulsions homogeneous during treatments.
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 doubly distilled water + Cremophor EL for a period of at least 7 days at room temperature were carried out before the study was initiated.
Samples of the test substance emulsions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analyses at the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 600 mg/kg body weight/day). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
The test substance emulsions were analyzed by GC. The analytical values of the samples corresponded to the expected values within the limits of the
analytical method, i.e. were always above 90% of the nominal concentration.
Details on mating procedure:
The animals were mated by the breeder (time-mated) and supplied on day 0 post coitum (= detection of vaginal plug / sperm).
Duration of treatment / exposure:
day 6 through day 19 p.c.
Frequency of treatment:
daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 600 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check for mortality 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.
Corrected body weight gain (net maternal body weight change):
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.)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: With the exception of day 0, the consumption of drinking water was determined on the same days as was body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Weight of liver, kidneys and spleen, Weight of the unopened uterus
One test group 1 animal and two test group 3 animals that died or had to be sacrificed during the study period were examined according to the same procedures as the females killed on schedule (exception: no weight determinations of uterus, liver, kidneys and spleen).

Examinations of the dams at termination:
- Clinical Pathology:
Blood was taken from the retroorbital venous plexus in the morning from non-fasted animals without 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 animals per test group at the end of the administration period.
Hematology: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count;
Furthermore, differential blood smears were prepared and stained according to Wright without being evaluated.
Clotting analyses: prothrombin time (Hepato Quick's test)
Clinical chemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
Ovaries and uterine content:
The 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: Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
- Food consumption, water consumption, 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: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- Organ weights (liver, kidneys, spleen): Non-parametric one-way analysis using KRUSKAL-WALLIS-test (two-sided). If the 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.
- Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
- Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Clinical pathology parameters, except 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 pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Indices:
In the present study the glossary of WISE et al. (Wise et al., 1997) was used as much as, possible to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (Phahoud et al., 1999; Solecki et al., 2001):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that occurs also in fetuses of control animals and is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that has otherwise followed a normal pattern of development.
Moreover, the terms "unclassified observation" or "unclassified cartilage observation" were used for those fetal findings, which could not be classified as malformations or variations (e.g. focal liver necrosis in fetuses, isolated cartilage findings without any impact on the respective bony structure).
Historical control data:
The results were compared with historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: (at mid and high dose animals)

Details on maternal toxic effects:
There were no substance-related mortalities in any of the groups. However, a number of low and high dose females either died or had to be sacrificed after gavage error. Those were animals Nos. 31 (50 mg/kg body weight/day) and 93 and 99 (600 mg/kg body weight/day). No relationship of these deaths to the test compound is assumed.

Test group 3 (600 mg/kg body weight/day):
- clinically observed salivation and urine smeared fur
- significantly reduced food consumption (11 % below control)
- significantly increased water consumption (34% above control)
- significantly reduced final body weight (7% below control )
- significantly reduced absolute (23% below control) and net (52% below control) body weight gain
- decreased platelets, sodium, chloride, total protein, globulins and cholesterol
- increased inorganic phosphate and urea
- increased absolute (12% above control) and relative (20% above control) liver weight (due to peroxisomal proliferation)

Test group 2 (200 mg/kg body weight/day):
- clinically observed salivation
- significantly reduced final body weight (6% below control)
- significantly reduced absolute (15% below control) and net (19% below control) body weight gain
- increased relative (5% above control) liver weight (due to peroxisomal proliferation)

Test group 1 (50 mg/kg body weight/day):
- no test substance related findings

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Test group 3 (600 mg/kg body weight/day):
- statistically significantly decreased fetal weights (11 % below control)
- increased incidence of skeletal variations, in particular incompletely or non-ossified morphologically unchanged skeletal structures or unspecific variants of ossification (supernumerary thoracic vertebrae, supernumerary and/or wavy ribs)
Though slight signs of a retardation of prenatal development (lower fetal weights, delay of ossification) were observed at high dose level (600 mg/kg body weight/day) they were considered to be secondary to the clear disturbance of maternal homeostasis during pregnancy and not relevant in terms of developmental toxicity.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dose (mg/kg bw) 0 50 200 600
Mated females 25 25 25 25
Pregnant females 22 22 24 22
Mortality of dams 0 1 0 1
Pregnant on C section 22 22 24 22
Clinical symptoms --- --- + +
Food consumption
day 6-19 p.c. (g)
20.6 19.9 19.7 18.4
Body weight day 0 (g) 174.4 171 170.9 175.5
Body weight day 6 (g) 204.3 268.8 259.6* 261.2*
Body weight gain (days 6-19), g 71.8 69.2 61.2** 55.5**
Uterus weight (g) 52.4 51.2 45.8 48.3
Liver weight rel. (%) 4.15 4.13 4.33* 4.97**
Kidney weight rel. (%) 0.15 0.51 0.52 0.53
Kidney weight rel. (%) 0.17 0.18 0.19* 0.16*
Platelets (giga/l) 788 797 761 682**
Na (mmol/l) 141 140.6 140.4 139.4*
Cl (mmol/l) 100.1 100.1 99.8 98.1**
Inorganic phosphate (mmol/l) 1.8 1.84 1.91 2.13**
Urea (mmol/l) 7 7.36 7.54 8.01**
Total protein (g/l) 66.1 65.1 65.3 60.7**
Globulin (g/l) 30.5 29.8 29.6 25.8**
Cholesterol (mmol/l) 2.29 2.15 5.15 1.84**
+ = salivation, urine smeared fur (high dose only)
* = p</=0.05; ** = p</= 0.01, Dunnett test or Kruskal-Wallis and Wilcoxon 
Caesarian section / fetal examination:
Dose (mg/kg bw) 0 50 200 600
Mated females 25 25 25 25
Pregnant on C section 22 22 24 22
Corpora lutea (mean) 10.8 10.8 10.8 11
Implantation sites (mean) 9.8 9.8 8.8 10
Post-implantation loss (mean %) 3.4 4.5 8.2 5.1
Dead fetuses/litter (mean %) 0 0 0 0
Resorption sites/litter (mean) 0.3 0.5 0.4 0.5
Mean No. of live fetuses/litter 9.5 9.3 8.8 9.5
Mean fetal weights, males (g) 3.6 3.7 3.6 3.2**
Mean fetal weights, females (g) 3.5 3.5 3.4 3.1**
% of fetuses with external malformations 0 0.5 0 0
% of fetuses with visceral malformations 0 0 1.1 0
% of fetuses with skeletal malformations 0 2.8 1.9 3.6
% of fetuses with external variations 0 0 0 0
% of fetuses with visceral variations 9.3 10 9.5 6.1
% of fetuses with skeletal variations 96 95 99 100

Applicant's summary and conclusion