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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:
other: OECD guideline study, GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Inhalation exposure according to OECD 412
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany.
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-dioxepane
- Physical state: liquid
- Analytical purity: 99.76 area-%
- Lot/batch No.: 09/0372-3
- Expiration date of the lot/batch: 22 Apr 2015
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Time-mated Wistar rats (Crl:WI[Han]) supplied in GD0, Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 10-12 weeks
- Housing:
Polycarbonat cages type III supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area about 800 cm²). During exposure: up to 4 animals were housed together in wire cages, type DK III (BECKER & Co., Castrop-Rauxel, Germany), divided into 4 separate space. Dust-free wooden bedding was used in this study, for enrichment, wooden gnawing blocks were offered.
- Diet (e.g. ad libitum): Kliba laboratory diet, mouse/rat maintenance “GLP”, 10 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland
- Water (e.g. ad libitum): yes
- Acclimation period: for adaptation to the exposure conditions the animals were placed into exposure cages from GD 3 – GD 5

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:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

-Equipment: Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Germany); Atomization vaporizer (glass) with thermostat (BASF SE, Germany); Thermostat (JULABO Labortechnik GmbH, Germany)
-Generation technique: for each concentration, constant amounts of the substance to be tested were supplied to the thermostated vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and passed into the inhalation systems.
-Whole-body inhalation system: The animals were housed singly in wire cages (DK III) that are located in a glass-steel inhalation chambers, V approx. 1.1 m³. The inhalation atmospheres are passed into the inhalation chambers with the supply air and are removed by an exhaust air system. For same exposure conditions, the cages with the animals were rotated between the levels within each chamber.
- Measurement and recording of technical conditions in the exposure systems: The air flow rates of supply and exhaust air, pressure conditions inside the chambers, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also recorded by means of this system.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentrations were analyzed both by off-line gas chromatography of absorption samples and by on-line total hydrocarbon analyzers (FID, abbreviation for flam ionization detector).
The constancy of concentrations in the inhalation atmospheres were surveyed continuously with total hydrocarbon analyzers (FID, Testa), which are calibrated with certificated test gas propane.
Absorption samples were analyzed during the first five study days to confirm the identity of the test substance in the test atmosphere. The control atmosphere will not be sampled. Atmospheric concentrations are determined by online FID measurement (sampling velocity in the sampling probe: 1.25 m/sec; sampling frequency: three samples per concentration during the exposure period).
- Samples taken from breathing zone: yes
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
All test groups were exposed for 6 hours over a time period of 14 consecutive days, on GD 6
through 19. The number of exposure days was 14.
Frequency of treatment:
daily over a time period of 14 consecutive days, on GD 6 through 19
Duration of test:
14 consecutive days of exposure
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 3000, 10000 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
921.2, 3018.2, 9453.4 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Mortality twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).
Clinical symptoms during the pre-exposure period and on the day of necropsy at least once a day. During the exposure period, at least three times a day (before, during and after exposure).
Food consumption 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 data on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.
Clinical Pathology on study termination, Blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane.
Hematology: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT),mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), differential blood count, Reticulocytes (RET).
Clinical chemistry: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
Organ weights: Anesthetized animals, Adrenal glands, Brain, Heart, Kidneys, Liver, Lungs, Spleen, Thymus
Organ/tissue fixation: All gross lesions, Adrenal glands, Brain, Heart, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Nose, Pharynx, Spleen, Thymus, Trachea.
Ovaries and uterine content:
Cesarean section:
On GD 20, the dams were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava.
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, Dead implantations (Early resorptions, Late resorptions, 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 C W (1955) multiple comparison procedure for Food 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.
Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (onesided) for 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 (onesided) for proportions of fetuses with malformations,
variations and/or unclassified observations in each litter.
WILCOXON-test (two-sided) for blood parameters.
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided) for weight parameters.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical examinations
2, 3 and 1 damn of test groups 0, 3000, or 10000 mg/m3, respectively, were not pregnant and were excluded from further assessments.
There were no mortalities.
During the exposure period, some females (12 out of 25) of test group 10000 mg/m³ showed transient salivation after exposure. During the post-exposure observation day no clinical signs or findings wer observed.
The dams in test group 10000 mg/m³ consumed about 11% less food in comparison to the concurrent control group if calculated for the entire treatment period (GD 6-19).

The mean body weights (BW) and the average body weight gain (BWC) of the dams in test group 10000 mg/m³ were statistically significantly reduced from GD 8-20 (BW) and on GD 6-8 (BWC). If calculated for the entire treatment period (GD 6-19) these dams gained 18% less body weight than the concurrent control animals. The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 10000 mg/m³ (about 29% below the concurrent control value). Furthermore, the carcass weight of the high-concentration dams was also statistically significantly reduced in comparison to the control group (about 4% below controls). These effects are assessed as
test substance-related signs of maternal toxicity.
The body weights of test groups 1000 and 3000 mg/m³ revealed no difference of any biological relevance to the corresponding control group.

Hematology
At gestation day 20 in dams of test group 10000 mg/m3, hemoglobin and hematocrit values as well as relative reticulocyte counts were higher compared to controls. However, all mentioned parameter values were within historical control ranges. Therefore, these alterations in the red blood cell parameters were regarded as incidental and not treatment-related.
In dams of test group 10000 mg/m3 absolute and relative eosinophil cell counts were decreased.

Clinical chemistry
At gestation day 20 in dams of test group 10000 mg/m3, cholesterol levels were increased. Apart from this change, in the same individuals alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities as well as urea, total bilirubin and inorganic phosphate levels were higher and triglyceride as well as creatinine values were lower compared to controls. However, all mentioned parameter values were within historical control ranges. Therefore, these alterations were regarded as incidental and not treatment-related.

Uterus weight
The mean gravid uterus weight of the animals of test group 10000 mg/m³ was slightly reduced (without attaining statistical significance) in comparison to the control group (about 7% below controls), which is likely to be a subsequent effect of reduced mean fetal weights in this test group. The mean gravid uterus weights of the animals of test groups 1000 and 3000 mg/m³ 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.

Reproduction data
The conception rate was 88% in the mid-concentration group (3000 mg/m³), 92% in the control, 96% in the high-concentration group (10000 mg/m³) and 100% in the low concentration group (1000 mg/m³). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between test groups 0, 1000, 3000 and 10000 mg/m³ in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, 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.

Relative organ weights
See Table 1.
The significant absolute and relative thymus weight decrease was considered to be treatment-related.
Since the absolute kidneys weight was not altered and only a minimal increase of the relative weight (+9%) was recorded at 10000 mg/m3, this was attributed to the decreased terminal body weight. The relative liver weight increase at 3000 and 10000 mg/m3 (+6 % and +12%) although not very high, was most likely treatment-related.

Gross lesions
No treatment related findings.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
3 000 mg/m³ air
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:
Sex distribution of the fetuses
The sex distribution of the fetuses in test groups 1000, 3000 and 10000 mg/m³ was comparable to the control fetuses.

Weight of the placentae
The mean placental weights of the low-, mid- and high-concentration groups were comparable to the corresponding control group.

Weight of the fetuses
The mean fetal weights of test group 10000 mg/m³ were statistically significantly reduced (about 8% below control - both sexes combined).
The mean fetal weights of test groups 1000 and 3000 mg/m³ were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Fetal external malformations/variations
One external malformation was recorded for one control fetus (mandible malformed). No external variations were recorded.

Fetal external unclassified observations
Two unclassified external observations, i.e. blood coagulum around placenta and pointed lower jaw, were recorded in two fetuses of the high-concentration group. These findings were not considered biologically relevant.

Fetal soft tissue malformations/variations
Soft tissue malformations occurred in one fetus, each, of the low- and mid-concentration group. Both fetuses had malformations affecting the urinary tract (hydronephrosis, hydroureter) which are present in the historical control data. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Three soft tissue variations, i.e. short innominate, dilated renal pelvis and dilated ureter, were detected in all test groups including the control. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant. All of
them can be found in the historical control data at comparable incidences.
No unclassified soft tissue observations were recorded.

Fetal skeletal malformations/variations
Some skeletal malformations were detected in test groups 0, 1000 or 10000 mg/m³. One control fetus had an associated external finding.
The incidences of these malformations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
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 in the majority of cases without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
A few variations – affecting some elements of the vertebral column and ribs were, at the top concentration, were present at incidences above the historical control range. Those minor changes either represent slight delays of ossification which did not affect morphology, or minor departures from normal morphology.
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. However, the incidence of bipartite processus xiphoideus was statistically significantly increased in test 1000 mg/m³. As a consequence of this occasional increase, also the incidence of total fetal skeletal unclassified cartilage observations was statistically significantly
increased in this test group. However, this finding showed no doseresponse and is therefore assessed to be without biological relevance.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
3 000 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxiciy

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/m³ air
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Relative organ weights 

[%]

Female animals

mg/m³

1000

3000

10000

Kidneys

102

102

109**

Liver

102

106**

112**

Thymus

103

97

81**

* : p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Executive summary:

In a prenatal developmental toxicity study the test substance 1,3-dioxepane was administered to pregnant Wistar rats via inhalation (whole-body exposure) from implantation to one day prior to the expected day of parturition (GD 6-19) at target concentrations of 1000, 3000 and 10000 mg/m³ to evaluate its potential maternal and prenatal developmental toxicity.

During the exposure period, 12 out of 25 females of test group 10000 mg/m³ showed transient salivation after exposure. Salivation persisted in the respective animals only for some minutes after exposure and was initially observed on GD 6. During the post-exposure observation day, all females were free from clinical signs or findings, which could be attributed to the test substance. Mean food consumption as well as gross and net body weights/body weight gain of the dams in test group 10000 mg/m³ were statistically significantly reduced. The high-concentration dams consumed about 11% less food and gained 18% (gross) or 29% (net) less body weight than the concurrent control animals. These effects are assessed as test substance-related signs of maternal toxicity. Regarding clinical pathology in dams of test group 10000 mg/m3 at gestation day 20, lower absolute and relative eosinophil counts were due to an increased stress situation. Higher cholesterol levels indicated an altered liver cell metabolism.

Regarding pathology, the terminal body weight was minimally but statistically significantly decreased (-4%) in the females at 10000 mg/m3, which was regarded as treatment-related. The significant absolute (-22%) and relative (-19%) thymus weight decrease in the same test group was considered to be associated with stress and was consistent with the relative decrease of eosinophils in the hematologic evaluation. The significant relative liver weight increase at 3000 mg/m3 (+6%) and 10000 mg/m3 (+12%) was regarded as treatment-related. However, a conclusive interpretation of adversity in the liver cannot be made without further histopathological examination and should rely on clinical pathology only.

No differences of toxicological relevance between the control and the treated groups (1000, 3000 and 10000 mg/m³) 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 influence of the test compound on sex distribution of the fetuses was noted at any concentration. The mean fetal weights of the high-concentration group (10000 mg/m³) were slightly but statistically significantly reduced (about 8% below control). This slight reduction is considered to be subsequent to a consistently lower food consumption and body weight gain of the dams in the high-concentration group, and thus not considered to be evidence of developmental toxicity on their own.

Fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to a concentration of 3000 mg/m³. At 10000 mg/m³ a few variations affecting some elements of the vertebral column and ribs were present at incidences above the historical control range. Incidences of incomplete ossifications of various skeletal elements represent temporary delays in development which have no permanent effect on morphology and function of the affected organs or structures. They are neither considered to be evidence of developmental toxicity on their own nor to be adverse. Litter and affected fetuses/litter incidences of supernumerary thoracic vertebrae and 14th ribs were distinctly increased above the inherently high background rate, thus an association to the test substance cannot be excluded. However, taking into account the considerable capacity of the postnatal skeleton to remodel (Holmbeck and Szabova, 2006) and the low impact of these findings on postnatal physical health, their toxicological significance is considered to be rather low. Thus, the test item is not teratogenic in rats.