Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 9 weeks
- Housing:
Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany / up to 6 animals,
for Motor Activity Measurement: polycarbonate cages (floor area about 800 cm², Ehret, Emmendingen, Germany) / 1 animal,
during Exposure: Wire cages , type DK III (BECKER & Co., Castrop-Rauxel, Germany) / 2 animal.
- 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: 2 weeks

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:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Analytical concentrations were 857.5 +/-122.2, 3008.1 +/-82.7, 9570.5 +/-434.5 mg/m3
Details on inhalation 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:
90 days
Frequency of treatment:
5 times a week (65 exposures in total)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 3000, 10000 mg/m3
Basis:
other: Target concentrations
Remarks:
Doses / Concentrations:
857.5, 3008.1, 9570.5 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1119.4, 3372.5, 11879.0 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, mortality (twice/day), general clinical symptoms (3 times on exposure days, once during pre- and post-exposure), observation of abnormalities, particularly the following parameters: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.

OPEN FIELD OBSERVATION:
animals were removed from their cages and placed in a standard arena (50 x 50 x 25 cm). Besides other abnormalities, the following parameters listed were assessed: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypes, Gait, Activity/arousal level, Feces (consistency/ color) within two minutes, Urine (amount/ color) within two minutes, Rearing within two minutes, Other findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: outside cages once before the beginning of the administration period (day 0) and on study days 42 and 84 (in the morning). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in PDS ToxData® software and included but is not limited to the following parameters: Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/ arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/ consistency), Assessment of the urine discharged during the examination, Pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to pre-exposure period, at the start of exposure period (day 0) and twice weekly thereafter. The body weight change of the respective week was calculated as the difference of Friday to the previous Monday.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION: Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the beginning of administration, the eyes of all animals will be examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic (Mydrum, Firma Chauvin ankerpharm GmbH, Rudolstadt, Germany). Against the end of the exposure period, the eyes of all animals of the control and high concentration group will be examined. The eyes of the animals of the other animals will be examined only if there is a striking discrepancy between the high concentration group and the control group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 92
- Anaesthetic used for blood collection: Yes, from the retrobulbar venous plexus in the morning from fasted animals using isoflurane as anesthesia.
- Animals fasted: Yes
- Parameters checked: Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Differential blood count, Reticulocytes, Preparation of blood smears (only evaluated blood smears will be archived), Prothrombine time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 92
- Animals fasted: Yes
- Parameters checked: Alanine aminotransferase; Aspartate aminotransferase, Alkaline phosphatase, Serum g glutamyl transferase, Sodium, Potassium, Chloride, Inorg. phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol.


URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- The FOB was carried out in all male and female animals for each test group once on study day 90. On the day of FOB the examined animals as well as the remaining animals of each test group were not exposed to the test substance. The examinations generally started in the morning.
At least one hour before the start of the FOB the animals were transferred individually to polycarbonate cages (floor area about 800 cm²). Drinking water was provided ad libitum whereas no food was offered during the measurements.
The FOB will started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Sensory motor tests / Reflex tests. Animals were removed from the open field and will be subjected to the sensory motor and reflex tests including: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordiantion of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test:
- Measurement of motor activity (MA) was carried out in all male and female animals at the end of the exposure period (in a randomized sequence on each examination day). On the day of MA the examined animals were not exposed.

Sacrifice and pathology:
Necropsy was performed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava.

GROSS PATHOLOGY: Yes, Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus.

HISTOPATHOLOGY: Yes (see table)
Statistics:
Mean values and standard deviations were calculated. The following statistical analyses were carried out, additionally:
Body weights, body weight change, Food consumption: DUNNET
Feces, rearing, grip strength fore and hindlimbs, foot-splay test, motor activity, Blood examinations, Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS and WILCOXON Test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs and mortality:
No deaths were recorded throughout the study. In comparison to the concurrent control group, the animals of the low (1000 mg/m³), mid concentration (3000 mg/m³) groups showed no treatment-related clinical signs of toxicity during the exposure period. In animals of the high concentration (10000 mg/m³) group, apathy was observed during the exposure on the first few exposure days indicating a narcotic effect of the test substance. After cessation of the exposure, the animals showed unsteady gait during their movement. From study day 7 onward, apathy and unsteady gait were not observed any further, probably due to adaptation to the exposure of the test substance. In addition, salivation was observed intermittently in 9 of the 10 males and in all females. Moreover, nose and eye discharge and encrusted eyes were observed occasionally in individuals of male and female animals. All female animals showed hyperexcitability at the end of exposure period.

Body weight and weight gain:
The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
At the initial phase of the exposure period, the male animals of the test group 3 gained less body weight than the concurrent controls. Due to its transient
nature, and since the body weight was in a comparable range with the concurrent controls, the decreased body weight gain in the high concentration group was considered not adverse.
In female animals from study day 46 to 50, the mean body weight changes of all test groups were higher than the control group. This transient finding is likely to be related to a low control value.

Neurobehaviour:
No substance related effects were observed in quantitative parameters, Home cage observations, Open field observations, and Sensorimotor tests/reflexes.

Regarding the overall motor activity, the mean value was reduced with statistical significance in the male animals in the high concentration (10000 mg/m³) on day 90. There were no test substance-related deviations for male and female rats of test groups 1 and 2, as well as females of test group 3. The increased overall motor activity in test group 3 as isolated single interval variances were considered incidental, because there were no concentration-response relationship.

Clinical chemistry:
Chloride levels were decreased and inorganic phosphate levels were increased in both sexes of test group 3 (10000 mg/m3) .
Inorganic phosphate levels were already higher in females of test group 2 (3000 mg/m3) compared to controls, but this mean was within the historical control range and therefore, the alteration in these individuals was regarded as incidental and not treatment-related.
In females of test group 3 (10000 mg/m3) urea levels were increased and in males of the same test group triglyceride levels were decreased.
In females of test group 1 (1000 mg/m3) total protein and albumin values were lower compared to controls, but the alteration was not concentration-related.
In females of test group 3 (10000 mg/m3), alanine aminotransferase (ALT) activities were higher compared to controls, but the mean was only marginally above the historical control range. In males of the same test group glucose levels were lower compared to controls. Glucose levels were already lower in males of test group 2 (3000 mg/m3). However, the glucose means were within the historical control range. Therefore, observations for ALT and glucose were regarded as incidental and not treatment-related.

Relative organ weights (see table 1):
In the kidneys of males at 10000 mg/m³, a significant absolute (+20%) and relative (+19%) weight increase was regarded as treatment-related and was consistent with an increase of eosinophilic droplets in the proximal convoluted tubules. Eosinophilic droplets represent a male and rat-specific poor soluble protein (α2u-globulin) synthesised in the male rat liver, filtered by the glomerulus and reabsorbed in the proximal tubules where it is slowly hydrolysed by lysosomal digestion. Reversible binding of the test substance or their metabolites to α2uglobulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible histologically in form of characteristic eosinophilic droplets. Although the increase of eosinophilic droplets is considered treatmentrelated, since no signs of tissue injury were detected in the renal tubules, it was regarded as not adverse. Furthermore, this finding does not represent a risk for humans since they do not synthesise this protein (Durham and Swenberg, 2013).
The significant increase of the relative weight of the kidneys (0.613%) in males of test group 2 (3000 mg/m3) was within the historical control range and had no histopathological correlate and was therefore regarded as incidental.
The significant absolute and relative liver weight increase in males and females of test group 3 correlated with histopathological findings and
was considered treatment-related. Since only one clinical chemistry liver parameter was altered in each sex (urea in females and triglycerides in
males), the weight and histological changes of the liver were considered treatment-related and adaptive.
The absolute (119.9 mg) and relative (0.052%) weight of the ovaries in females of test group 3 was above the maximal historical control values (absolute: 104.4 mg, relative: 0.049%). However, histopathological findings were not detected either in the ovaries or in the uterus and vagina that could indicate altered ovarian function. Although a treatment-related effect cannot be ruled out, this was regarded as not adverse. Though the absolute weight of the ovaries in test groups 1 and 2 (1000 and 3000 mg/m3) were over the maximal control values, since they were not accompanied by significant increases of the relative weights, they were regarded as incidental and not related to treatment.
The significant weight changes of the heart and kidneys in females at 10000 mg/m3 had no histopathological correlate; furthermore they were
all within the historical control range and were therefore regarded as incidental and not treatment-related.

Effect levels

Dose descriptor:
NOAEC
Effect level:
3 000 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical pathology, salivation, nose and eye discharge, and transient apathy

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Relative change of relative organ weight

[%]

Male animals

Female animals

mg/m³

1000

3000

10000

1000

3000

10000

Kidneys

103

111**

119**

99

102

111

Liver

97

100

116**

99

98

118

Ovaries

 

 

 

109

112

130

Heart

 

 

 

97

95

107

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

Applicant's summary and conclusion