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Diss Factsheets

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
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Mean weight at study initiation: 295 g for males and 199 g for females
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) as environmental enrichment. Initially, all animals were housed five per cage, separated by sex. However, from 2 July 2010 onwards, because of their increased weight, male animals were housed 2 or 3 animals per cage.
- Diet: Rat and Mouse No. 3 Breeding Diet RM3 (SDS Special Diets Services, Witham, England) was available ad libitum, except during periods of exposure to test substance
- Water: Tap water was available ad libitum via polypropylene bottles, except during periods of exposure to test substance.
- Acclimation period: 8 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
CHAMBER DESCRIPTION
- Exposure equipment: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The units were illuminated externally by normal laboratory TL-lighting.

GENERATION OF TEST ATMOSPHERE
- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 27 and 28 L/min for all groups except during the period from 7 June to 1 July 2010 (the period when also animals of a prenatal developmental inhalation toxicity study were exposed) when it was between 54 and 55 L/min and on 28 June 2010 where it was between 29 and 30 L/min. At all times the flow was higher than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).

MEASUREMENT OF TEMPERATURE AND HUMIDITY
- The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes.
- Mean temperature (± standard deviation) during exposure was 23.8 (± 0.4), 23.5 (± 0.5), 23.4 (± 0.5) and 23.6 (± 0.5) °C for the control, low, mid and high concentration exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 25.1 °C, respectively. The temperature ranged between 25.0 and 25.1 °C during 70 minutes on 11 June for the control group, during 23 minutes on 9 July for the low concentration group and during 10 minutes on 9 July for the high concentration group.
- Mean relative humidity (± standard deviation) during exposure was 40 % (± 3), 40 % (± 3), 38 % (± 2) and 37 % (± 3), respectively. Measured minimum and maximum relative humidity was 28 and 74%, respectively. Relative humidity was less than 30 % (minimum 28 %) during periods ranging from 1 to 9 minutes on 27 May, 1, 15, 20 and 21 July and 19 and 26 August for the high concentration group and on 1 July for the control group. Relative humidity was higher than 70% for group 1 on 1 June (duration 5 min, maximum 74 %), for group 3 on 6 July (duration 3 min, maximum 71.5 %) and for group 4 on 23 July (duration 1 min, maximum 71 %) and on 5 August (duration 5 min, maximum 72.5 %).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test substance in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analysers. The response of the analysers was recorded on a pc every minute using a CAN transmitter. The daily mean response was calculated by averaging values read every minute.
The total carbon analysers were calibrated in the period 18 - 21 May 2010. Calibrations were done by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyser were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material. The calibrations were checked weekly by measuring the concentration from a freshly prepared sample bag with a concentration around the target concentration. The measured concentrations from the sample bags did not deviate more than 5 % from the calculated concentration in the sample bags during the study (maximum difference 2.1 %, 4.4 % and -2.5 % for the low, mid and high concentration, respectively). At the end of the study calibration was also checked with a sample bag with a concentration around the target concentration. Results were 0.5 %, 5.7 % and -0.7 % deviations for the low, mid and high concentration, respectively. Because the deviation of the mid concentration was above 5 %, the measurement was repeated with a newly prepared sample bag. The result was a deviation of the calculated concentration of 3.7 %.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week (i.e., 65 exposure days over a 91-day study period)
Dose / conc.:
4 000 ppm
Remarks:
Group 2: Low dose. Corresponding with an analytical concentration of 3987 ppm.
Dose / conc.:
10 000 ppm
Remarks:
Group 3: Mid dose. Corresponding with an analytical concentration of 9974 ppm and 21240 mg/m³ air.
Dose / conc.:
15 000 ppm
Remarks:
Group 4: High dose. Corresponding with an analytical concentration of 14903 ppm.
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on results of a sub-acute (28-day) inhalation toxicity study with this test substance in rats.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway during each exposure day. On working days, all cages were checked again in the afternoon. At weekend days and public holidays only one check per day was carried out.

BODY WEIGHT: Yes
The body weight of each animal was recorded once during the acclimatisation period (one or two days before the start of the study; nominal day -1/-2 for males and females respectively), at initiation of treatment prior to the first exposure (Nominal day 0), and weekly thereafter. In addition, animals were weighed before sacrifice for calculation of relative organ weights..

FOOD CONSUMPTION:
Food consumption of the main groups was measured per cage, over successive weekly periods (starting on Nominal day 0). The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were made prior to the first exposure in all animals and during the last week of exposure in animals of the control and high concentration group (groups 1 and 4). Because no treatment-related changes were observed in animals of the high concentration group, the eye examinations were not extended to the animals of the intermediate concentration groups (groups 2 and 3). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY:
At scheduled necropsy (Nominal day 91) blood samples of all surviving animals were taken from the abdominal aorta of (overnight) fasted rats whilst under pentobarbital anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
Clinical chemistry determinations were conducted on blood plasma of all surviving animals. Blood samples were collected in heparinised plastic tubes at the same time blood samples for haematology were collected. Plasma was prepared by centrifugation. The following measurements were made: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate

URINALYSIS: Yes
On the day before necropsy, animals were transferred to metabolism cages (one animal per cage) for overnight urine collection. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. The following determinations were carried out in individual samples: volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS NECROPSY
At the end of the exposure period, all surviving animals were killed in such a sequence that the average time of killing was approximately the same for each group. Animals were killed on Nominal day 91. The animals were killed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes. The following organs were weighed (paired organs together) as soon as possible after dissection (to avoid drying): adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, thyroids (with parathyroids), lungs with trachea and larynx, ovaries, uterus, epididymides

Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10 % solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation. adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, epididymides, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs/trachea/larynx, lungs/trachea/larynx, mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth), nerve-peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (three levels), spleen, sternum with bone marrow, stomach (glandular, non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all relevant gross lesions.

HISTOPATHOLOGY:
- Preparation of slides: All tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
- Histopathological examination: The organs of the list above were examined in animals of the high concentration group (group 4) and control group (group 1). The nasal tissues were examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and each lung lobe at 1 level. A few gross lesions and the hearts of the animals of the low and mid concentration groups were also examined microscopically.
Statistics:
- Body weight data, clinical pathology data measured on continuous or semi-continuous scales, and organ weights: data were analysed using one-way analysis of variance (Anova), after checking for homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data not normally distributed, the data were stepwise log or rank transformed prior to the Anova. If the Anova yields a significant effect (p<0.05), intergroup comparisons with the control group were made by Dunnett’s multiple comparison test.
- Food consumption: no statistics were applied on food intake (only two cages per sex).
- Incidences of histopathological changes: Fisher’s exact probability test.
Tests generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 or p<0.01. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.
Clinical signs:
no effects observed
Description (incidence and severity):
The individual observations in the mornings, before the start of each day’s exposure did not indicate exposure-related clinical abnormalities. The group-wise observations during each day’s exposure did not reveal abnormalities.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weights between the groups were seen.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar among the groups throughout the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Near the end of the exposure period no abnormalities were seen in groups 1 and 4, therefore the animals of groups 2 and 3 were not investigated.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The concentration of thrombocytes was statistically significantly decreased in the female animals of the low and mid concentration group. Since a concentration-response relation was not seen, these small differences were not considered related to the treatment. Other parameters associated with the red blood cells were not different among the groups. The relative concentration of the lymphocytes was decreased in male animals of the high concentration group. The decrease in the relative concentration of the lymphocytes and the increase of the relative concentration of the neutrophils in female animals of the low concentration group was considered not related to the treatment because of the absence of a relation with the concentration.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The concentration of ASAT and ALAT was significantly increased in plasma of the male animals of the high concentration group. The isolated small significantly decreased concentration of ASAT in male animals of the low concentration group was not considered to be treatment-related. In female animals of the mid and high concentration groups, glucose and urea were significantly increased. Potassium was significantly increased and triglyceride was slightly, but significantly decreased in female animals of the high concentration group.
Endocrine findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative and absolute weight of the heart was significantly decreased in male animals of the high concentration group. In female animals of that group relative weight was also significantly increased. In male animals of the high concentration group, relative liver weight was slightly, but significantly increased. The significant increase in relative kidney weight of the male animals of the low concentration group was considered an isolated incidental finding, not related to the treatment. Other significant differences in organ weight were not found.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal treatment-related gross changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed treatment related histopathological changes in the hearts of the treated females and males. The changes comprised very slight to moderate multifocal (diffuse in one high-dose male) mononuclear cell infiltrates in the ventricular part of the heart muscle. Several animals showed very slight to slight focal mononuclear cell infiltrates in the heart, but this was not considered treatment related because this finding is a common background change and occurred also in some control animals. The multifocal inflammatory infiltrations were observed in 9/10 high-dose males, in 5/10 high-dose females, in 7/10 mid-dose males and in 1/10 low-dose males. In several cases the inflammatory changes were accompanied by vacuolisation of cardiac muscle cells. The males were clearly more affected than the females. Because of the presence of moderate multifocal mononuclear cell infiltrates with concomitant muscle cell vacuolation in the heart of one male animal of the low-concentration group, a No Adverse Effect Level could not be established. The other histopathological changes observed are common findings in rats of this age and strain. Furthermore, they were about equally distributed amongst the groups or they occurred in only one or a few animals.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
LOAEC
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Equivalent to 21240 mg/m³ air
Key result
Critical effects observed:
not specified
Conclusions:
Under the conditions of the test, a NOAEC in rats exposed to the test substance for 6 hours/day, 5 days/week for a period of 90 days could not be derived. Therefore the lowest concentration was considered to be the LOAEC: 4000 ppm (21240 mg/m3).
Executive summary:

In a 90-day repeated dose toxicity study, performed according to OECD Guideline 413 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at concentrations of 4000, 10000, and 15000 ppm to groups of 10 Sprague-Dawley rats per sex each. The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. Daily observation of the animals did not reveal treatment-related clinical abnormalities. Treatment-related differences in body weight gain or food consumption were not seen. Ophthalmoscopic examination near the end of the exposure period did not reveal any abnormalities. Treatment-related effects in red blood cell parameters were not seen. In male animals of the high concentration group the relative concentration of lymphocytes was decreased. No other treatment-related effects were seen in any of the other haematology parameters. In the high concentration group, the concentration of ASAT and ALAT in blood plasma was significantly increased in male animals. In female animals of the high and mid concentration groups glucose and urea were increased. Female animals of the high concentration group also showed increased potassium and decreased triglyceride levels. Other treatment-related changes in clinical chemistry parameters were not seen. Occult blood in urine was significantly increased in male animals of the high concentration group. The relative and absolute weight of the heart was significantly decreased and the relative weight of the liver was significantly increased in male animals of the high concentration group. Relative weight of the heart was also significantly decreased in female animals of the high concentration group. No other treatment-related changes in absolute or relative organ weights were detected. Macroscopic examination at necropsy did not show exposure-related gross changes. Microscopic examination revealed that exposure to the test substance induced multifocal mononuclear cell infiltrates in the ventricular part of the heart muscle. It was seen in males and females of the high-concentration group and with a lower incidence in male animals of the mid and low concentration groups. Because the histopathological changes in the heart of one male animal of the low concentration group was considered to be related to the exposure, a NOAEC can not be given and the lowest concentration tested was considered to be the LOAEC: 4000 ppm (21240 mg/m3).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
gas under pressure: liquefied gas

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charlese River Deutschland, Sulzfeld, Germany
- Age at study initiation: females were 11 weeks old at mating; males were 13 weeks old at mating
- Weight at study initiation: Female weights ranged from 196.90 to 201.16 g on gestation day 0.
- Fasting period before study: None
- Housing: The rats were housed in macrolon cages with a bedding of wood shavings and strips of paper (Enviro-dri) as environmental enrichment. During the quarantine and acclimatisation period, the males were housed individually and the females were housed in groups of 4. For mating, one male and two females were housed together in type 3 macrolon cages. Mated females were housed individually in macrolon cages which were placed in a separate cage rack. The location of the mated females in the new cage racks were determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females was housed in the order of animal number).
During exposure periods, the rats were individually housed in the exposure unit.
- Diet: The rats received a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure.
- Water: The drinking water (tap-water) was available ad libitum and given in polypropylene bottles, which were cleaned weekly and filled as needed. Drinking water was provided from the arrival of the rats until the end of the study, except during exposure.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
EXPOSURE EQUIPMENT:
Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The location of the rats was changed weekly by rotating the animals 5 places clockwise and moving the animals from the upper section to the middle section, the animals from the middle section to the lower section and the animals from the lower section to the upper section. The units were illuminated externally by normal laboratory TL-lighting.

GENERATION OF TEST ATMOSPHERE:
- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 54 and 55 L/min for all groups except on one day, where it was between 29 and 30 L/min. At all times the flow was more than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ACTUAL CONCENTRATIONS:
- The actual concentration of the test material in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analyzers. The response of the analyzers was recorded on a pc every minute using a CAN transmitter.
- The daily mean response was calculated by averaging values read every minute.
- The total carbon analyzers were calibrated by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyzer were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material.
Details on mating procedure:
At the start of mating, 2 nulliparous females were caged with each male for mating until a sperm positive vaginal smear was detected. Every consecutive morning vaginal examinations were made to ascertain copulation by detection of sperm cells in a vaginal smear. Upon evidence of copulation, positive females were housed individually. The day a sperm-positive smear is detected was considered as gestation day 0. The mated females were distributed over the 4 experimental groups in such a way that the animals from the same day of pregnancy were, as far as possible, equally distributed over all groups. Females mated by the same male were placed in different groups.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
From gestation day 6 up to and including gestation day 19.
Duration of test:
In-life termination of female rats occurred on gestation day 21.
Doses / concentrationsopen allclose all
Dose / conc.:
4 000 ppm
Remarks:
Group 2: Low dose. Corresponding with an analytical concentration of 3988 ppm.
Dose / conc.:
10 000 ppm
Remarks:
Group 3: Mid dose. Corresponding with an analytical concentration of 9981 ppm.
Dose / conc.:
15 000 ppm
Remarks:
Group 4: High dose. Corresponding with an analytical concentration of 14906 ppm.
No. of animals per sex per dose:
24 pregnant female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: The concentration levels were selected in consultation with the sponsor and were the same as used in the sub-chronic (90-day) inhalation toxicity study in rats (TNO Triskelion study V8964).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily and, if necessary, the animal was handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

CLINICAL OBSERVATIONS DURING EXPOSURE:
- Time schedule: A group-wise observation was made halfway through each exposure day.

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation days 0, 3, 6, 9, 12, 15, 19 and 21.

FOOD CONSUMPTION: Yes
- The food consumed for each mated female was measured over the periods: gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-19 and 19-21.
- The results were expressed in g per animal per day and g per kg body weight per day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri (including the fetuses), ovaries and placentas of all females.
Ovaries and uterine content:
The uteri (including the fetuses), ovaries and placentas of all females killed on gestation day 21 were examined for the following parameters: number of corpora lutea, number of implantation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities, weight of ovaries, weight of uterus, containing placentas and fetuses, weight of empty uterus, weight of live fetuses, weight of the placentas of live fetuses, gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: [all]
- Soft tissue examinations: Yes [half per litter ]
- Skeletal examinations: Yes [half per litter ]
Statistics:
The resulting data were analysed using the methods mentioned below. As a level of significance was considered: p< 0.05.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live fetuses.
- Number of corpora lutea, implantation sites, live and dead fetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
- Mortality data, data of the pathology of parent females and the fetopathological screening were evaluated by the Fisher’s exact probability test.
Indices:
As far as applicable for each group the following indices were recorded:
- female fecundity index = (number of pregnant females/number of females mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation sites)/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Daily clinical observations during gestation did not reveal any remarkable findings in animals’ appearance, general condition or behaviour among the test and control groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in body weight and body weight change were observed between the females of the test groups and the control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (both g/kg body weight/day and g/animal/day) of the test groups did not differ from the control group
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean weight of the gravid uterus, carcass, net weight change from day 0, empty uterus and ovaries of the groups exposed to the test substance did not differ from the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant differences were observed in the incidence of parental necropsy observations among the groups. The findings observed were incidental and not related to treatment
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
One female of the control group had no viable foetuses including a late resorption. No statistically significant differences were observed in the gestation index and female fecundity index among the groups. No statistically significant differences were observed in the number of corpora lutea, implantation sites, pre- and post-implantation loss, live and dead foetuses, resorptions and sex ratio between test groups and control group.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
In each group, 24 females were mated. A total of 18, 17, 20 and 18 females of the control, low-, mid-, and high-concentration test substance groups, respectively, appeared to be pregnant.

Effect levels (maternal animals)

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Key result
Dose descriptor:
NOEC
Remarks:
Maternal toxicity
Effect level:
>= 15 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: Equivalent to 79650 mg/m3
Key result
Dose descriptor:
NOEC
Remarks:
Maternal developmental toxicity
Effect level:
10 000 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: Equivalent to 53100 mg/m3

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed on placenta and fetal weight among the groups.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related statistically significant differences in fetal external observations. One female of the low-concentration test substance group showed a fetus with a soft skull, malformed ears and subcutaneous oedema. Because no similar effect was observed in the mid- and high-dose groups, these findings were considered as incidental and not related to treatment. At skeletal examination of the one female fetus of the low-concentration test substance group, only incomplete ossification of the frontal, parietal, interparietal and supra occipital bones was observed. Other fetal external observations that occurred at low incidences in all groups included small fetuses and small subcutaneous haemorrhages/petechiae on the skin. These incidental observations are common for this strain and not related to treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
- No skeletal malformations were observed.
- Skeletal anomalies: No skeletal anomalies were observed.
- Skeletal variations: Skeletal variations were seen in parietal and interparietal bones (supernumerary), supraoccipital bones (holes), ribs (accessory lumbar ribs), and sternebrae (irregular ossification of one or irregular shape of one or more) and caudal bodies (one or two supernumerary). No statistically significant differences were observed in skeletal variations among the groups.
- Skeletal retardations: A few statistically significant differences were observed in the incidences of fetuses and/or litters showing skeletal retardations after treatment with the test substance: (1) Decreased fetal incidence of three or more incompletely ossified caudal arches in the low- and mid-concentration groups. (2) Decreased fetal incidence of 1-2 incompletely ossified metacarpals in the low-concentration group. The observed statistically significant differences in retardations of fetal skeletons were incidental, inconsistent and/or not dose-related. This kind of variation in skeletal ossification is considered as normal developmental variability. No other indications of developmental toxicity were observed. Therefore, these variations are considered to be not treatment-related.
Visceral malformations:
no effects observed
Description (incidence and severity):
- No visceral malformations were observed.
- Visceral anomalies: A statistically significant increase was observed in the incidence of a dilated urinary bladder in foetuses of the high-concentration test substance group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses of the test substance. Therefore this finding is considered to be a treatment-related effect. One foetus of the mid-concentration group showed diverticulum of the intestines. This finding occurred in the mid-concentration group only; therefore, this finding was considered not to be a treatment-related effect. No other statistically significant effects in the incidence of visceral anomalies were observed among the groups.
- Visceral variations: Visceral variations in all groups included haemorrhagic areas in the oral cavity, haemorrhagic areas in the nasal cavity, folded retinas, not well-defined soft lenses, pericard and stomach filled with haemorrhagic fluid, and kinked and bent ureters. The incidences of folded retina and stomach containing haemorrhagic fluid were statistically significantly decreased in the high-concentration test substance group and the low-concentration group, respectively. Overall, the total incidence of foetal visceral variations was decreased in the low- and high-concentration groups compared to the control group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Findings of the placenta: In one female of the control group, a large placenta and an amnion sac filled with red fluid were observed with a late resorption. This female had no viable fetuses. No treatment-related findings of the placenta were observed.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEC
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group (15000 ppm or 79650 mg/m3) was considered to be a treatment-related effect. Therefore, the NOEL for developmental toxicity was determined to be 10000 ppm (53100 mg/m3). Maternal toxicity was not observed up to and including the highest dose tested. Therefore, the NOEL for maternal toxicity was determined to be 15000 ppm.
Executive summary:

In a developmental toxicity study performed according to OECD 414 and GLP, 24 pregnant Wistar rats per dose group were exposed by nose-only inhalation from Gestation Day (GD) 6 to GD 19. The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 4000, 10000 or 15000 ppm for 6 hours per day. Inhalation of the test substance did not induce any relevant changes in the condition or macroscopic findings of the dams. No treatment-related effects were observed on body weight, body weight gain and food consumption of the females exposed to the test substance. Gestation index, female fecundity index, the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead foetuses, resorptions and sex ratio after exposure to the test substance did not differ from none-treated controls. Parental necropsy did not reveal any differences in reproductive organ weights and macroscopic findings. Foetal external- and placental observations and weights did not reveal any treatment-related effects. A statistically significant increase in the incidence of dilated urinary bladders was observed in foetuses of the high-dose group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses. Therefore this finding is considered to be a treatment-related effect. No treatment-related effects were observed on visceral malformations and variations, and on skeletal malformations, anomalies, variations, and retardations. In conclusion, the increased incidence of dilated urinary bladders in foetuses of the high-dose group was considered to be a treatment-related effect. Therefore, under the conditions of this study, inhalation of the test substance at concentrations up to 10000 ppm during gestation of females was tolerated without obvious signs of developmental toxicity. Under the conditions of this study, inhalation of the test substance at concentrations up to 15000 ppm during gestation of females was tolerated without obvious signs of maternal toxicity.