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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Aug 2002 - 04 Dec 2003 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Aug 2002 - 04 Dec 2003 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)
Version / remarks:
712-C-00-368; July, 2002
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
712-C-98-204; August, 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG)
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG)
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L
Duration of treatment / exposure:
- males: 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.106 mg/L air (nominal)
Remarks:
equals 20 ppm
Dose / conc.:
0.319 mg/L air (nominal)
Remarks:
equals 60 ppm
Dose / conc.:
0.956 mg/L air (nominal)
Remarks:
equals 180 ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
106 mg/m3 (ca. 20 ppm) : as the expected no observed adverse effect level
319 mg/m3 (ca. 60 ppm) : as the intermediate dose level
956 mg/m3 (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

URINALYSIS:
- Time schedule for collection of urine: not reported
- Analysis only performed in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Sacrifice and pathology:
GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter . Testes, epididymides and ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group
Statistics:
Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups delivered per litter. Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy and urine analysis except volume, colour, turbidity and specific gravity. Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations. Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the study period one male animal of the control group showed injuries laterally at right flank and on the right head. This injury was of mechanic matter and was therefore not related to the study. One female animal of the low concentration group showed alopecia on dorsal body region and on both forelimbs. This was most likely to be incidental, because alopecia was not observed in other animals of the low and mid concentration group. At the high concentration (0.956 mg/L) male and female animals showed various clinical abnormalities comprising slight to moderate visually increased respiration, eyelid closure, salivation, eye discharge (red) indicating that the test substance was irritating to eyes and upper respiratory tract at this high concentration. Other findings like aggressiveness, apathy (1 female), as well as alopecia and piloerection were more of general nature indicating the bad general state of the animals.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy)
Mortality:
mortality observed, treatment-related
Description (incidence):
Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development of the high concentration animals was substantially impaired by the exposure to the test substance. Although the mean body weights on some examination days were not of statistical significance, the significantly reduced mean body weight gains proved the existence of this effect. The retardation of the body weight development is not only of secondary matter due to the reduced food consumption.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Test group 3 (0.956 mg/l = 180 ppm):
Retarded body weight development of the males
- mean body weight: - 8.1 % to - 15.5 % of the control from study day 9 onward (statistically significant to a level of 99 %)
- mean body weight gain: -23.2 % to -36.3 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Retarded body weight development of the females
- mean body weight: - 3.0 % to - 8.0 % of the control from study day 9 onward (statistically significant on study day 51 to a level of 95 %)
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Significantly (to a level of 99 %) decreased mean terminal body weight in males and in females (- 17.2 % and -9.4 %, resp.)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.
Food efficiency:
no effects observed
Description (incidence and severity):
The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There are no treatment-related changes in the haematological parameters measured. Clotting analysis revealed prolonged prothrombin times in the blood of the males of the high concentration group at the end of the study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm): :
-Increased prothrombin times, urea*, magnesium*, urinary specific gravity and urinary casts* in the males
-Decreased triglycerides** and urinary volume in the males
-Decreased chloride, creatinine*, total bilirubin, total protein**, albumin* and globulins** in the females
(* statistically significant to a level of 95 %
** statistically significant to a level of 99 %)

Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding clinical pathology findings, treatment-related effects were observed only in the high concentration groups. The investigations revealed mild impairment of renal function in the males, substantiated by slightly increased urea concentrations in the serum, excretion of decreased urinary volume with increased specific gravity and the presence of urinary casts.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related findings were observed in the functional observation battery and motor activity examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The administration of the test article had significantly decreased the mean terminal body weight of male and female rats of the high dose group. As a consequence of the treatment-related body weight loss, a few absolute organ weights were significantly decreased in males of the high dose group (liver and thymus), while the mean relative weights were significantly increased in males (lungs, kidneys, testes, epididymides, brain and adrenal glands) and in females (kidneys and brain) of the high dose group. No morphologic alterations were noted in the organs with significant weight changes that may account for them. Therefore, these organ weight changes were regarded not to be treatment-related per se but to be the consequence of the significantly decreased mean terminal body weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Two female animals of the high dose group died prematurely, revealing non-specific organ changes (atrophy) in the lymphoid tissues of spleen and thymus (both animals) or prefinal erosion/ulcer in the mucosa of the glandular stomach (one animal). Although these findings rather reflect the consequence of a longer story of illness than a treatment-related effect and although none of the findings in thymus, spleen and/or glandular stomach were recorded in the animals killed at scheduled dates, a relationship of the premature death of both animals to the administration of the test article could not be excluded.

Most of the gross lesions recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence in control and high dose males and/or females. Hence, they were all regarded to have developed by chance and unrelated to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology detected treatment-related hyperplasia of the respiratory epithelium in the anterior part of the nasal cavity (cut level I) of males (higher incidence) and of females (higher grades of severity). One male of the high dose group showed hyperplasia of the respiratory epithelium also in cut level III of the nasal cavity.
Hyperplasia of the respiratory epithelium in the nasal cavity was interpreted as an adaptive, reversible reaction to the inhaled test article.

All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence and graded severity in control and high dose males and/or females. Hence, they were all regarded to have developed fortuitously and unrelated to treatment.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm):
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only)
Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.
Histopathological findings: neoplastic:
not examined
Details on results:
Summary of the treatment-related findings:
Test group 2 (0.319 mg/L = 60 ppm): no treatment-related findings
Test group 1 (0.106 mg/L = 20 ppm): no treatment-related findings
Key result
Dose descriptor:
NOAEC
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
urinalysis
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Aug 2002 - 04 Dec 2003 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)
Version / remarks:
712-C-00-368; July, 2002
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
712-C-98-204; August, 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG); from day 18 post coitum until sacrifice, the pregnant animals and their litters were housed in Markolon M III cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy.
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: from study day 69 - study day 80
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)
Frequency of treatment:
6 hours/day, 5 days/week
Duration of test:
- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)
Dose / conc.:
0.106 mg/L air (nominal)
Remarks:
equals 20 ppm
Dose / conc.:
0.319 mg/L air (nominal)
Remarks:
equals 60 ppm
Dose / conc.:
0.956 mg/L air (nominal)
Remarks:
equals 180 ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
106 mg/m3 (ca. 20 ppm) : as the expected no observed adverse effect level
319 mg/m3 (ca. 60 ppm) : as the intermediate dose level
956 mg/m3 (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter. Ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
All surviving pups (after sacrifice on day 4 p.p. by means of C02), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy and urine analysis except vlume, color, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.
Indices:
- Viability index (%) = number of live pups on day 4 after birth / number of liveborn pups on the day of birth x 100
- Sex ratio (%) – number of live male or female pups on day (0/4) / number of live male and female pups on day (0/4) x 100
Historical control data:
not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity in the high dose group (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (0.956 mg/L = 180 ppm):

Retarded body weight development of the females
- mean body weight: - 3.0 % and -8.0 % of the control from study day 9 onward
(statistically significant on study day 51 )
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant )

- Decreased mean terminal body weight in females.

Reduced body weight development in the dams during pregnancy and lactation
- Average weight gain 61 % less than the control between days 0-20 of pregnancy
- Mean body weight on day 20 of pregnancy 24% below control
- Body weights persisted being 19-23% below control during lactation days 0-4, though weight gain was about 60% above control in these females after cessation of exposure on gestation day 18.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.
Slightly to moderately reduced food consumption of dams during pregnancy (about 16% below control) and lactation days 0-4 (about 39% below control)
Food efficiency:
no effects observed
Description (incidence and severity):
The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There are no treatment-related changes in the haematological parameters measured.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related findings were observed in the functional observation battery and motor activity examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Due to the significant decrease of the mean terminal body weight in animals (females 9,4 %) of the high dose group, some mean relative organ weights were increased. No morphologic alterations were noted in those organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Two female animals of the high dose group died prematurely, revealing non-specific organ changes (atrophy) in the lymphoid tissues of spleen and thymus (both animals) or prefinal erosion/ulcer in the mucosa of the glandular stomach (one animal). Although these findings rather reflect the consequence of a longer story of illness than a treatment-related effect and although none of the findings in thymus, spleen and/or glandular stomach were recorded in the animals killed at scheduled dates.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only) Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
In high dose group lower live birth index (80% vs 100% in the control). Lower gestation index (67% vs 100% in the control), 4 out of 6 surviving dams with
liveborn pups, 2 dams with only stillborn pups
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Insufficient maternal care in 3 females of the high dose group with subsequent complete litter loss until day 3 post delivery
Key result
Dose descriptor:
NOAEC
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose group: Lower pup body weights on day 1 p.p. (28% below control). In addition, average pup body weights and body weight gain significantly below control (42% on day 4 p.p. and 67% for days 1-4 p.p., respectively)
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
In high dose group: lower live birth index (80% vs 100% in the control ). Lower gestation index (67% vs . 100% in the control), 4 out of 6 surviving dams with
liveborn pups, 2 dams with only stillborn pups
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
High dose group: markedly lower viability index (20% vs 97% in the control). Insufficient maternal care in 3 females of the high dose group with subsequent complete litter loss until day 3 post delivery.
External malformations:
no effects observed
Description (incidence and severity):
At pup necropsy, no malformations were found at any dose level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
At pup necropsy, no malformations were found at any dose level.
Visceral malformations:
no effects observed
Description (incidence and severity):
At pup necropsy, no malformations were found at any dose level.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
At the highest dose level (0.956 mg/L) the pre- and postnatal development of the offspring was substantially impaired. At test concentrations where no maternal toxicity was observed, no embryotoxic,
Key result
Dose descriptor:
NOAEC
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in postnatal survival
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
0.956 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Dose group  (mg/L)           0     0.106   0.319    0.956
maternal body weight 
change gd 0 - 20 (g)         91.6  91.5    90.2     35.7**
maternal body weight
lactation day 0 - 4 (g)      10    10      9        6 
maternal mortality during 
gestation (%)                0     0       0        20      
duration of gestation (days) 21.8  21.9    21.9     21.8
gestation index (%)          100   100     100      67
pups delivered               11.0  9.7     10.4     8.3
live birth index (%)         100   100     100      80**
Stillborn (%)                0     0       0        20**    

pups died (%)                0.9   0       1.1      36**
pups cannibalized (%)        1.8   1.0     0        28**
pups viability index         97    99      99       20**
pups weight day 1 (g)        6.0   6.2     6.2      4.3**
pups weight day 4 (g)        8.9   9.0     9.2      5.2**


Reason / purpose:
reference to same study
Reference
Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Aug 2002 - 04 Dec 2003 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)
Version / remarks:
712-C-00-368; July, 2002
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
712-C-98-204; August, 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG)
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
other: clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG)
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L
Duration of treatment / exposure:
- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.106 mg/L air (nominal)
Remarks:
equals 20 ppm
Dose / conc.:
0.319 mg/L air (nominal)
Remarks:
equals 60 ppm
Dose / conc.:
0.956 mg/L air (nominal)
Remarks:
equals 120 ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
0.106 mg/L (ca. 20 ppm) : as the expected no observed adverse effect level
0.319 mg/L (ca. 60 ppm) : as the intermediate dose level
0.956 mg/L (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams.
The littering behavior of the dams was also inspected on each workday in the afternoons in
addition to the evaluations in the mornings.

BODY WEIGHT: Yes
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well
as prior to gross necropsy.

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

URINALYSIS:
- Time schedule for collection of urine: not reported
- Analysis only performed in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment
Neurobehavioural examinations performed and frequency:
NEUROBEHAVIOURAL EXAMINATION:
Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable.
A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals. Attention was paid to:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations :
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes
19. other findings

Sensorimotor Tests/Reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response" )
7. coordination of movements ("righting response")
8. behaviour during "handling "
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Sacrifice and (histo)pathology:
GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter . Testes, epididymides and ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group
Positive control:
none
Statistics:
Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy and urine analysis except volume, colour, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the study period one male animal of the control group showed injuries laterally at right flank and on the right head. This injury was of mechanic matter and was therefore not related to the study. One female animal of the low concentration group showed alopecia on dorsal body region and on both forelimbs. This was most likely to be incidental, because alopecia was not observed in other animals of the low and mid concentration group. At the high concentration (0.956 mg/L) male and female animals showed various clinical abnormalities comprising slight to moderate visually increased respiration, eyelid closure, salivation, eye discharge (red) indicating that the test substance was irritating to eyes and upper respiratory tract at this high concentration. Other findings like aggressiveness, apathy (1 female), as well as alopecia and piloerection were more of general nature indicating the bad general state of the animals.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy)
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development of the high concentration animals was substantially impaired by the exposure to the test substance. Although the mean body weights on some examination days were not of statistical significance, the significantly reduced mean body weight gains proved the existence of this effect. The retardation of the body weight development is not only of secondary matter due to the reduced food consumption.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Test group 3 (0.956 mg/l = 180 ppm):
Retarded body weight development of the males
- mean body weight: - 8.1 % to - 15.5 % of the control from study day 9 onward (statistically significant to a level of 99 %)
- mean body weight gain: -23.2 % to -36.3 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Retarded body weight development of the females
- mean body weight: - 3.0 % to - 8.0 % of the control from study day 9 onward (statistically significant on study day 51 to a level of 95 %)
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Significantly (to a level of 99 %) decreased mean terminal body weight in males and in females (- 17.2 % and -9.4 %, resp.)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.
Food efficiency:
no effects observed
Description (incidence and severity):
The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There are no treatment-related changes in the haematological parameters measured. Clotting analysis revealed prolonged prothrombin times in the blood of the males of the high concentration group at the end of the study.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related findings were observed in the functional observation battery and motor activity examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The administration of the test article had significantly decreased the mean terminal body weight of male and female rats of the high dose group. As a consequence of the treatment-related body weight loss, a few absolute organ weights were significantly decreased in males of the high dose group (liver and thymus), while the mean relative weights were significantly increased in males (lungs, kidneys, testes, epididymides, brain and adrenal glands) and in females (kidneys and brain) of the high dose group. No morphologic alterations were noted in the organs with significant weight changes that may account for them. Therefore, these organ weight changes were regarded not to be treatment-related per se but to be the consequence of the significantly decreased mean terminal body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Two female animals of the high dose group died prematurely, revealing non-specific organ changes (atrophy) in the lymphoid tissues of spleen and thymus (both animals) or prefinal erosion/ulcer in the mucosa of the glandular stomach (one animal). Although these findings rather reflect the consequence of a longer story of illness than a treatment-related effect and although none of the findings in thymus, spleen and/or glandular stomach were recorded in the animals killed at scheduled dates, a relationship of the premature death of both animals to the administration of the test article could not be excluded.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology detected treatment-related hyperplasia of the respiratory epithelium in the anterior part of the nasal cavity (cut level I) of males (higher incidence) and of females (higher grades of severity). One male of the high dose group showed hyperplasia of the respiratory epithelium also in cut level III of the nasal cavity.
Hyperplasia of the respiratory epithelium in the nasal cavity was interpreted as an adaptive, reversible reaction to the inhaled test article.

All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence and graded severity in control and high dose males and/or females. Hence, they were all regarded to have developed fortuitously and unrelated to treatment.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only)
Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEC
Effect level:
0.319 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
urinalysis
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Effect level:
>= 0.956 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Key result
Critical effects observed:
no

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD guideline 413 (Sub-Chronic Inhalation Toxicity: 90-day Study)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental)
Version / remarks:
712-C-00-368; July, 2002
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Version / remarks:
712-C-98-204; August, 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tert.- Butylacrylat
- Physical state: colorless liquid
- Analytical purity: 99.62% (Batch No. 2-011002-15/1) - 99.8% (Batch No. B602)
- Purity test report: Analytical report 02L00206 (Batch No. B602); reanalysis (Batch No. 2-011002-15/1)
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: refrigerator, protected from light and stored under air

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: Males: 106.1-108.3 g; Females: 92.7-94.7 g (groupwise)
- Housing: individually in type DK II stainless steel wire mesh cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Underneath the DK III cages, waste trays were fixed containing bedding material (type 3/4 dust free embedding (SSNIFF, Soest, FRG); from day 18 post coitum until sacrifice, the pregnant animals and their litters were housed in Markolon M III cages (BECKER & CO., Castrop-Rauxel, Germany), floor area about 800 cm2. Pregnant females were provided with nesting material (cellulose wadding) towards the end of pregnancy.
- Diet: ad libitum, "GLP" (Provimi Kliba SA, Kaiseraugst, CH)
- Water: ad libitum, tap water
- Acclimation period: ca. 2 weeks

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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps (Sarstedt DESAGA) and glass vaporizers with thermostat (BASF AG)
- Method of holding animals in test chamber: The animals were kept singly in wire cages located in a glass-steel inhalation chamber, volume of 1.4 m3 (BASF AG).
- Method of conditioning air: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump. The vapor was generated with conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C) and passed into the inhalation system.
- Temperature, pressure in air chamber: 25 ± 3°C, -10 Pa
- Air flow rate: 27.5-28.5 m3/h

TEST ATMOSPHERE
- Brief description of analytical method used:  The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control (Hewlett-Packard 5840 A). Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean  concentrations and standard deviations for the entire study were derived. The concentration constancy in each inhalation system was continuously monitored by means of a total hydrocarbon analyzer.
To ensure, that no liquid aerosols were formed at concentration levels as high as 180 ppm, a scattered light photometer was used to monitor the test atmosphere of the high dose group.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: from study day 69 - study day 80
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Target concentrations were: 0.106, 0.319, and 0.956 mg/L (corresponding to 20, 60, and 180 ppm)
- Measured concentrations were: 0.107 ± 0.0061, 0.317 ± 0.0211, 0.958 ± 0.0481 mg/L
Duration of treatment / exposure:
- males: ca. 13 weeks (10 weeks premating, 3 weeks mating and post mating)
- females: ca. 15 weeks (10 weeks premating, during mating and gestation through day 4 after delivery)
Frequency of treatment:
6 hours/day, 5 days/week
Details on study schedule:
- Age at mating of the mated animals in the study: 16 weeks
- After ten weeks of exposure, the parental animals were mated to produce a litter. Mating pairs were formed from the same concentration group. The parental animals were examined for their mating and reproductive performances.
- The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups were necropsied on day 4 post partum and were examined macroscopically for external and visceral findings.
Doses / concentrationsopen allclose all
Dose / conc.:
0.106 mg/L air (nominal)
Remarks:
equals 20 ppm
Dose / conc.:
0.319 mg/L air (nominal)
Remarks:
equals 60 ppm
Dose / conc.:
0.956 mg/L air (nominal)
Remarks:
equals 180 ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
0.106 mg/L (ca. 20 ppm) : as the expected no observed adverse effect level
0.319 mg/L (ca. 60 ppm) : as the intermediate dose level
0.956 mg/L (ca. 180 ppm): as the dose level where toxic effects were expected

Preflow period of 4 days
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on each workday in the afternoons in addition to the evaluations in the mornings.

BODY WEIGHT:
- Time schedule for examinations: day -7, on day -4 (start preflow period), on day 0(start exposure period) and then in weekly intervals as well as prior to gross necropsy.

FOOD CONSUMPTION:
- Time schedule: day -7, on day -4 (start preflow period), on day 0 (start of exposure period) and then in weekly intervals.
- It was calculated as mean food consumption in grams per animal and day.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

HAEMATOLOGY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular, haemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anaesthesia.
- Animals fasted: Yes
- How many animals: all
- Following parameters were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium, bile acids

URINALYSIS:
- Time schedule for collection of urine: not reported
- Analysis only performed in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION:
- Detailed clinical observation (DCO) were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The following parameters were examined: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine, pupil size
- A functional observational battery (FOB) was carried out on the assigned animals (5 males and 5 females/ test group) on study days 56 and 57 for males and females, respectively. On the days of neurofunctional tests there was no exposure of the concerning animals as well as the other 5 animals of the same test group. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. During the home cage observations attention was paid to posture, tremor, convulsions, abnormal movements, impairments of gait and other findings. During the open field observations the following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within 2 minutes and other findings. After the open field test animals were subjected to the following sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behaviour during “handling”, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test and other finding. All findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Motor activity (MA) was measured on the same day and with the same animals as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order . The measurements started at about 14:00 p.m. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages ; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Oestrous cyclicity (parental animals):
not reported
Sperm parameters (parental animals):
not reported
Litter observations:
PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring: number and sex of pups, weight gain (The pups were weighed on the day after birth (day 1 p.p.) and on day 4 after birth), stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 3 weeks after mating
- Maternal animals: All surviving animals after d4 of gestation

GROSS PATHOLOGY:
The animals were sacrificed under narcoren® anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. To prevent post mortem autolysis, the animals that died intercurrently were necropsied as soon as possible after death.

ORGAN WEIGHTS:
The following weights parameters from all animals sacrificed were determined: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, thymus, spleen, brain, heart, lungs

HISTOPATHOLOGY:
-The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumber cord), sciatic nerve, pituitary gland, salivary glands (glandula mandibularis and glandula sublingualis), thyroid glands/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (mandibular and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), skull (with nasal cavities, larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lacrimal glands. Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution and embedded in paraplast, thereafter . Testes, epididymides and ovaries of animals that died intercurrently were fixed in 4% formaldehyde solution.
- After the organs were fixed, histotechinical processing and examination was were performed as follows: Nasal cavities (level I- IV), Larynx (level I- III), Trachea (longitudinal, with carina), Lungs (5 lobes) and thyroid glands/parathyroid glands in all animals; all gross lesions in all affected animals; evaluations of all other organs and tissues fixed were only performed in animals of the control and high dose group
Postmortem examinations (offspring):
GROSS NECROPSY
All surviving pups (after sacrifice on day 4 p .p . by means of C02), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
Two-sided Dunnett test for food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups
delivered per litter.
Pairwise comparison by the Fisher´s exact test for male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy and urine analysis except vlume, color, turbidity and specific gravity.
Pairwise comparison by the Wilcoxon test for the proportions of affected pups per litter with necropsy observations.
Non-parametric Kruskal-Wallis test (two-sided)/Wilcoxon test for feces, rearing, grip strength forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity for the different time intervals, clinical pathology parameters except differential blood count and organ weights.
Reproductive indices:
- Male mating index (%) = number of males with confirmed mating / number of males places with females x 100
- Male fertility index (%) = number of males proving their fertility / number of males place with females x 100
- Female mating index (%) = number of females mated / number of females placed with males x 100
- Female fertility index (%) = number of females pregnant / number of females mated x 100
- Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100
Offspring viability indices:
- Viability index (%) = number of live pups on day 4 after birth / number of liveborn pups on the day of birth x 100
- Sex ratio (%) – number of live male or female pups on day (0/4) / number of live male and female pups on day (0/4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the study period one male animal of the control group showed injuries laterally at right flank and on the right head. This injury was of mechanic matter and was therefore not related to the study. One female animal of the low concentration group showed alopecia on dorsal body region and on both forelimbs. This was most likely to be incidental, because alopecia was not observed in other animals of the low and mid concentration group. At the high concentration (0.956 mg/L) male and female animals showed various clinical abnormalities comprising slight to moderate visually increased respiration, eyelid closure, salivation, eye discharge (red) indicating that the test substance was irritating to eyes and upper respiratory tract at this high concentration. Other findings like aggressiveness, apathy (1 female), as well as alopecia and piloerection were more of general nature indicating the bad general state of the animals.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Unspecific clinical symptoms indicative for some irritation and systemic toxicity (comprising visually increased respiration, salivation, piloerection, eyelid closure, eye discharge, alopecia, aggressiveness, hyperactivity and apathy)
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two female animals exposed to the high concentration died on study day 88 and 91 (day 18 and 20 of gestation), respectively. Both animals were found pregnant at death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development of the high concentration animals was substantially impaired by the exposure to the test substance. Although the mean body weights on some examination days were not of statistical significance, the significantly reduced mean body weight gains proved the existence of this effect. The retardation of the body weight development is not only of secondary matter due to the reduced food consumption.

Summary of the treatment-related findings, test group 3 (0.956 mg/L = 180 ppm):
Test group 3 (0.956 mg/l = 180 ppm):
Retarded body weight development of the males
- mean body weight: - 8.1 % to - 15.5 % of the control from study day 9 onward (statistically significant to a level of 99 %)
- mean body weight gain: -23.2 % to - 36.3 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Retarded body weight development of the females
- mean body weight: - 3.0 % to -8.0 % of the control from study day 9 onward (statistically significant on study day 51 to a level of 95 %)
- mean body weight gain: -19.9 % to - 41.8 % of the control from study day 9 onward (statistically significant to a level of 99 %)
Significantly (to a level of 99 %) decreased mean terminal body weight in males and in females (- 17.2 % and -9.4 %, resp.)
Reduced body weight development in the dams during pregnancy and lactation
- Average weight gain 61 % less than the control between days 0-20 of pregnancy
- Mean body weight on day 20 of pregnancy 24% below control
- Body weights persisted being 19-23% below control during lactation days 0-4, though weight gain was about 60% above control in these females after cessation of exposure on gestation day 18
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the high concentration animals was either decreased only marginally and of transient matter, or increased slightly compared to the control.
Food efficiency:
no effects observed
Description (incidence and severity):
The food efficiency of the high concentration animals was, when compared with the control, only reduced transiently at the beginning of the exposure (day 9).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There are no treatment-related changes in the haematological parameters measured. Clotting analysis revealed prolonged prothrombin times in the blood of the males of the high concentration group at the end of the study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding clinical pathology findings, in the high concentration group females, indications of a reduced general state was seen, characterized by significantly decreased serum creatinine, total protein, albumin and globulin levels. No treatment-related effects were observed in the clinical pathology parameters of the animals of the low and mid concentration groups.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm): :
-Increased prothrombin times, urea*, magnesium*, urinary specific gravity and urinary casts* in the males
-Decreased triglycerides** and urinary volume in the males
-Decreased chloride, creatinine*, total bilirubin, total protein**, albumin* and globulins** in the females
(* statistically significant to a level of 95 %
** statistically significant to a level of 99 %)

Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Regarding clinical pathology findings, treatment-related effects were observed only in the high concentration groups. The investigations revealed mild impairment of renal function in the males, substantiated by slightly increased urea concentrations in the serum, excretion of decreased urinary volume with increased specific gravity and the presence of urinary casts.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related findings were observed in the functional observation battery and motor activity examinations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology detected treatment-related hyperplasia of the respiratory epithelium in the anterior part of the nasal cavity (cut level I) of males (higher incidence) and of females (higher grades of severity). One male of the high dose group showed hyperplasia of the respiratory epithelium also in cut level III of the nasal cavity.
Hyperplasia of the respiratory epithelium in the nasal cavity was interpreted as an adaptive, reversible reaction to the inhaled test article.

All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at low or at comparable incidence and graded severity in control and high dose males and/or females. Hence, they were all regarded to have developed fortuitously and unrelated to treatment.

Summary of the treatment-related findings, test group 3 (0.956 mg/l = 180 ppm): :
Hyperplasia of the respiratory epithelium in the nasal cavity at level I in male (incidence) and in female rats (graded severity, only)
Hyperplasia of the respiratory epithelium in the nasal cavity at level III in one male rat.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Male cohabitation data
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index reached 100% in all groups, including the controls.
Fertility could be proven for nearly all F0 parental males within the scheduled mating interval for F1 litter. One mid dose male and one high dose male did not generate F1 pups. Thus, the male fertility index ranged between 89% and 100%. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. For none of the affected male rats conclusive histopathological findings were gathered which could account for the observed impaired fertility.

- Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% for all groups.
The mean cohabitation time (duration until sperm was detected (i.e. day 0 p.c.)) amounted to 3.3 days/2.3 days/2.5 days/2.0 days (0, 0.106, 0.319 and 0.956 mg/l). These values reflect the normal range of biological variation inherent in the strain used in this study. Consequently, the differences between the groups are assessed as spontaneous in nature and without any biological relevance.
All sperm positive rats delivered pups with the following exceptions: one mid dose F0 parental female and one high dose female did not become pregnant. Therefore, the fertility indices ranged between 89% and 100%.

The mean duration of gestation was very similar in the test groups 0-3 (between 21.8 and 21.9 days).

The gestation index was 100% in test groups exposed to 0, 0.106 and 0.319 mg/l, but was markedly reduced to 67% in the high dose group (0.956 mg/L). Only 4 out of 6 surviving presumed pregnant females had liveborn pups in the high dose group.

The number of liveborn and stillborn pups was comparable between the control and test groups 1 and 2, while there were a statistically significantly decreased number of liveborn and an increased number of stillborn pups in test group 3. Thus, the live birth index amounted to 100% in test groups exposed to 0, 0.106 and 0.319 mg/l and 80% in test group 3 (0.956 mg/L).

Details on results (P0)

Dose group (mg/L): 0 - 0.106 - 0.319 - 0.956
cohabitation time (days): 3.3 - 2.3 - 2.5 - 2.0
male mating index (%) : 100 - 100 - 100 - 100
male fertility index (%): 100 - 100 - 90 - 89
female mating index (%): 100 - 100 - 100 - 100
female fertility index (%) 100 100 90 89
duration of gestation (days) 21.8 21.9 21.9 21.8
gestation index (%) 100 100 100 67
pups delivered 11.0 9.7 10.4 8.3
live birth index (%) 100 100 100 80**
Stillborn (%) 0 0 0 20**

(** significant to a level of 99 %)

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
urinalysis
histopathology: non-neoplastic
Remarks on result:
other: marked maternal toxicity, including mortality
Key result
Dose descriptor:
NOAEC
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Test group 3 (956 mg/m3 ): markedly lower viability index (20% vs 97% in the control)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (956 mg/m3 ): lower pup body weights on day 1 p .p . (28% below control ). Average pup body weights and body weight gain significantly below control (42% on
day 4 p.p. and 67% for days 1-4 p.p., respectively)
Sexual maturation:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.319 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
0.956 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion