tert-butyl acrylate

Endpoint summary

Description of key information

NOAEC = 60 ppm (equivalent to 0.319 mg/L) (Wistar rat, vapour inhalation, OECD TG 413, 422)

The inhalation of 180 ppm tert-butyl acrylate vapours (equivalent to 0.956 mg/L) by male and female Wistar rats in a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test caused slight irritation of the eyes and upper respiratory tract, retarded body weight development, mild impairment of renal function in the males, a reduced general health status and two deaths during gestation in the females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

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

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

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

712-C-98-204; August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)

Version / remarks:

May 30, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

mortality

urinalysis

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

319 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

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

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

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

712-C-98-204; August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC L133 (Sub-Chronic Inhalation Toxicity Study)

Version / remarks:

May 30, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

mortality

urinalysis

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

319 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Since vapour inhalation is the most likely route of human exposure to tert-butyl acrylate due to its vapour pressure, this was the route chosen for animal testing.

Tert-butyl acrylate was tested by inhalation in a combined sub-chronic toxicity study with a reproduction / developmental toxicity screening test (based on OECD-guidelines 413 and 422) (BASF 2004). Groups of ten male and ten female Wistar rats were exposed to vapours of tert-butyl acrylate for 6 hours/day and 5 days/week to concentrations of 20, 60 and 180 ppm (corresponding to 0.106, 0.319 and 0.956 mg/L). A concurrent control group was exposed to clean air. The males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment lasted from 10 weeks premating, during mating and gestation through day 4 after delivery (approx. 15 weeks). After ten weeks of exposure, the parental animals were mated to produce a litter. Mating pairs were formed from the same concentration group. No treatment related findings were observed in the low and mid dose group. Two female animals of the high dose group died prematurely (day 88 and day 91 of the study), 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). These findings rather reflect the consequence of a prolonged deteriorated state of general health than a treatment-related organ specific effect because none of the findings in thymus, spleen and/or glandular stomach were recorded in the animals killed at scheduled dates. In the high concentration group, the mean body weight development in females and males was significantly retarded, the mean terminal body weight was significantly decreased in both sexes. The animals showed 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). Histopathology detected treatment related hyperplasia of the respiratory epithelium in the nasal cavity of males and of females, which was interpreted as an adaptive, reversible reaction to the inhaled test article. Also changes in haematological and clinicochemical parameters as well as urinalysis were observed (decreased triglycerides and urinary volume in the males; decreased chloride, creatinine, total bilirubin, total protein, albumin and globulins in the females; increased prothrombin times, urea, magnesium, urinary specific gravity and urinary casts in the males). Thus, the lowest observed adverse effect concentration was found to be 180 ppm (equivalent to 0.956 mg/L) the no observed adverse effect concentration was 60 ppm (equivalent to 0.319 mg/L).

Justification for classification or non-classification

Based on the available data, classification for repeated dose toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and GHS classification (GHS UN rev.6, 2015).