Registration Dossier
Registration Dossier
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EC number: 209-548-7 | CAS number: 585-07-9
Endpoint summary
Administrative data
Description of key information
RA oral:
OECD 408, GLP compliant, K1; rats, oral (gavage): NOAEL = 120 mg/kg bw/d
RA inhalation:
OECD 412, GLP compliant, K1; rats, inhalation (vapour): NOAEC systemic 11175 mg/m3, NOAEC local 1832 mg/m3
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May 27, 2008 - October 02, 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY) Rheinlandpfalz; date of inspection: 08.06.2005 and 25. to 27.07.2005
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Batch number of test material: not specified
- Purity: 99.8%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (RT) ; temperatures > 30°C were avoided
- Stability: The stability of the test substance in drinking water containing 1% Carboxymethylcellulose + Cremophor EL + one drop Hydrochloric Acid (1mol/L) was demonstrated over a period of 4 h at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
- Homogeneity: Considering the standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in 1% Carboxymethylcellulose + Cremophor EL under the addition of one drop Hydrochloric Acid (1 mol/L).
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration: 60, 120 and 360 mg/kg bw/day
FORM AS APPLIED IN THE TEST
- emulsion
OTHER SPECIFICS
- Physical state: liquid / colorless, clear - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:WI (Han)
- Details on species / strain selection:
- Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 34 ± 1 day (males and females)
- Weight at study initiation: not specified
- Fasting period before study: No
- Housing: in groups of 5 animals per cage
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes (per hr): not specified
- Photoperiod: 12 / 12 (6.00 pm - 6.00 am dark; 6.00 am - 6.00 pm light)
IN-LIFE DATES: From: May 27, 2008 To: October 02, 2008 - Route of administration:
- oral: gavage
- Details on route of administration:
- The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
- Vehicle:
- other: drinking water containing 1% carboxymethylcellulose
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as an emulsion. The appropriate amount of test substance was weighed out depending on the desired concentration. Thereafter, the vehicle (drinking water containing 1% carboxymethyl cellulose) was filled up to the desired volume + Cremophor EL + one drop Hydrochloric Acid (1mol/L) and subsequently mixed using a magnetic stirrer.
DIET PREPARATION
- Rate of preparation (frequency): The test-substance preparations were produced daily.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: drinking water containing 1% carboxymethylcellulose
- Amount of vehicle (if gavage): not specified - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Concentration control analyses of the test-substance preparations in samples of all concentrations were performed at the start of the administration period and thereafter weekly. Thus, 13 analytical results were obtained for each dose group throughout the entire study. Taken together these results of the low dose group, the measured concentration was 84.2% of the nominal value. For the mid dose group, the corresponding value was 87.0% and for the high dose group 91.7% of the nominal concentration were determined. Independently from the obtained variations of the analytical series, these mean values are assessed to demonstrate the correctness of the test-substance preparations within the current study.
- Duration of treatment / exposure:
- a period of 3 consecutive months
- Frequency of treatment:
- administered daily, 7 days each week
- Dose / conc.:
- 360 mg/kg bw/day (actual dose received)
- Remarks:
- the measured concentration was 91.7 % of the nominal value.
- Dose / conc.:
- 120 mg/kg bw/day (actual dose received)
- Remarks:
- the measured concentration was 87.0 % of the nominal value.
- Dose / conc.:
- 60 mg/kg bw/day (actual dose received)
- Remarks:
- the measured concentration was 84.2% of the nominal value
- No. of animals per sex per dose:
- Control and high dose groups: 15 animals per sex per group (thereof 5 animals per sex per group for 28-day recovery)
Low and mid dose groups: 10 animals per sex per group. - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard
- Rationale for animal assignment (if not random): Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not
exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
- Fasting period before blood sampling for clinical biochemistry: Fasting period (withdrawal of food) of about 16 to 20 hours before blood sampling/necropsy
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: 28 days (5 rats/sex of control and high dose groups only)
- Section schedule rationale (if not random): Sep. 04, 2008 (study day 92) necropsy of the main groups (male); Sep.05, 2008 (study day 93) necropsy of the main groups (female); Oct. 02, 2008 (study day 120) necropsy of the recovery groups - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- MORTALITY
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
CLINICAL OBSERVATIONS
All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.
DETAILED CLINICAL OBSERVATIONS (DCO)
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high).The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size
FOOD CONSUMPTION
Group food consumption was determined weekly for each cage. The average food consumption/cage was used to estimate the mean food consumption in grams per animal per day.
FOOD EFFICIENCY
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption:
[(BWx - BWy)/FCy to x] x 100 = Food efficiency for day x,
with BWx = body weight on day x (g), BWy = body weight on day y (last weighing date before day x) (g), FCy to x = mean food consumption from day y to day x; calculated as mean daily food consumption on day x, multiplied with the number of days from day y to day x (g).
WATER CONSUMPTION
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.
BODY WEIGHT DATA
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.
FUNCTIONAL OBSERVATION BATTERY (FOB)
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 am. At least one hour before the start of the FOB the animals were transferred singly to cages. Drinking water was provided ad libitum whereas no food was offered during the measurements. 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 behavior of the animals. Attention was paid to posture, tremor, convulsions, abnormal movements, impairment of gait, 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: behavior 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, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes.
Sensorimotor Tests/Reflexes: The animals were removed from the open field and subjected to 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"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.
MOTOR ACTIVITY (MA) ASSESSMENT
MA measurements were carried out in all animals toward the end of the administration period. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. 18 beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals for 5 minutes in each case. The animals were randomly placed in the cages. Motor activity measurements were carried out from 2.00 pm onwards. On account of the measuring variant "staggered", the starting time varied by the time which is needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was given to the animals during measurements. After the transfer of the last animal in each case, the room of measurement was darkened. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.
OPHTHALMOSCOPY
Prior to the administration period, the eyes of all animals were examined using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany). At the end of the administration period the eyes of the control (0 mg/kg bw/day) and high dose animals (360 mg/kg bw/day) were examined for any changes, again.
HAEMATOLOGY
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoba, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: All animals per test group and sex. At the end of the recovery period only clotting analyses were examined in the remaining animals of the control and high dose group.
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test; HQT). The clotting analyses were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
CLINICAL CHEMISTRY
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoba, Essex GmbH Munich, Germany)
- Animals fasted: Yes
- How many animals: All animals per test group and sex. At the end of the recovery period only blood chemistry parameters were examined in the remaining animals of the control and high dose group.
- An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. The activities of the following enzymes were determined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT). The following blood chemistry parameters were determined: sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), magnesium (MG).
URINALYSIS
- Time schedule for collection of urine: at the end of the administration period overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Investigated parameters: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume. With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips (Combur 9 test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- NECROPSY
All animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
WEIGHT PARAMETERS
The following weight determinations were carried out on all animals: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus, thyroid glands.
HISTOPATHOLOGY
After the organs were fixed, processing, examination by light microscopy and evaluation of findings in the following tissues of all control and high-dose animals of the main groups were performed: salivary glands (mandibular and sublingual glands), esophagus, stomach (forestomach and glandular stomach), duodenum, ileum, jejunum (with Peyer’s plaques), cecum, colon and rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cords), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, nose (nasal cavity/level III), aorta, heart, bone marrow (femur), lymph nodes (mesenteric and axillary lymph nodes), spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus and vagina, prostate, seminal vesicle and epididymides, female mammary gland, skin.
Additionally, nose tissue (nasal cavity/level III) was examined in all low- and mid-dose animals of the main groups and in all control and high-dose animals of the recovery groups. Furthermore, all gross lesions were examined in all affected animals of all groups. - Statistics:
- Statistics of clinical examinations: Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were DUNNETT's test (two-sided) for body weight and body weight change, and KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, and motor activity.
Statistics of clinical pathology: Means, medians and standard deviations of each test group were calculated for several parameters. Further statistical analyses were KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for clinical pathology parameters, urine volume, and urine specific gravity, and FISHER's exact test for urinalysis, except color, turbidity, volume and specific gravity.
Statistics of pathology: KRUSKAL-WALLIS test (two-sided)/Wilcoxon-test (two-sided) for weight parameters. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- During clinical examinations, only Microphthalmia left was observed in 1 female of test group 2 (120 mg/kg bw/day). This isolated finding only observed in one mid dose female animal was assessed as spontaneous in nature and therefore not substance related.
- Mortality:
- no mortality observed
- Description (incidence):
- No animal died prematurely in the present study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weight was significantly decreased in male animals of test group 3 (360 mg/kg bw/day) towards the end of the study (-7% on days 84 and 91). The corresponding body weight change was also significantly decreased in these animals from day 77 till the end of the administration period (-12.1% on day 84 and 91). This impairment of body weight data in high dose males was assessed as related to treatment with the test substance and indicates general systemic toxicity. During the recovery period, no significant influence on body weight data was measured anymore. The body weight of the female animals in all test groups was not significantly influenced by the test article during the administration period. However, in female animals of test group 3 (360 mg/kg bw/day) a significantly lower (-7.8%) weight was noted on day 98, at the beginning of the recovery period, only. This significant difference evolved from an apparent above-average "increase" (by 9 grams) of the mean control body weights in the recovery animals, right after cessation of the treatment. The control group rats designated at the start of the study for recovery, by chance, weighed more than the control group rats sacrificed at the end of the administration period. Because of this, the body weight of the test group 3 (360 mg/kg bw/day) recovery rats appeared to be significantly decreased compared to the control group recovery rats at the beginning of the recovery period. Thus, the apparent weight decrease in the high-dose recovery females is not related to the test article and is considered to be toxicologically irrelevant.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food consumption was significantly increased in males of group 1 (60 mg/kg bw/day) on day 91. This finding, which occurred only once in low dose males, was assessed as incidental in nature and therefore not substance related.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- No substance-related findings were observed.
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No substance-related overt changes in water consumption were observed.
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The observed corneal stipplings and remainders of the pupillary membrane were equally distributed between the dosed animals and the controls. Therefore, these findings were clearly not related to treatment and were without any toxicological relevance.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the administration period no treatment-related adverse changes were found in the rats of both sexes concerning the complete blood cell counts. In the male rats of the 360 mg/kg bw/d dose group the relative reticulocyte counts were significantly lower compared to the controls. This change was not regarded as toxicological relevant, because no other red blood cell parameter were affected. After substance administration, the prothrombin time (Hepatoquick’s time) was significantly prolonged in male and female rats of the 360 mg/kg bw/d dose group. This change was no longer present after the 4 weeks recovery period.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the administration period in rats of both sexes of the 360 mg/kg bw/d dose group the inorganic phosphate levels were significantly increased and the calcium levels were significantly decreased. The changes were no longer present after the recovery period apart from weakly significantly increased inorganic phosphate levels in males. The urea levels were significantly increased in male and female rats of the 360 mg/kg bw/d dose group, and additionally in males of the 120 mg/kg bw/d dose group. This increased urea level was the only clinical pathology value affected in the mid dose group. Therefore, it was regarded as treatment-related but not adverse. After the recovery period, the urea level was still slightly significantly increased in female rats of the high dose group. After the last administration day, the triglyceride concentrations were significantly decreased in rats of both sexes of the 360 mg/kg bw/d dose group. Additionally, in rats of the high dose group the total bilirubin levels in males as well as the glucose levels in females were significantly increased and the globulin values were significantly decreased in females. All these changes were not present after the recovery period, anymore. But in male rats of the high dose group the glucose levels were weakly significantly decreased after the recovery period. These lower glucose levels were regarded as incidentally, because in these animals the glucose levels were not changed at the end of the administration period, and in female rats the glucose levels were increased during the substance administration. At the end of the administration period, the inorganic phosphate and urea levels were statistically significantly higher in females of the 60 mg/kg bw/d dose group. These were the only findings in this dose group, and they were not dose-dependent. Therefore, theses changes were regarded as incidentally rather than treatment-related. At the end of the administration period, the sodium levels were significantly lower in males of the 360 mg/kg bw/d dose group. This change was not accompanied by any other electrolyte level change, and was not present in females. Therefore, this deviation was regarded as incidentally rather than treatment-related.
- Endocrine findings:
- not examined
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the administration period no treatment-related adverse effects were found in the dosed rats regarding the urinalysis parameters. In the males of the 360 mg/kg bw/d dose group there were found less phosphate crystals in the urine sediment compared to the controls. Regarding the individual pH values of the urine samples, there appeared to be slightly lower pH values in the dosed rats. Therefore, it was assumed, that the phosphate crystals were dissolved in the urine of the dosed males. Nevertheless, this effect was regarded as treatment-related, but not adverse.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Deviations from "zero values" were obtained in several animals. However, as all findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. Besides this, during functional observational battery the following examinations were carried out and have to be assessed individually: Home cage observations: No substance-related findings were observed. Open field observations: No substance-related findings were observed. Sensorimotor tests/reflexes: No substance-related findings were observed. Quantitative parameters: No substance-related findings were observed.
- Motor activity (MA) measurement: Regarding the overall motor activity, no substance-related findings were observed. Comparing the single intervals, the motor activity was significantly decreased in female animals of group 3 at interval 10, compared to the control value. This single occurrence only observed towards the end of the period of measurement was without any significant influence on the overall motor activity and therefore assessed as toxicologically irrelevant. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The increased kidney weights (+ 11%) in female animals of the 360 mg/kg body weight group are regarded as treatment-related, although there was no histopathologic correlate which could explain this weight increase. The decrease in adrenal gland weights in males of the mid-dose (- 11%) and high-dose (- 10%) groups is regarded as a consequence of the decreased terminal body weights, although these decreases were not statistically significant. It is therefore not regarded as a treatment-related effect. All other mean absolute weight parameters did not show significant differences when compared to the control group. The significant increase in kidney weights of the male animals of the recovery group (+ 17%) is not treatment-related, as the kidney weights of the main group males of the 360 mg/kg dose group were within the range of the control group. All other mean absolute weight parameters did not show significant differences when compared to the control group.
- Relative organ weights: The increased relative brain, kidney, and liver weights in the 360 mg/kg body weight group of male animals are regarded as a consequence of the reduction in terminal body weight. In females, the significant increase in relative kidney and liver weights are regarded as treatment-related. Due to the fact that the terminal body weight was not changed in treated animals and the absolute liver weight was increased up to +9%, although not statistically significant, a substance relationship can not be excluded. This effect is not regarded to be adverse in nature as no histopathologic finding were observed. The decrease in the uterus weight of the 60 mg/kg body weight group is assessed as incidental, because there is no dose-response relationship and the uterus weights in the higher dose groups were greatly than that of the control level. All other mean relative weight parameters of the main groups and the recovery groups did not show significant differences when compared to the control groups. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- All gross lesions occurred singly or they were biologically equally distributed between control and treatment groups.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males and females of the 360 mg/kg body weight group degeneration and regeneration of the olfactory epithelium was observed. The nasal cavities of animals of the recovery groups (control and 360 mg/kg body weight) were examined. There were no abnormalities detected. Mainly the nasal septum and the ethmoid turbinates were affected. In the more severe cases, there was extension to almost all parts of the nasal cavity. The finding was always observed at the transition of the respiratory to the olfactory epithelium at the nasal septum and extended upwards until up to 2/3 of the septum was affected. There was a mixture between degenerative findings (loss of epithelial cells, reduction in epithelium height, irregular architecture, apoptosis) and regenerative findings (hyperplasia/hypertrophy of basal cells, nest-like accumulation of basal cells, occasionally extending into the submucosa, mitotic figures). In animals diagnosed with grade 1, the hyperplasia/hypertrophy was often the most evident finding. All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them are considered to be incidental or spontaneous in origin and without any relation to treatment.
- Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- Functional observational battery (FOB)
- Other effects:
- not examined
- Details on results:
- please refer to tables 1-4 in section "Any other information on results incl. tables"
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 60 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: multifocal degenerative and regenerative olfactory epithelium of the nasal cavity
- Remarks on result:
- other: completely reversible, as no animal of the recovery group showed any finding in the nose after 28 days after cessation of exposure.
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 120 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- other: Effects on the liver activity (increased liver weight, prolonged prothrombin time, lower serum globulin and triglyceride levels in males and/or females)
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- In conclusion, the oral administration of the test substance by gavage over a period of 3 months with a recovery period of 28 days revealed toxicologically relevant signs of systemic toxicity at the high dose level of 360 mg/kg bw/d, limited to effects on the liver activity (increased liver weight, prolonged prothrombine time, lower serum globuline and triglyceride levels in males and/or females) and kidneys weight (increased absolute weight in females). The NOAEL for these effects was 120 mg/kg body weight/day in both males and females.
- Executive summary:
In a study conducted according to OECD TG 408 in compliance with GLP, the test substance was administered daily to male and female Wistar rats by gavage at dose levels of 0, 60, 120 and 360 mg/kg body weight per day over a period of 3 consecutive months. Control and high dose groups consisted of 15 animals per sex per group, whereas low and mid dose groups consisted of 10 animals per sex per group. After 3 months of treatment, 10 animals per sex of all dose groups were sacrificed (main groups). The remaining 5 animals per sex of control and high dose groups were maintained for another 28 days without administration of the test substance (recovery groups).
At 360 mg/kg bw/day, body weight change significantly decreased in male animals from day 77 onwards (-12.1% on days 84 and 91). Prothrombin time was significantly prolonged (+10% males; +12% females) and inorganic phosphate (+14% males; +30% females), total bilirubin levels (+17% in males), glucose levels (+13% in females) as well as urea levels (+36% males; 21% females) significantly increased. Calcium (-3% in males and females), globulin levels (-4% in females) and triglyceride levels (-24% in males and females) significantly decreased. Absolute kidney weight (+11% in females), relative kidney weight (+13% males and females) and liver weight (+9% males; +11% females) significantly increased. Multifocal degeneration/regeneration of olfactory epithelium was observed in males (5 out of 10) and females (7 out of 10). After the recovery period, phosphate levels (+7% in males) and urea concentrations (+22% in females) were significantly increased. At 120 mg/kg bw/day, multifocal degeneration/regeneration of olfactory epithelium was observed in males (4 out of 10) and females (2 out of 10). At 60 mg/kg bw/day, no substance-related adverse findings were observed. Multifocal degenerative and regenerative olfactory epithelium of the nasal cavity was observed in high dose (360 mg/kg bw/day) and mid dose (120 mg/kg bw/day) (4 out of 10 males and 2 out of 10 females). At 60 mg/kg bw/day, no substance-related adverse findings in the olfactory tissues were observed. Considering the short half life of n-BMA in blood (99.7 % removed in first pass by the liver, Jones 2002) it is unlikely that these effects were of systemic origin, but were rather local effects as a consequence of the dosing technique. This substance-related effect was completely reversible, as no animal of the recovery group showed any finding in the nose after 28 days after cessation of exposure. In conclusion, the oral administration of n-butyl methacrylate by gavage over a period of 3 months with a recovery period of 28 days revealed toxicologically relevant signs of systemic toxicity at the high dose level of 360 mg/kg bw/day, limited to effects on the liver activity (increased liver weight, prolonged prothrombin time, lower serum globulin and triglyceride levels in males and/or females) and kidneys weight (increased absolute weight in females). The NOAEL for these effects was 120 mg/kg body weight/day in both males and female Wistar rats.
Reference
Table 1: ABSOLUTE ORGAN WEIGHTS (main groups).
Main groups:
| Male animals | Female animals | ||||
Group (mg/kg bw) | 60 | 120 | 360 | 60 | 120 | 360 |
Adrenal glands | - 4% | - 11%* | - 10%* |
|
|
|
Kidneys |
|
|
| + 4% | + 3% | + 11%** |
*: p ≤ 0.05, **: p ≤ 0.01; Kruskal-Wallis H and Wilcoxon test, two-sided
Table 2: ABSOLUTE ORGAN WEIGHTS (recovery groups).
Recovery groups:
| Male animals |
Group (mg/kg bw) | 360 |
Kidneys | + 17%* |
*: p ≤ 0.05, **: p ≤ 0.01; Wilcoxon test, two-sided
Table 3: RELATIVE ORGAN WEIGHTS (main groups).
Main groups:
| Male animals | Female animals | ||||
Group (mg/kg bw) | 60 | 120 | 360 | 60 | 120 | 360 |
Brain | + 1% | + 1% | + 11%** |
|
|
|
Kidneys | 0% | - 2% | + 13%** | + 1% | + 2% | + 13%** |
Liver | + 4% | + 7% | + 9%** | - 3% | + 4% | + 11%** |
Uterus |
|
|
| - 23%* | + 3% | + 9% |
*: p ≤ 0.05, **: p ≤ 0.01; Kruskal-Wallis H and Wilcoxon test, two-sided
Table 4: INCIDENCE AND GRADING OF THE OBSERVED DEGENERATION AND REGENERATION OF THE OLFACTORY EPITHELIUM.
Nasal cavity level III | Male animals | Female animals | ||||||
Dosemg/kg bw | 0 | 60 | 120 | 360 | 0 | 60 | 120 | 360 |
Organs examined | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Degeneration/regen-eration olf. epithelium | 0 | 0 | 4 | 5 | 0 | 0 | 2 | 7 |
Grade 1 |
|
| 3 | 3 |
|
|
| 2 |
Grade 2 |
|
|
| 1 |
|
| 1 | 3 |
Grade 3 |
|
| 1 |
|
|
| 1 | 2 |
Grade 4 |
|
|
| 1 |
|
|
|
|
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 120 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and guideline compliant read-across study
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 15 - November 13, 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Version / remarks:
- 1981
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed on July 27, 1993
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Supplier: Dupont, Belle plant, Belle, West Virgina
- Physical state: liquid
- Analytical purity: > 99%
- Impurities (identity and concentrations): iso-butyl methacrylate max 1%, MEHQ max. 0.002%
- Purity test date: no data
- Lot No.: SWF90319
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- vapour - Species:
- rat
- Strain:
- other: Crl:CD BR
- Details on species / strain selection:
- The rat is being utilized for this study because of the large amount of comparative toxicological and physiological data on this species.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Kingston, Stone Ridge, NY, USA
- Age at study initiation: 35-day old
- Weight at study initiation: ca 260 g for males and ca 195 g for females
- Housing: individually in suspended wire-mesh cage in the inhalation chamber rooms
- Diet: Purina certified rodent chow 5002 (excepted during exposure), ad libitum (withheld during exposure)
- Water: tap water (excepted during exposure), ad libitum (withheld during exposure)
- Acclimation period: two weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27
- Humidity (%): 40 - 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- other: unchanged (no vehicle)
- Details on inhalation exposure:
- All chambers were operated at an air flow rate of 400 L/min, resulting in a calculated 99% aerosol equilibrium time of 23 minutes or 6.4% of the exposure time. Vapours of the test material were generated from the liquid form, introduced at a constant flow into heated flasks (approx. 102-105°C) through which a constant flow of air was metered. The inhalation chambers were stainless steel, approximately2000 L. Chamber temperature was 22.8-25.5°C; chamber relative humidity was 62.5-67.6%. Fresh air was mixed with the test chemical vapours to achieve the desired concentration.
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals received a whole-body inhalation exposure under dynamic conditions in 2000-L stainless steel, glass, and Plexiglas chambers.
- Method of holding animals in test chamber: Each animal was individually housed in a suspended wire-mesh cage without food or water during the exposure. Cage positions within the chambers were rotated each day to insure that the animals were exposed at several different locations within each chamber.
- Source and rate of air: Calibrated flowmeters (Fisher-Porter Inc., Warminster, PA) delivered compressed air at a rate of 30 L/min into the air inlet of each flask
- Temperature, humidity, pressure in air chamber: The mean temperatures and relative humidities for all four chambers ranged from 22.8 to 25.5°C and 62.5 to 67.6%, respectively.
- Air flow rate: The chamber volume and air flow rate were adjusted to reach 99% of the maximum vapor concentration (t99) within 30 minutes of the total exposure time (Silver, 1946). The volume of animals loaded into the chamber was Iess than 7% of the total chamber volume.
- Air change rate: no data
- Generation of the test Atmosphere: The test material vapour concentrations were generated by metering the test material with calibrated Fluid Metering Pumps into 500 or 5000 ml three-necked round bottom flasks. The flasks were located in hemispherical heating mantles.
- Method of particle size determination: not appropriate, only vapours were generated
- Treatment of exhaust air: no data
TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test substance in the chambers was determined using a Miran 1A gas analyzer attached to a strip chart recorder. Before the initiation of the exposure, the Miran 1A gas analyzer was calibrated using precisely measured amounts of test material delivered into a dynamic vapour generator. A calibration curve was generated to convert the readings obtained from the Miran 1A gas analyzer into parts-per-million (ppm) butyl methacrylate.
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical concentrations only of test material were determined throughout the study. Test concentrations were measured during each daily exposure period (every 80 minutes) and the vapours analyzed by GC to confirm purity.
- Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- 6 h/day, 5 days/week
- Dose / conc.:
- 310 ppm (analytical)
- Remarks:
- 1832 mg/m3
- Dose / conc.:
- 952 ppm (analytical)
- Remarks:
- 5626 mg/m3
- Dose / conc.:
- 1 891 ppm (analytical)
- Remarks:
- 11175 mg/m3
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Fasting period before blood sampling for clinical biochemistry: yes
- Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed before and after each daily exposure for general condition and signs of toxicity, and once during the post-exposure observation period.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at the day of first exposure and then weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured weekly
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: during necropsy at termination of the study
- Anaesthetic used for blood collection: Yes, sodium pentobarbital
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: hematocrit, platelet count, hemoglobin, mean cell volume, red blood cell, mean ceil hemoglobin, white blood cell count, mean cell hemoglobin conc.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during necropsy at termination of the study
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- Parameters examined: triglyceride, cholesterol, blood urea nitrogen, glucose, alkaline phosphatase, total protein, chloride, albumin, sodium, potassium, globulin, glutamic pyruvic transaminase/alanine aminotransferase activity, glutamic oxaloacetic transaminase/aspartate aminotransferase activity, gamma-glutamyl transferase activity, albumin : globulin ratio, creatinine, bilirubin (total)
alkaline phosphatase, calcium, inorganic phosphorus
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- ORGAN WEIGHTS: Absolute and relative organ weights (organ-body), measured at necropsy, included: adrenals, brain, kidneys, liver, lungs, and testes.
GROSS PATHOLOGY: Full compliment of tissues, including all male and female reproductive organs, were fixed and examined in all animals.
HISTOPATHOLOGY: The following tissues and organs were examined histologically in the high dose and control groups only: adrenals, gross lesions, heart, kidneys, larynx, liver, lungs, nasal cavity, spleen and trachea. Only identified target organs were to be examined in mid and low dose groups. - Statistics:
- Distribution of body weight, body weight changes, and organ weight were inspected for normality and homogeneity of variance across all dose groups. Analysis of variance models were used to assess the presence or absence of an overall compound effect. Separate one-way models were used within the male and female data to assess overall treatment group effects. Two-way models were used for treatment group, sex and interaction between groups. Pairwise comparisons of least square means between control and treated groups were evaluated using Dunnett'st-test. Hematology and clinical chemistry values were inspected for normality and homogeneity using ANOVA. Statistical significance was demonstrated at the p < 0.05.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- The only treatment-related signs of toxicity observed were inactivity, lacrymation, eye squinting, and labored breathing. These signs were seen only during the six-hour exposure. Slight inactivity occurred in all exposure groups on days 1, 2, and 3. Inactivity to slight inactivity occurred at concentrations of 952 and 1891 ppm on days 4 through 20. Lacrymation was observed only twice during the first three days, once at 952 ppm and once at 1891 ppm. Eye squinting occurred only at the highest concentration from day 3 to day 20, with the exception of day 4, were no eye squinting was observed. Labored breathing occurred once during day 1, at 1891 ppm.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant increase in the body weight for the 310 ppm females was observed during Week 3. However, because this was an isolated finding, and no concentration-related trend was observed, it was judged not to be treatment-related.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant decrease in the feed consumption for the 1891 ppm females was observed during Week 1. However, because this was an isolated finding, and no effect on body weight was observed, it was judged not to be treatment-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The clinical chemistry data showed two parameters with statistically significant findings. A decrease in the alkaline phosphatase concentration for 310, 952, and 1891 ppm female animals, was observed. A statistically significant decrease in the triglyceride concentration for 952 ppm and 1891 ppm female animals, was also observed. Although these parameters were statistically different from the controls in a concentration-related trend, these findings were judged not to be of toxicological significance.
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The organ weight data showed a statistically significant increase in kidney weight to body weight ratio in males and females exposed at 1891 ppm. Absolute kidney weights for this group were not statistically different from the controls.
In the absence of any histologic effects, hematology, and clinical chemistry findings this organ weight effect was judged to be not toxicological significant. - Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathologic evaluation revealed treatment-related observations in the nasal cavities. Microscopic examination of the nasal cavities of male and female rats exposed to 1891 ppm had slight and localized bilateral degeneration of olfactory epithelium lining the dorsal meati. One male rat and one female rat exposed to 952 ppm had similar changes in the olfactory epithelium. Rats exposed to 310 ppm had no exposure related nasal cavity microscopic changes.
- Histopathological findings: neoplastic:
- no effects observed
- Dose descriptor:
- NOAEC
- Remarks:
- local toxicty
- Effect level:
- 1 832 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- LOAEC
- Effect level:
- 5 626 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEC
- Remarks:
- systemic toxicity
- Effect level:
- 11 175 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no systemic toxicity up to and including the highest tested dose
- Critical effects observed:
- not specified
- Executive summary:
In an OECD Guideline 412 Repeated Dose 28-day inhalation study, 5 male and 5 female rats were exposed by whole body to 0, 310, 952 and 1891 ppm (0,1832, 5626, 11175 mg/m3) of the test item as a vapour for 6 h/day, 5 days/week for 4 weeks. Treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 952 and 1891 ppm (5626 and 11175 mg/m3) concentration groups throughout the study. These signs were observed sporadically during exposure throughout the study. There were no treatment-related effects on body weight or feed consumption, and no deaths occurred. Haematological measurements and clinical chemistry values generally were unaffected by treatment. At necropsy, the only organ weight effect was a statistical increase in the kidney weight to body weight ratio of the 1891 ppm males and females. In the absence of corresponding histologic effects, hematology or clinical chemistry findings, this increase in relative kidney weight was judged not to be toxicologically significant. Microscopic examination of the nasal cavities of rats exposed to 1891 ppm revealed a slight and localized bilateral degeneration of olfactory epithelium lining the dorsal meati. One male and one female rat exposed to 952 ppm had similar changes in the olfactory epithelium. Rats exposed to 31 0 ppm had no exposure related nasal cavity microscopic changes.
Based on the histopathologic changes seen in the nasal cavities, the LOAEL for local toxicity for a four-week whole-body inhalation exposure was 952 ppm. The NOAEL for local toxicity was 310 ppm. As no systemic toxicity was observed under the conditions of this study, the NOAEL for systemic toxicity was the highest tested dose of 1891 ppm.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 11 175 mg/m³
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 30
- Species:
- rat
- Quality of whole database:
- GLP and guideline compliant read-across study
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 15 - November 13, 1992
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP compliant
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Version / remarks:
- 1981
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- signed on July 27, 1993
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Supplier: Dupont, Belle plant, Belle, West Virgina
- Physical state: liquid
- Analytical purity: > 99%
- Impurities (identity and concentrations): iso-butyl methacrylate max 1%, MEHQ max. 0.002%
- Purity test date: no data
- Lot No.: SWF90319
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- vapour - Species:
- rat
- Strain:
- other: Crl:CD BR
- Details on species / strain selection:
- The rat is being utilized for this study because of the large amount of comparative toxicological and physiological data on this species.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Kingston, Stone Ridge, NY, USA
- Age at study initiation: 35-day old
- Weight at study initiation: ca 260 g for males and ca 195 g for females
- Housing: individually in suspended wire-mesh cage in the inhalation chamber rooms
- Diet: Purina certified rodent chow 5002 (excepted during exposure), ad libitum (withheld during exposure)
- Water: tap water (excepted during exposure), ad libitum (withheld during exposure)
- Acclimation period: two weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27
- Humidity (%): 40 - 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- other: unchanged (no vehicle)
- Details on inhalation exposure:
- All chambers were operated at an air flow rate of 400 L/min, resulting in a calculated 99% aerosol equilibrium time of 23 minutes or 6.4% of the exposure time. Vapours of the test material were generated from the liquid form, introduced at a constant flow into heated flasks (approx. 102-105°C) through which a constant flow of air was metered. The inhalation chambers were stainless steel, approximately2000 L. Chamber temperature was 22.8-25.5°C; chamber relative humidity was 62.5-67.6%. Fresh air was mixed with the test chemical vapours to achieve the desired concentration.
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals received a whole-body inhalation exposure under dynamic conditions in 2000-L stainless steel, glass, and Plexiglas chambers.
- Method of holding animals in test chamber: Each animal was individually housed in a suspended wire-mesh cage without food or water during the exposure. Cage positions within the chambers were rotated each day to insure that the animals were exposed at several different locations within each chamber.
- Source and rate of air: Calibrated flowmeters (Fisher-Porter Inc., Warminster, PA) delivered compressed air at a rate of 30 L/min into the air inlet of each flask
- Temperature, humidity, pressure in air chamber: The mean temperatures and relative humidities for all four chambers ranged from 22.8 to 25.5°C and 62.5 to 67.6%, respectively.
- Air flow rate: The chamber volume and air flow rate were adjusted to reach 99% of the maximum vapor concentration (t99) within 30 minutes of the total exposure time (Silver, 1946). The volume of animals loaded into the chamber was Iess than 7% of the total chamber volume.
- Air change rate: no data
- Generation of the test Atmosphere: The test material vapour concentrations were generated by metering the test material with calibrated Fluid Metering Pumps into 500 or 5000 ml three-necked round bottom flasks. The flasks were located in hemispherical heating mantles.
- Method of particle size determination: not appropriate, only vapours were generated
- Treatment of exhaust air: no data
TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test substance in the chambers was determined using a Miran 1A gas analyzer attached to a strip chart recorder. Before the initiation of the exposure, the Miran 1A gas analyzer was calibrated using precisely measured amounts of test material delivered into a dynamic vapour generator. A calibration curve was generated to convert the readings obtained from the Miran 1A gas analyzer into parts-per-million (ppm) butyl methacrylate.
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical concentrations only of test material were determined throughout the study. Test concentrations were measured during each daily exposure period (every 80 minutes) and the vapours analyzed by GC to confirm purity.
- Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- 6 h/day, 5 days/week
- Dose / conc.:
- 310 ppm (analytical)
- Remarks:
- 1832 mg/m3
- Dose / conc.:
- 952 ppm (analytical)
- Remarks:
- 5626 mg/m3
- Dose / conc.:
- 1 891 ppm (analytical)
- Remarks:
- 11175 mg/m3
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Fasting period before blood sampling for clinical biochemistry: yes
- Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed before and after each daily exposure for general condition and signs of toxicity, and once during the post-exposure observation period.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed at the day of first exposure and then weekly
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured weekly
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: during necropsy at termination of the study
- Anaesthetic used for blood collection: Yes, sodium pentobarbital
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: hematocrit, platelet count, hemoglobin, mean cell volume, red blood cell, mean ceil hemoglobin, white blood cell count, mean cell hemoglobin conc.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during necropsy at termination of the study
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- Parameters examined: triglyceride, cholesterol, blood urea nitrogen, glucose, alkaline phosphatase, total protein, chloride, albumin, sodium, potassium, globulin, glutamic pyruvic transaminase/alanine aminotransferase activity, glutamic oxaloacetic transaminase/aspartate aminotransferase activity, gamma-glutamyl transferase activity, albumin : globulin ratio, creatinine, bilirubin (total)
alkaline phosphatase, calcium, inorganic phosphorus
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- ORGAN WEIGHTS: Absolute and relative organ weights (organ-body), measured at necropsy, included: adrenals, brain, kidneys, liver, lungs, and testes.
GROSS PATHOLOGY: Full compliment of tissues, including all male and female reproductive organs, were fixed and examined in all animals.
HISTOPATHOLOGY: The following tissues and organs were examined histologically in the high dose and control groups only: adrenals, gross lesions, heart, kidneys, larynx, liver, lungs, nasal cavity, spleen and trachea. Only identified target organs were to be examined in mid and low dose groups. - Statistics:
- Distribution of body weight, body weight changes, and organ weight were inspected for normality and homogeneity of variance across all dose groups. Analysis of variance models were used to assess the presence or absence of an overall compound effect. Separate one-way models were used within the male and female data to assess overall treatment group effects. Two-way models were used for treatment group, sex and interaction between groups. Pairwise comparisons of least square means between control and treated groups were evaluated using Dunnett'st-test. Hematology and clinical chemistry values were inspected for normality and homogeneity using ANOVA. Statistical significance was demonstrated at the p < 0.05.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- The only treatment-related signs of toxicity observed were inactivity, lacrymation, eye squinting, and labored breathing. These signs were seen only during the six-hour exposure. Slight inactivity occurred in all exposure groups on days 1, 2, and 3. Inactivity to slight inactivity occurred at concentrations of 952 and 1891 ppm on days 4 through 20. Lacrymation was observed only twice during the first three days, once at 952 ppm and once at 1891 ppm. Eye squinting occurred only at the highest concentration from day 3 to day 20, with the exception of day 4, were no eye squinting was observed. Labored breathing occurred once during day 1, at 1891 ppm.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant increase in the body weight for the 310 ppm females was observed during Week 3. However, because this was an isolated finding, and no concentration-related trend was observed, it was judged not to be treatment-related.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistically significant decrease in the feed consumption for the 1891 ppm females was observed during Week 1. However, because this was an isolated finding, and no effect on body weight was observed, it was judged not to be treatment-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The clinical chemistry data showed two parameters with statistically significant findings. A decrease in the alkaline phosphatase concentration for 310, 952, and 1891 ppm female animals, was observed. A statistically significant decrease in the triglyceride concentration for 952 ppm and 1891 ppm female animals, was also observed. Although these parameters were statistically different from the controls in a concentration-related trend, these findings were judged not to be of toxicological significance.
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The organ weight data showed a statistically significant increase in kidney weight to body weight ratio in males and females exposed at 1891 ppm. Absolute kidney weights for this group were not statistically different from the controls.
In the absence of any histologic effects, hematology, and clinical chemistry findings this organ weight effect was judged to be not toxicological significant. - Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathologic evaluation revealed treatment-related observations in the nasal cavities. Microscopic examination of the nasal cavities of male and female rats exposed to 1891 ppm had slight and localized bilateral degeneration of olfactory epithelium lining the dorsal meati. One male rat and one female rat exposed to 952 ppm had similar changes in the olfactory epithelium. Rats exposed to 310 ppm had no exposure related nasal cavity microscopic changes.
- Histopathological findings: neoplastic:
- no effects observed
- Dose descriptor:
- NOAEC
- Remarks:
- local toxicty
- Effect level:
- 1 832 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- LOAEC
- Effect level:
- 5 626 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEC
- Remarks:
- systemic toxicity
- Effect level:
- 11 175 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no systemic toxicity up to and including the highest tested dose
- Critical effects observed:
- not specified
- Executive summary:
In an OECD Guideline 412 Repeated Dose 28-day inhalation study, 5 male and 5 female rats were exposed by whole body to 0, 310, 952 and 1891 ppm (0,1832, 5626, 11175 mg/m3) of the test item as a vapour for 6 h/day, 5 days/week for 4 weeks. Treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 952 and 1891 ppm (5626 and 11175 mg/m3) concentration groups throughout the study. These signs were observed sporadically during exposure throughout the study. There were no treatment-related effects on body weight or feed consumption, and no deaths occurred. Haematological measurements and clinical chemistry values generally were unaffected by treatment. At necropsy, the only organ weight effect was a statistical increase in the kidney weight to body weight ratio of the 1891 ppm males and females. In the absence of corresponding histologic effects, hematology or clinical chemistry findings, this increase in relative kidney weight was judged not to be toxicologically significant. Microscopic examination of the nasal cavities of rats exposed to 1891 ppm revealed a slight and localized bilateral degeneration of olfactory epithelium lining the dorsal meati. One male and one female rat exposed to 952 ppm had similar changes in the olfactory epithelium. Rats exposed to 31 0 ppm had no exposure related nasal cavity microscopic changes.
Based on the histopathologic changes seen in the nasal cavities, the LOAEL for local toxicity for a four-week whole-body inhalation exposure was 952 ppm. The NOAEL for local toxicity was 310 ppm. As no systemic toxicity was observed under the conditions of this study, the NOAEL for systemic toxicity was the highest tested dose of 1891 ppm.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 1 832 mg/m³
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- GLP and guideline compliant read-across study
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
Reliable studies of the test item and the close analogous substance n-butyl methacrylate (n-BMA) were used to assess the potential of test item for repeated dose oral toxicity.
In addition, n-BMA has been tested by inhalation in a subacute inhalation study (OECD 412). A chronic study does not have to be performed, because the substance is rapidly metabolised, and by analogy to MMA ultimately metabolised to carbon dioxide and water. Therefore, there is no concern for lesions due to accumulative toxicity.
Repeated dose toxicity oral route:
Key study:
In a 90-day repeated dose oral toxicity study conducted according to OECD TG 408 in compliance with GLP (reliability 1, 2009), n-butyl methacrylate was administered daily to male and female Wistar rats by gavage at dose levels of 0, 60, 120 and 360 mg/kg body weight per day over a period of 3 consecutive months. Control and high dose groups consisted of 15 animals per sex per group, whereas low and mid dose groups consisted of 10 animals per sex per group. After 3 months of treatment, 10 animals per sex of all dose groups were sacrificed (main groups). The remaining 5 animals per sex of control and high dose groups were maintained for another 28 days without administration of the test substance (recovery groups).
There were no local effects in the oral cavity or in the intestinal tract – the site of first contact on oral dosing. On the other hand, multifocal degenerative and regenerative olfactory epithelium of the nasal cavity was observed in high dose (360 mg/kg bw/day) and mid dose (120 mg/kg bw/day) males and females. Multifocal degeneration/regeneration of olfactory epithelium was observed in males (5 out of 10) and females (7 out of 10). At 120 mg/kg bw/day, multifocal degeneration/regeneration of olfactory epithelium was observed in males (4 out of 10) and females (2 out of 10). At 60 mg/kg bw/day, no substance-related adverse findings were observed. However, considering the study design, it is not possible to determine if this effect is of a systemic or local origin. The NOAEL for these effects was 60 mg/kg body weight/day in both males and females. This substance-related effect was completely reversible, as no animal of the recovery group showed any finding in the nose after 28 days after cessation of exposure.
At 360 mg/kg bw/day, prothrombin time was significantly prolonged (+10% males; +12% females) and inorganic phosphate (+14% males; +30% females), total bilirubin levels (+17% in males), glucose levels (+13% in females) as well as urea levels (+36% males; 21% females) significantly increased. Calcium (-3% in males and females), globulin levels (-4% in females) and triglyceride levels (-24% in males and females) decreased significantly. Absolute kidney weight (+11% in females), relative kidney weight (+13% males and females) and liver weight (+9% males; +11% females) significantly increased. After the recovery period, phosphate levels (+7% in males) and urea concentrations (+22% in females) were significantly increased. The NOAEL for systemic effects was 120 mg/kg body weight/day in both males and females. At 360 mg/kg bw/day, body weight change significantly decreased in male animals from day 77 onwards (-12.1% on days 84 and 91). The NOAEL for body weight effects was 360 mg/kg body weight/day in females and 120 mg/kg body weight/day in males.
Summary
In conclusion, the oral administration of n-butyl methacrylate by gavage over a period of 3 months with a recovery period of 28 days revealed toxicologically relevant signs of systemic toxicity at the high dose level of 360 mg/kg bw/day, limited to effects on the liver activity (increased liver weight, prolonged prothrombin time, lower serum globulin and triglyceride levels in males and/or females) and kidneys weight (increased absolute weight in females). The NOAEL for systemic toxicity was 120 mg/kg body weight/day in both males and females.
Key study:
A 28-day repeated dose toxicity oral study similar to OECD TG 407 was conducted with tert. butyl methyacrylate using concentrations of 0, 20, 100, 500 mg/kg bw.
There were no deaths throughout the course of the study. Transient salivation after administration was observed in both sexes given 500 mg/kg bw/day. The body weights and food consumption revealed no differences between the control and treated groups. There were no observed effects of the test substance on hematological findings. Blood chemical examination showed increases in total cholesterol and total protein in both sexes given 100 and 500 mg/kg bw/day, an increase in albumin in females given 100 mg/kg bw/day and both sexes given 500 mg/kg bw/day, and a decrease in alkaline phosphatase in males given 100 mg/kg bw/day and both sexes given 500 mg/kg bw/day. Urinalysis showed an increase in protein in both sexes given 500 mg/kg bw/day, an increase in occult blood in males given 100 and 500 mg/kg bw/day and an increase in bilirubin in males given 500 mg/kg bw/day. In addition, microscopic examination of urinary sediment revealed an increase in erythrocytes in males given 500 mg/kg bw/day and an increase in epithelial cells in females given 500 mg/kg bw/day. Absolute and relative liver and kidneys weight were increased in both sexes given 500 mg/kg bw/day, and relative weight of the liver increased in males given 100 mg/kg bw/day. Necropsy revealed hypertrophy of the liver in three males and five females given 500 mg/kg bw/day. Histopathological examination showed centrilobular hypertrophy of hepatocytes in four males given 100 mg/kg bw/day and all animals of both sexes given 500 mg/kg bw/day. Increase in the amounts of hyaline droplets of proximal renal tubules was observed in two males given 100 mg/kg bw/day and four males given 500 mg/kg bw/day. These form of hyaline droplets are considered specific to male rats and have no relevance for the humans species. Basophilic change of renal tubules was observed in one male given 100 mg/kg bw/day and three males given 500 mg/kg bw/day. These changes disappeared or tended to recover during the recovery period. After a review of this study, the NOAEL for the repeat dose toxicity is considered to be 100 mg/kg bw/day for both sexes.
Key study:
The repeated dose
toxicity and reproductive and developmental toxicity were investigated
according to a OECD TG 421 study (reliability 2, 2008).The tert. butyl
methacrylate was administered at 0 (control group: aqueous 0.5 w/v%
carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100,
300 and 1000 mg/kg bw/day to Sprague-Dawley SPF rats, for 14 days before
mating and throughout the mating period until the day before autopsy in
males (42 days), and for 14 days before mating and throughout the mating
period and gestation period until day 4 of nursing in females (42-48
days). No animal deaths or abnormality in general condition were
observed in any administration group. No effects of test substance
administration on body weight or feed intake were observed in any
administration group.On pathological examination, effects of test
substance administration were found in the livers and
kidneys of males and females in the ≥100 mg/kg bw/day groups. In the
livers there was visible hypertrophy, and histologically, centrilobular
hepatocyte hypertrophy, brightening of hepatocytes and localized
necrosis of hepatocytes were observed. The centrilobular hepatocyte
hypertrophy was thought to be an adaptive change accompanying
extracorporeal excretion of the present agent; the brightening of
hepatocytes was attributed to glycogen accumulation under satiation; and
the necrosis of hepatocytes was a localized lesion in each case. The
histological characteristics and degrees of these changes were similar
to those of naturally occurring changes in rats of the same strain, and
no organic disorders were observed males or females, and so these
changes were thought to have no toxicological significance. In the
kidneys there was visible hypertrophy, and histologically, glomerular
hyaline droplets, renal tubule hyaline droplets, basophilic change,
dilatation, necrosis and vacuolation were observed; hyaline cast and
erythrocyte cast were also observed. All renal tubule necrosis was
accompanied by vacuolation. It was determined from these results that
the no-observed effect dose with respect to the repeated administration
toxicity of the test substance in males and females was less than 100
mg/kg bw/day.
Supporting study:
n-Butyl methacrylate was studied for oral toxicity in rats in an OECD TG 422 combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 30, 100, 300 and 1000 mg/kg bw/day.
Local effects
There were no local effects in the oral cavity or in the intestinal tract – the site of first contact on oral dosing or any other tissue investigated. Systemic effects
Body weight effects in both males and females: there were weight gain depression and a decrease in food consumption at a dose of 1000 mg/kg bw/day. At 1000 mg/kg bw/day relative kidney weights were increased. Urinary examination revealed increases in ketone bodies and occult blood, and haematological and blood chemical examinations showed increases in prothrombin time and urea nitrogen and atrophy of the red pulp in the spleen was also observed histopathologically. Absolute and relative weights of the spleen were decreased at a dose of 100 mg/kg bw/day or more, and relative kidney weights were increased at a dose of 1000 mg/kg bw/day. Histopathological examination revealed atrophy of the splenic red pulp at doses of 100 mg/kg bw/day or more. The kidney showed no histopathological abnormalities attributable to the test substance. The NOELs for repeat dose toxicity were considered to be 30 mg/kg bw/day for males and 300 mg/kg bw/day for females. This study has been independently reviewed (RSA, 2008). Based on published historical control data from the same test laboratory, effects seen at 100 and 300 mg/kg bw/day were not statistically significant when compared with historical controls. In conclusion, the oral administration of n-butyl methacrylate by gavage in an OECD 422 study revealed toxicologically relevant signs of systemic toxicity at the high dose level of 1000 mg/kg bw/day, The NOAEL in this study was 300 mg/kg bw/day in both males and females.
Repeated dose toxicity inhalation route:
In an OECD TG 412 Repeated Dose 28-day inhalation study, 10 male and 10 female rats were exposed by whole body to 0, 310, 952 and 1891 ppm (0,1832, 5626, 11175 mg/m3) n-BMA for 6 hr/day, 5 days/week for 4 weeks. Treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 5626 and 11175 mg/m3 concentration groups throughout the study. There were no treatment-related effects on body weight or feed consumption, and no deaths occurred. Haematological measurements and clinical chemistry values generally were unaffected by treatment.
Local effects
Treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 5626 and 11175 mg/m3 concentration groups throughout the study. The only treatment-related histopathological finding was localised bilateral degeneration of olfactory epithelium lining the dorsal meatus of the nasal cavity at 5626 and 11175 mg/m3 in both sexes.
Systemic effects
Haematological measurements and clinical chemistry values generally were unaffected by treatment. Despite increased relative kidney weights at the high concentration (11175 mg/m3) in both sexes, and slight increases in serum BUN values (resulting in increased BUN:creatinine ratio), histopathology of the kidneys was normal. There were no treatment-related effects on body weight or feed consumption.
The no-observable adverse effect concentration (NOAEC) for systemic effects was 11175 mg/m3. Based on histopathological changes seen in the nasal cavities, the no-observable adverse effect concentration (NOAEC) for local effects was 1832 mg/m3 while the LOAEL for local effects was 5626 mg/m3.
Justification for classification or non-classification
The available experimental test data from oral and inhalativ studies of the close analogous substance n-butyl methacrylate (n-BMA) are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.
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