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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03/2013- 12/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-ethylpyrrolidin-2-one
EC Number:
220-250-6
EC Name:
1-ethylpyrrolidin-2-one
Cas Number:
2687-91-4
Molecular formula:
C6H11NO
IUPAC Name:
1-ethylpyrrolidin-2-one
Test material form:
other: vapor
Details on test material:
- Name of test material (as cited in study report): N-Ethyl-2-pyrrolidone
- Physical state: liquid
- Analytical purity: 99.8%
- Lot/batch No.: 52195875L0. 03 Nov 2011 date of production
- Expiration date of the lot/batch: 02 Nov 2013
- Storage condition of test material: room temperature
Specific details on test material used for the study:
Batch-no. 52195875L0
Purity: 99.8% corr. area %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test species and strain: Wistar rats, Crl:WI(Han)
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
Sex: Male / female
Age at supply: about 7 weeks / start of pre-exposure 9 weeks, start of exposure 9 weeks
Air conditions: Temperature 20-24°C, relative humidity 30-70%. 15 air changes per hour.
Illumination period: 06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark
Type of cage / No. of animals per cage: Female animals: Makrolon cages type M III / 1 animal. Male animals: Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany / up to 5 animals
For motor activity measurement: polycarbonate cages (floor area about 800 cm2, Ehret, Emmendingen, Germany) / 1 animal
Enrichment: Wooden gnawing blocks (Type NGM E-022); Abedd Lab. and Vet. Service GmbH Vienna, Austria and play tunnel, large, Plexx b.v. Elst. Netherlands.
Type of diet: Males: Kliba laboratory diet, mouse/rat maintenance “GLP”, 10 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland; ad libitum
Females: Ground Kliba mouse/rat maintenance diet “GLP”, meal supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
Bedding: dustfree wooden bedding
Watering: Drinking water ad libitum
Acclimatization: During the acclimatization period the animals are accustomed to the surroundings of the study and to the diet.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose/head only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF THE INHALATION ATMOSPHERES

Generator systems:
- Continuous infusion pumps PHD Ultra (Harvard Apparatus, Inc., Holliston, Massachusetts, U.S.A.)
- Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)

Generation procedure:
The test substance was used unchanged. For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air mixed with conditioned dilution air and passed into the inhalation system. Conditioned supply air is activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C. Compressed air is filtered air pressurized to about 6 bar. The nozzle pressure was between 1.2 and 2.0 bar. The control group was exposed to conditioned air.

The following test substance flow and air flows and were scheduled:

Test group Substance flow (g/h) Supply air 1 conditioned (m³/h) Supply air 2 compressed (m³/h) Exhaust air 1 (m³/h)
0 - 5.4 – 6.6 5.1 – 5.7 -
1 0.1 – 0.4 4.2 – 4.8 5.1 – 5.7 1.2 – 1.8
2 0.3 – 0.7 4.2 – 4.8 5.1 – 5.7 1.2 – 1.8
3 1.0 – 2.0 4.2 – 4.8 5.1 – 5.7 1.2 – 1.8


ANALYSES
Calculation of nominal concentrations:
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

Analytical determination of concentrations:
The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, were converted to concentration of the test substance. FID monitored continuously the constancy of the concentrations in the chamber. FID measures total hydrocarbon in the atmosphere, it is not a specific method. Therefore, the identity of the test substance in the atmosphere was confirmed by gas chromatography of absorption samples. For this purpose, two samples (of the same day) per concentration and week were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups.

The measured concentrations in the inhalation chambers were calculated as daily mean of the FID measurements, less the mean value of the back ground during the study. From the daily means, the study means were calculated. Study means of the off-line analyses served as control for FID analyses. The retention time of the peak in GC chromatograph served as a control that the atmospheres consisted of the test substance.

Real time monitoring of constancy of concentrations:
Total hydrocarbon analyzers (Testa 123) were used to continuously monitor the constancy of concentrations of test substance vapors in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded using line recorders and transferred to the automated measuring system.

Particle size analysis:
Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321.
Frequency: Once (3 repeats) per concentration during the first week and once (3 repeats) per concentration during the second week of exposure.
No further analyses were performed, because measured particle concentration was in all concentration groups around the background level.

EXPERIMENTAL PROCEDURE
Acclimatization and exposure period: The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings. Prior to the pre-exposure period, 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.

Exposure systems; exposure of the animals:

Head nose exposure systems:
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V  90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the vapor. The exposure systems were located in exhaust hoods in an air conditioned room.

Exposures:
The head nose exposure technique was preferably selected for this inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole body exposure. Furthermore, by using the dynamic mode of operation with a low volume chamber , the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99 % of the final target concentration) is shorter as compared to whole body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.

Measurements of the exposure conditions:
Principles of recording with the automated measuring system: Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0-20 or 4-20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.
Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data. Relevant disturbances were reported.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, were converted to concentration of the test substance. FID monitored continuously the constancy of the concentrations in the chamber. FID measures total hydrocarbon in the atmosphere is not a specific method. Therefore, the identity of the test substance in the atmosphere was confirmed by gas chromatography of absorption samples. For this purpose, two samples (of the same day) per concentration and week were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups.
See table 1 for results of exposure measurements
Duration of treatment / exposure:
90 days
Frequency of treatment:
65 exposures, during the week (5-times every week, Monday to Friday)
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 60, 200 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The objective of the study was to assess the toxicity of the test substance after multiple exposures to vapors/aerosols. Special emphasis was laid on possible neurotoxic effects. Test concentrations were selected based on the results of a preceded 28d inhalation study (BASF 40/I0033/04I021, see above).
Wistar rats, 10 male and 10 female animals per test group, were head-nose exposed to vapor for 6 hours per day, on 5 consecutive days per week for 13 weeks (65 exposures). The target concentrations were 30, 60 and 200 mg/m3. A concurrent control group was exposed to air. On each exposure day a clinical examination was performed before, during and after exposure. Detailed clinical observation was performed at the beginning, midterm and end of the study. Ophthalmology was performed before the beginning of the exposure in all test groups and at the end of the end of the exposure in the control and high concentration group animals. Body weights and food consumption of the animals were determined weekly. At the end of the exposure period, functional observation battery and motor activity tests were performed. Against the end of the exposure period, urine were collected in all animals and were analyzed according to the guidelines. On the day after the last exposure, blood was sampled and examined for a range of hematology and clinical chemical parameters as indicated in the guideline. After blood sampling the animals were sacrificed and subject to necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). In addition, sperm motility and total sperm head count (testis and caudal epididymides) was assessed. Selected tissues were processed histopathologically and were evaluated by light microscopy according to the OECD guideline.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS

Mortality
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

Clinical observations
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.

Detailed clinical observation (DCO)
All animals were subject to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and on study days 42 and 84 (same time in the morning). For observation, the animals were therefore removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed were based on the current index of findings in PDS ToxData® and includes but is not limited to the following parameters listed:
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

Body weight data
The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, once a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change of the respective week was calculated as the difference between body weight on Friday and the previous Monday. Group means were derived from the individual differences.

Food consumption
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.

Ophthalmology
Before the start of the exposure period (day -1/ -2) the eyes of all main group animals (groups 0 to 4), and at the end of the study (day 83) the eyes of the animals of test group 0 (control group), test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

Functional observational battery
A functional observation battery (FOB) was carried out on assigned animals. At least one hour before the start of the FOB the animals were transferred singly to Polycarbonate cages (floor area about 800 cm²). The cages were placed in the racks in a randomized order (randomization based upon animals number). 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 home cage, and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. Another technician documented all findings and values obtained. The findings were ranked according to the degree of severity, if applicable.

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

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, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypies, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency) within two minutes, urine (amount/color) 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: Aapproach 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

Motor activity measurements (MA)
Motor activity 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 supplied by Columbus Instruments Int. Corp. U.S.A. 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. 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.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The results of clinical pathology examinations were expressed in International System (SI) units.
The following examinations were carried out in 10 animals per test group and sex.
Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET)

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

Clinical chemistry
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters:

Enzyme (systematic name and system number): Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP)(orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.), -Glutamyltransferase (GGT) ( -glutamyl) peptide: aminoacid--glutamyl-transferase; EC 2.3.2.2.)

Blood Chemistry Parameter: 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)

Urinalysis
The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Parameter investigated: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume
Sacrifice and pathology:
PATHOLOGY
Necropsy: All animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (modified Davidson’s solution), Esophagus, Extraorbital lacrimal gland , Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary glan, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testis, left (modified Davidson’s solution), Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

In case of macroscopic findings in the right testis (animal no. 25), this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm parameters.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
Organs
Test group 0 1 2 3
All gross lesions A2 A2 A2 A2
Adrenal glands A1 A1
Aorta A1 A1
Bone marrow (femur) A1 A1
Brain A1 A1
Cecum A1 A1
Colon A1 A1
Duodenum A1 A1
Epididymis, left A1 A1 A1 A1
Esophagus A1 A1
Eyes with optic nerve A1 A1
Extraorbital lacrimal gland A1 A1
Femur with knee joint A1 A1
Harderian gland A1 A1
Heart A1 A1
Ileum A1 A1
Jejunum A1 A1
Kidneys A1 A1
Larynx (3 levels)a A1 B1 B1 A1
Liver A1 A1
Lung A1 A1
Lymph nodes
(Tracheobronchial,
mediastinal, mesenteric) A1 A1
Mammary gland (female) A1 A1
Nasal cavity (4 levels)b A1 A1 A1 A1
Ovaries A1 A1
Pancreas A1 A1
Parathyroid glands A1 A1
Pharynx A1 B1 B1 A1
Pituitary gland A1 A1
Prostate A1 A1
Rectum A1 A1
Salivary glands
(Mandibular and
sublingual glands) A1 A1
Sciatic nerve A1 A1
Seminal vesicles A1 A1
Skeletal muscle A1 A1
Skin A1 A1
Spinal cord
(cervical, thoracic and
lumbar cord) A1 A1
Spleen A1 A1
Sternum with marrow A1 A1
Stomach
(forestomach and
glandular stomach) A1 A1
Teeth A1 A1
Testis, left A1 A1 A1 A1
Thymus A1 A1
Thyroid glands A1 A1
Tracheac A1 A1
Urinary bladder A1 A1
Uterus A1 A1

Abbreviations:
A = Hematoxylin and eosin (H&E) stain
B = Paraplast embedding
1 = all animals/test group
2 = all animals affected/test group
a one level does include the base of the epiglottis
b one level will include nasopharyngeal duct; the 4 levels allow adequate examination of the squamous, transitional, respiratory and olfactory epithelium, and the draining lymphatic tissue (NALT)
c one transverse section and one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al 2003, Kittel et al 2004 and Morawietz et al 2004). A correlation between gross lesions and histopathological findings was attempted.

Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed by a third pathologist including left testes, left epididymides and nasal cavity of all animals. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Other examinations:
Sperm parameters
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis and cauda epididymis) were evaluated in control and high dose group, only. Morphology was counted in control and high dose group of the F0 generation and in all groups of the F1 generation.

Parameters and methods of sperm examination: Sperm motility, Sperm morphology, Sperm head count (cauda epididymis), Sperm head count (testis)
Statistics:
Statistical analyses see table below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional abservational battery and the moror activity examination did no reveal any substance related abnormalities.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Description (incidence and severity):
see below
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the olfactory epithelium at different levels of the nasal cavity, a minimnal to moderate degeneration and/or regeneration was observed in all males and females of the highest concentration (200 mg/m3).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CONCENTRATION MEASUREMENTS

See table 1
The vapor generation effectiveness was as expected for these high concentrations. Some loss of concentration probably occurred by condensation on the wall of the stainless steel chamber.

Offline GC analysis of absorption samples showed that the atmosphere consisted of the vapor of the test substance. The measured concentrations of GC analyses were generally slightly lower than those of FID analyses, probably due to substance loss during sampling.

CLINICAL EXAMINATIONS
Mortality
No deaths were recorded throughout the study.

Clinical observations
During the pre-exposure period the animals showed no clinical signs and findings different from normal.

During the exposure period several animals of each test group showed discoloration of the fur. This finding was distributed evenly in the control and test groups, therefore it is considered not to be substance-related. No other clinical signs of toxicity were observed.

Detailed clinical observations
The detailed clinical observations generally did not reveal any clinical signs of toxicity in all test animals. However, in a few animals of each group, discoloration of fur was observed. As discoloration of the fur was observed in the controls, it was considered not to be substance-related and was not adverse.

Body weight data
Body weight during the exposure period:
The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
Body weight change during the exposure period was decreased in the following cases:

Test group 3 (male):
Study day 0 to 2, p < 0.01
Study day 40 to 44, p < 0.05
Test group 3 (female)
Study day 4 to 8, p < 0.01

Test group 2 (male)
Study day 5 to 9, p < 0.05
Study day 40 to 44, p < 0.05

Test group 1 (male)
Study day 40 to 44, p < 0.05

However, these changes were transient, without concentration-relationship. Therefore, they are considered to be substance-related.

Food consumption
No substance-related changes of food consumption were observed during the whole study period.

Ophthalmology
The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

Functional observational battery
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substance.
Deviations from "zero values" were obtained in several animals. However, as most 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, the following groups of parameters (study day 90) have to be assessed individually:

Quantitative parameters:
No substance-related findings were observed.

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

Motor activity
Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female rats.

CLINICAL PATHOLOGY
Hematology
No treatment-related changes among hematological parameters were observed.

Clinical chemistry
No treatment-related changes among clinical chemistry parameters were observed.

At the end of the study in females of test groups 1, 2 and 3 (30, 60 and 200 mg/m3), creatinine values were lower and potassium levels were higher compared to controls. Additionally, in females of test group 1 (30 mg/m3), aspartate aminotransferase (AST) activities were decreased. All mentioned parameters were not dose-dependently changed. In females of test groups 2 and 3 (60 and 200 mg/m3), globulin values were decreased, but the means were within the historical control range (globulins 22.51-29.31 g/L). Therefore, all mentioned alterations among clinical chemistry parameters were regarded as incidental and not treatment-related.

Urinalyses
No treatment-related changes among urinalysis parameters were observed.

Sperm parameters
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

In males of test group 2 (60 mg/m3) total sperm head counts in the testis were lower and the incidence of abnormal sperms in males of test group 1 (30 mg/m3) were higher compared to controls. Both parameters were not dose-dependently changed. Therefore, these alterations were regarded as incidental and not treatment-related.

PATHOLOGY
Weight parameters

Absolute organ weights
When compared with control group 0 (=100%), the mean absolute weight of the ovaries was significantly decreased in test group 2. All other mean absolute weight parameters did not show significant differences when compared to the control group 0. (see table 2 below)

Relative organ weights
When compared with control group 0 (=100%), the mean relative organ weights of liver and ovaries were significantly increased or decreased in one or more test groups (see table 3 below).
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
There was no histopathological correlate for the decreased mean relative liver weight in females of test group 3 (200 mg/m³). Therefore, this weight decrease was regarded to be incidental. Because there was no concentration-response relationship, the decreased ovarian weights in females of test group 2 (60mg/m³) as well as the increased ovarian weights in females of test group 3 (200 mg/m³) were considered to be incidental.

Gross lesions
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY

Nasal cavity:
In the olfactory epithelium at different levels of the nasal cavity, a degeneration and/or regeneration was observed in all males and females of test group 3 (200 mg/m3). The degeneration/ regeneration of the olfactory epithelium was characterized by increased intercellular spaces, irregular epithelial architecture, dilation (ectasia) of nasal glands, necrotic or metaplastic epithelium and/or increased nuclear:cytoplasmic ratio. The finding occurred focal or multifocal and was located at the dorsal part of septum, nasoturbinate and/or ethmoid turbinate. The severity of degeneration/ regeneration of the olfactory epithelium varied from a single small area with clear epithelial changes or few small or single larger areas with only a minimal loss of the organization of the cell layers (diagnosed as grade 1, minimal) up to some large areas with clear irregular epithelial architecture and/or occurrence of necrosis or metaplasia (diagnosed as grade 3, moderate). The incidence and grading are given in table 4 below.

The occurrence of degeneration/ regeneration of the olfactory epithelium in males and females of test group 3 (200 mg/m3) was regarded to be treatment-related.

Left testicle and left epididymis:
A minimal to severe multifocal tubular degeneration was observed in left testes in control and treated males. Incidence and severity are given in the table 5 below:

In the left epididymis, debris and oligospermia was observed in control and treated males. Incidence and severity are given in table 6 below.

Multifocal tubular degeneration in the testes occurred in control and treated males without relation to the concentration resulting in debris and/or oligospermia in the epididymides in some of these males. These findings in testes and in the epididymides have been observed frequently in head-nose exposed control animals in the past in the same laboratory. The incidence of tubular degeneration in 20 repeated dose inhalation studies with the same rat strain was up to 100% (157 animals, range (degeneration) 0 - 100%, mean 23.57% ) with gradings from minimal (grade 1) to extreme (grade 5). Therefore, the occurrence of tubular degeneration in treated males as well as of the resulting debris and oligospermia in the epididymides was considered to be incidental. Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. In males of test group 2 (60 mg/m3) total sperm head counts in the testis were lower and the incidence of abnormal sperms in males of test group 1 (30 mg/m3) were higher compared to controls. Both parameters were not dose-dependently changed. Therefore, these alterations were regarded as incidental and not treatment-related.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. In particular there were no substance-related changes in the kidney and the liver.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
60 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: respiratory tract irritation
Key result
Dose descriptor:
LOAEC
Effect level:
200 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: minimal to light effects in nasal cavity

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/m³ air (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1 Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations

 

Test group

Target concentration
(mg/m³)

Measured concentration (mg/m³)

Nominal concentration (mg/m³)

Effectiveness of vapor generation
(%)

Mean

SD

1

30

29.8

2.9

33.3

89.7

2

60

62.6

4.7

69.8

89.7

3

200

197.5

23.6

216.1

91.4

 

The vapor generation effectiveness was as expected for these high concentrations. Some loss of concentration probably occurred by condensation on the wall of the stainless steel chamber.

 

Offline GC analysis of absorption samples showed that the atmosphere consisted of the vapor of the test substance. The measured concentrations of GC analyses were generally slightly lower than those of FID analyses, probably due to substance loss during sampling. Real time surveillance of the inhalation atmospheres with total hydrocarbon analyzers generally proved the constancy of each concentration throughout the daily exposures.

Table 2 Relative change of absolute organ weight in ovaries

* : p <= 0.05

 

Female animals

Test group

(mg/m³)

1

(30)

2

(60)

3

(200)

Ovaries

103%

86%*

111%

Table 3 Relative change of relative organ weight in liver and ovaries

 

 

Female animals

Test group

(mg/m³)

1

(30)

2

(60)

3

(200)

Liver

100%

96%

94%*

Ovaries

100%

87%*

110%*

* : p <= 0.05

Table 4 Histological findings in nasal cavity

 

Male animals

Female animals

Test group

Concentration (mg/m3)

0

 

1

(30)

2

(60)

3

(200)

0

1

(30)

2

(60)

3

(200)

Organs examined

10

10

10

10

10

10

10

10

Nasal cavity (level I)

 

 

 

 

 

 

 

 

Degeneration/ regeneration, olfactory epithelium

 

 

 

 

 

 

 

3

  • Grade 1

 

 

 

 

 

 

 

2

  • Grade 2

 

 

 

 

 

 

 

1

Nasal cavity (level II)

 

 

 

 

 

 

 

 

Degeneration/ regeneration, olfactory epithelium

 

 

 

7

 

 

 

7

  • Grade 1

 

 

 

5

 

 

 

4

  • Grade 2

 

 

 

2

 

 

 

2

  • Grade 3

 

 

 

 

 

 

 

1

Nasal cavity (level III)

 

 

 

 

 

 

 

 

Degeneration/ regeneration, olfactory epithelium

 

 

 

8

 

 

 

10

  • Grade 1

 

 

 

4

 

 

 

4

  • Grade 2

 

 

 

3

 

 

 

4

  • Grade 3

 

 

 

1

 

 

 

2

Nasal cavity (level IV)

 

 

 

 

 

 

 

 

Degeneration/ regeneration, olfactory epithelium

 

 

 

10

 

 

 

10

  • Grade 1

 

 

 

3

 

 

 

5

  • Grade 2

 

 

 

4

 

 

 

4

  • Grade 3

 

 

 

3

 

 

 

1

Table 5 Histological findings in left testes

Left testicle

Male animals

Test group

Concentration (mg/m3)

0

 

1

(30)

2

(60)

3

(200)

Organs examined

10

10

10

10

Degeneration, tubular, multifocal

6

8

6

8

  • Grade 1

4

4

 

3

  • Grade 2

1

1

5

3

  • Grade 3

1

2

1

2

  • Grade 4

 

1

 

 

 

 

Table 6 Histological findings in left epididymides

Left epididymis

Male animals

Test group

Concentration (mg/m3)

0

 

1

(30)

2

(60)

3

(200)

Organs examined

10

10

10

10

Debris

3

5

5

6

  • Grade 1

2

2

3

4

  • Grade 2

1

1

2

1

  • Grade 3

 

2

 

1

Oligospermia

 

2

2

2

  • Grade 2

 

2

2

1

  • Grade 3

 

 

 

1

 

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of rats to N-ethyl-2-pyrrolidone for 90 day (65 exposures) did not lead to any exposure-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology, urine and sperm parameters. Histological examination revealed some effect in nasal cavity at the highest tested concentration of 200 mg/m3. There were no findings in liver or kidneys and no other signs of a specific target organ toxicity. Under the current test conditions, the NOAEC for local effect in nasal cavity was 60 mg/m3.
Executive summary:

The study was conducted after the registrant's testing proposal for a 90d inhalation study following to the ECHA decision TPE-D-0000001949 -59 -05/F.

To determine the potential toxicity of N-Ethyl-2-pyrrolidoneafter inhalation exposure, a 90-day inhalation study was carried out according to OECD 413 and EC No 440/2008. Wistar rats, 10 male and 10 female animals per test group, were head-nose exposed to vapor for 6 hours per day, on 5 consecutive days per week for 13 weeks (65 exposures). The target concentrations were 30, 60 and 200 mg/m3. A concurrent control group was exposed to air. On each exposure day a clinical examination was performed before, during and after exposure. Detailed clinical observation was performed at the beginning, midterm and end of the study. Ophthalmology was performed before the beginning of the exposure in all test groups and at the end of the end of the exposure in the control and high concentration group animals. Body weights and food consumption of the animals were determined weekly. At the end of the exposure period, functional observation battery and motor activity tests were performed. Against the end of the exposure period, urine were collected in all animals and were analyzed according to the guidelines. On the day after the last exposure, blood was sampled and examined for a range of hematology and clinical chemical parameters as indicated in the guideline. After blood sampling the animals were sacrificed and subject to necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). In addition, sperm motility and total sperm head count (testis and caudal epididymides) was assessed. Selected tissues were processed histopathologically and were evaluated by light microscopy according to the OECD guideline.

When compared with the control group, the following treatment-related adverse findings were noted in Wistar rats after 90 days of inhalation: 

High concentration (200mg/m3):

Nasal cavity

- Degeneration and regeneration of the olfactory epithelium in all males and females

Mid concentration (60mg/m3) and low concentration (30 mg/m3):

 - No treatment-related adverse findings.

 

Conclusion

Inhalation exposure of rats to N-ethyl-2-pyrrolidone for 90 day (65 exposures) did not lead to any exposure-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology, urine and sperm parameters. Histological examination revealed some effect in nasal cavity at the highest tested concentration of 200 mg/m3. Under the current test conditions, the NOAEC for local effect in nasal cavity was 60 mg/m3.