Pyrrolidine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to OECD 422

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 Mar 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals; Section 4: Health Effects, No. 413, "Subchronic Inhalation Toxicity: 90-day Study" (7 September 2009) for the conduction of inhalation exposure and the examination of the respiratory tract.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency (EPA), Health Effects Test Guidelines OPPTS 870. 3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test EPA Document 712-C-00-368, July, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pyrrolidine

EC Number:

204-648-7

EC Name:

Pyrrolidine

Cas Number:

123-75-1

Molecular formula:

C4H9N

IUPAC Name:

pyrrolidine

Details on test material:

- Name of test material (as cited in study report): Pyrrolidine
- Physical state: Liquid / clear, colorless
- Analytical purity: 99.9 area-% (GC)
- Homogeneity: Homogenous
- Lot/batch No.: LBPRDA1004
- Storage condition of test material: Refrigerator; under N2

Test animals

Species:

rat

Strain:

other: Wistar rats, Cri:WI(Han)

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; Sulzfeld, Germany
- Age at study initiation: 10- 11 weeks
- Weight at study initiation:
- Housing: Makrolon cages type M Ill, 1 animal per cage (exceptions: during mating: 1 male/1 female per cage and during rearing up to PND 4: 1 dam with her litter)
- Diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Basel Switzerland; ad libitum
- Water: Drinking water ad libitum
- Feed and drinking water was withdrawn from the animals during exposure.
- Acclimation period: For adaptation to the exposure conditions the animals were exposed to fresh air under comparable flow conditions in head-nose inhalation systems on several days before start of the exposure period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20- 24
- Humidity (%): 30- 70
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head-nose inhalation systems: The test atmospheres were passed into the aerodynamic exposure apparatuses (INA 60, V ca. 90 L, BASF SE) with the supply air.
- Method of holding animals in test chamber: The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamber to inhale the atmosphere.
- Source and rate of air: The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air was lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones of the animals.
- Method of conditioning air: for each concentration, constant amounts of the substance was supplied to about 30°C thermostated vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and passed into the inhalation systems. As equipment continuous infusion pumps PERFUSOR (B. Braun Melsungen AG, Melsungen, Germany), glass vaporizers with thermostat (BASF SE, Ludwigshafen, Germany) and thermostat (JULABO Labortechnik GmbH, Seelbach, Germany) was used.
- Air flow rate: The air flow rates of supply and exhaust air were measured continuously.

The nominal concentration of the inhalation atmospheres were calculated from the amounts of test substance dosed and air-flow per unit time.

TEST ATMOSPHERE
- Brief description of analytical method used: total hydrocarbon analyzer, gas chromatography
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The constancy of concentrations in the inhalation atmospheres was surveyed continuously with total hydrocarbon analyzers (FID, Testa). The FID was calibrated with certificated test gas propane. Using specific response factor provided by the manufacture concentration of Pyrrolidine was derived. The measured values over the study day gave information about the constancy of the Pyrrolidine concentration over the exposure time.
As the measurements with FID present the sum of the hydrocarbon in the air, to confirm the composition of the test atmosphere, the test atmospheres were analyzed by gas chromatography of absorption samples.
Absorption samples were taken adjacent to the animals noses in order to confirm the identity of the test substance in the atmospheres (sampling velocity in the sampling probe = 1.25 m/sec). For this purpose, absorption vessels were connected in series, filled with appropriate solvent. Using a gas sampling station an appropriate volume of atmosphere was drawn through the absorption vessels, which was analyzed by gas chromatography.
Sampling frequency: two samples per concentration and week. The control atmosphere was sampled on one day during the exposure period.

Duration of treatment / exposure:

6 hours per day
Males:
a) 14 days premating
b) up to 14 days mating
c) Sacrifice after a minimum of 28 days after the first application
Females:
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19
d) after necropsy of the pups total 9 exposures on 9 consecutive days including the day before scheduled killing

Frequency of treatment:

6 hours per day on 7 consecutive days per week (no exposure on the day of FOB/MA)

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 50, 150 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

other: exposed to conditioned air

Details on study design:

- Dose selection rationale: Based on the results of a dose range finding study
- Reason for the selection of the route of administration: Most likely exposure route for man

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Further details: A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.
On exposure days, a clinical inspection was performed on each animal at least three times a day (before, during and after exposure).
On non-exposure days, a cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were subjected to detailed clinical observations (including palpation) outside their cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning). For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 x 25 em) for at least 20 seconds/animal. The following parameters were examined:
abnormal behavior during “handling”, fur, skin, posture, salivation, lacrimation, pupil size, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, exophthalmos, feces (appearance/consistency), other findings

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period. Afterwards, the body weight of the male and female parental animals was determined once a week

FOOD CONSUMPTION:
- Food consumption was determined once a week for the male and female parental animals.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken from the retrobulbar venous plexus towards the end of the administration period
- Anaesthetic used for blood collection: Yes, isoflurane as anaesthesia
- Animals fasted: Yes
- How many animals: first five parental males and females with litter (in order of delivery) per group.
- Parameters checked: Leukocyte, erythrocytes, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears (only evaluated blood smears will be archived), prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was be taken from the retrobulbar venous plexus towards the end of the administration period
- Animals fasted: Yes
- How many animals: first five parental males and females with litter (in order of delivery) per group.
- Parameters checked:
• Enzymes (systematic name and system number): Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), serum-γ-glutamyl transferase (GGT)
• Blood Chemistry Parameter: Sodium (NA), potassium, 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), bile acids

HORMONES
- Serum samples were frozen at -80°C for storage. Measurement of T3, T4 and TSH was carried out only if there was an indication for an effect on pituitary-thyroid axis. The determination is triggered based upon alterations of thyroid histopathology.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: For the males the Functional observational battery (FOB) was carried out at the end of the administration period, for the females at the end of the premating period.
- Dose groups that were examined: 5 surviving males and females in all dose groups
- Battery of functions tested: passive observations without disturbing the animals (home cage observations), followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. Motor activity was measured on the same day as FOB.
The observations were performed at random.
In detail, the following parameters were examined:
- Home cage observations: posture, tremors, convulsions, abnormal movements, gait abnormalities, other findings
- Open field observations: behavior when removed from cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypies, gait abnormalities, activity/arousal level, feces (number of fecal pellets/appearance/consistency excreted within two minutes, urine (color/amount) excreted within two minutes, rearing within two minutes
- Sensory motor tests/reflex tests: approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“auditory 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 measurement (MA): Measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. Animals received no food and water during the measurements.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology.
- Weight parameters (determined on all animals): anesthetized animals, testes, epididymides,
- Weight parameters (determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands, brain, heart, liver, kidneys, lung, thymus, spleen. Due to technical reasons, the weight measurement of adrenal glands and thymus of the first five female in all test groups failed and only single values were recorded. To reach the number of at least 5 measured organs in each test group, the adrenal glands and thymus of all remaining pregnant female animals per each test group were weighed.

- Organ / Tissue fixation
The following organs or tissues were fixed in 4% buffered formaldehyde solution or modified Davidson's solution: all gross lesions, adrenal glands, aorta, bone marrow, brain, cecum, cervix, coagulation glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbitallacrimal glands, epididymides (modified Davidson's solution), femur with knee joint, heart, ileum, jejunum (with Payer's patches), kidneys, larynx, liver, lungs, lymph nodes (tracheobronchial, mediastinal and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson's solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), target organs, testes (modified Davidson's solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

HISTOPATHOLOGY: Yes
(1) Hematoxylin-eosin (H&E) staining for all affected animals per group (all treatment groups) was performed for all gross lesions
(2) Hematoxylin-eosin (H&E) staining for all animals per test group (control and high dose group) was performed with uterus, vagina, cervix, ovaries, oviducts, testes, epididymides, prostate gland, coagulating glands, seminal vesicles, trachea (one transverse section and one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi), lungs (5 lobes), lymph nodes (tracheobronchial, mediastinal)
(3) Hematoxylin-eosin (H&E) staining for 5 animals per sex per test group, and for females with litters only, and same animals as used for clinical pathological examinations (control and high dose group) was performed with liver, kidney, heart, spleen, adrenal glands, thyroid glands, thymus, brain, sciatic nerve, spinal cord (cervical, thoracic and lumbar cords), duodenum, cecum, colon, rectum, ileum, jejunum, urinary bladder, bone marrow (femur), Peyer's patches, Lymph nodes (mesenteric), Stomach (forestomach and glandular stomach)
(4) Hematoxylin-eosin (H&E) staining for all animals per test group (control and high dose group) as well as paraplast embedding for all animals per dose group (low and mid dose group) was performed with larynx (3 levels, one level included the base of the epiglottis) and nasal cavity (4 levels, one level included nasopharyngeal duct; the 4 levels allow adequate examination of the squamous, transitional, respiratory and olfactory epithelium, and the draining lymphatic tissue (NALT).
(5) Due to pathological findings in the nasal cavity (level I and II) of male and female animals at high concentration (test group 3), nasal cavity (level I and II) of male and female animals of test groups 1 and 2 was examined by light microscopy.

Statistics:

Food consumption, body weight and body weight change: DUNNETT-test (two-sided);
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKALWALLIS test (two-sided) and WILCOXON-test (two-sided);
Clinical pathology parameters: KRUSKALWALLIS test and WILCOXON-test;
Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS and WILCOXON-test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups. During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal. During the post-mating day the male animals showed no clinical signs and findings different from normal. One female animal showed vaginal discharge. During the post-mating period one female animal of the control group showed vaginal discharge. During the gestation period six female animals of the control group, six female animals of the low concentration (15 mg/m³), six female animals of the mid concentration (50 mg/m³) and four female animals of the high concentration (150 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related. In addition, during gestation period, one female animal of the high concentration group showed salivation and smeared fur. This animal had implants but did not deliver pups. During the lactation period one animal of test group 2 showed reduced after-birth care. As this was not observed in high concentration group, this finding was considered to be incidental. During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal. One sperm positive high concentration female did not deliver F1 pups.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.

BODY WEIGHT AND WEIGHT GAIN
Premating period: The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.
Mating period: The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 were slightly lower than those of other groups.
Gestation: The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 20, though statistically not significant.
Lactation: The mean body weights of F0 female animals were not statistically significantly different from the control group 0 during lactation period.
After PND 4: The mean body weights of F0 female animals after PND 4 were not statistically different to the controls.
Body weight change during the exposure period: During pre-mating period, the mean body weight changes of male and female animals were not statistically different when compared with the control. The body weight change of the F0 female animals of the high concentration group (150 mg/m³) was statistically significantly lower than the controls during gestation period (-24.1 g, p < 0.05). This effect was probably because one female of this group was not pregnant and one female of this group had post-implantation loss.

FOOD CONSUMPTION
In high concentration (150 mg/m³) food consumption during gestation was significantly decreased in female animals between gestation days 0 to 20 (-7.8 %, p<0.05), which is mainly influenced by decreases in food consumption between GD 14-20 (-11.8 %, p<0.01). This finding was considered to be substance-related. No other findings were observed for male and female animals in test group 1 and 2 (15 and 50 mg/m³), as well as in male animals of test group 3 during the whole study period.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed. In females of test group 1 (15 mg/m³) absolute neutrophil counts were lower compared to controls. However, this alteration was not dose-dependent and therefore, it was regarded as incidental and not treatment-related.

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

NEUROBEHAVIOUR
Functional observational battery (FOB): On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances. Observations were performed on study day 12 in females and on study day 26 in males.
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
- Quantitative Parameters: In general no test substance-related impaired parameters were observed in male and female animals of all test groups. In F0 females of test group 1, slightly though significantly higher grip strength of forelimbs was determined. This parameter was not increased in other groups, it was considered to be incidental due to the lack of concentration-response relationship.
Motor activity measurement (MA):
- Overall motor activity (summation of all intervals): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.
- Single intervals: No abnormalities were detected.

ORGAN WEIGHTS
Absolute organ weights: All mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights: When compared to control group 0 (set to 100%), the mean relative weights of the liver was significantly increased (112%) in male animals of test group 3 (150 mg/m³). The increase in relative liver weight of male animals was regarded to be treatment related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
All 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.

HISTOPATHOLOGY
Treatment-related findings were observed in in the nasal cavity (levels I and II) in males and females (for incidences and grading see table below). Males and females of the test group 3 (150 mg/m³) revealed substance-related findings in the level I and II of the nasal cavity. In the very basal and frontal part of the nasal cavity at the transition from squamous to respiratory epithelium 4 animals showed a complete focal loss of the epithelium (ulcer). In nine animals of each sex there was a flattening of the epithelium (squamous and/or respiratory), the remaining cells were irregularly shaped and slightly more basophilic (regeneration). Whenever this term was used there was in addition to these findings a minimal to slight inflammation, edema and a slight reduction in nasal bone in this area (osteolysis) present. The findings in level II were attenuated when compared to level I.
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.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

50 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathology (pathological findings in the nasal cavity (Ievel I and II) of male and female animals at high concentration)

2

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

150 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Relative liver weights in male parental animals:

	Male animals
Test group (mg/m³)	1 (15)	2 (50)	3 (150)
Liver	99%	103%	112%*

* p <= 0.05

Histopathology findings:

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (15)	2 (50)	3 (150)	0 (0)	1 (15)	2 (50)	3 (150)
No. of animals	10	10	10	10	10	10	10	10
Nasal cavity, level I
Erosion,/ulcer	0	0	0	2	0	0	0	2
Degeneration/regeneration squamous/respiratory epithelium	0	0	0	8	0	0	0	9
Grade 1				2				2
Grade 2				1				1
Grade 3				5				5
Grade 4								1
Nasal cavity, level II
Degeneration/regeneration squamous/respiratory epithelium	0	0	0	6	0	0	0	5
Grade 1				2				1
Grade 2				1				3
Grade 3				3				1

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats the following no observed adverse effect concentration (NOAEC) of pyrrolidine were determined:
The NOAEC for general, local toxicity at the respiratory tract was 50 mg/m³ for the F0 females and males based on histological findings in nasal cavity.
The NOAEC for general, systemic toxicity was 150 mg/m³ for the F0 females and males.
The NOAEC for reproductive performance and fertility was 150 mg/m³ for the F0 parental rats.
The NOAEC for developmental toxicity in the F1 offspring was 150 mg/m³.

Executive summary:

The study was performed according to OECD guideline 422 in compliance with GLP.

To evaluate the toxicity profile of Pyrrolidine (PYR) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to dynamic atmosphere of PYR for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 9 consecutive days.

The target concentrations were 15, 50 and 150 mg/m³. A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the study and were exposed nose-only to fresh air on two days before start of the exposure period.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20,on the day after parturitionpostnatal day [PND] 1) and on PND 4. After the pups are sacrificed the females that were exposed for 9 consecutive days were weighed on study day 47, 49 and 54.

A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 12. The male animals were performed at the end of the exposure period on study day 26.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO₂, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and grouptowards the end of the exposure period.

A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines.

All F0 parental animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

During the exposure period, the target concentrations were met well and were maintained as constant and stable as could be provided with the presented generation techniques in the concentration range tested.

Test concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)
		Mean	Standard deviation
1	15	11.7	1.4
2	50	48.4	2.7
3	150	156.8	13.5

The following test substance-related adverse effects were observed:

Test group 3 (150 mg/m³):

- Ulceration of the squamous and/or respiratory epithelium in the nasal cavity (level I) in 2 male and 2 female animals

- Minimal to severe degeneration/regeneration of squamous and/or respiratory epithelium in the nasal cavity (level I) in 9 male and 9 female animals

- Minimal to moderate degeneration/regeneration of squamous and/or respiratory epithelium in the nasal cavity (level II) in 6 male and 5 female animals

 

Test group 2 (50 mg/m³):

- No adverse test substance-related histopathologic and macroscopic findings or weight changes

 

Test group 1 (15 mg/m³):

- No adverse test substance-related histopathologic and macroscopic findings or weight changes

Conclusion:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats the following no observed adverse effect concentration (NOAEC) of pyrrolidine were determined:

The NOAEC for general, local toxicity at the respiratory tract was 50 mg/m³ for the F0 females and males based on histological findings in nasal cavity.

The NOAEC for general, systemic toxicity was 150 mg/m³ for the F0 females and males.

The NOAEC for reproductive performance and fertility was 150 mg/m³ for the F0 parental rats.

The NOAEC for developmental toxicity in the F1 offspring was 150 mg/m³.