Pyrrolidine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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)

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 (if applicable):

nose/head only

Vehicle:

air

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: overnight until there was evidence of copulation or the maximum period of 14 days has elapsed. Throughout the mating period, each female was mated with a predetermined male.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)

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 was 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:

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)

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

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

Parental animals: Observations and examinations:

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 cm) 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.
The following exceptions are notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of sperm (GO 0) and on GO 7, 14 and 20.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4.
• Females without litter were weighed once a week.
• After weaning (PND 4), females were weighed once a week until sacrifice.
• After the pups were sacrificed the females were exposed for 9 consecutive days. The F0 females were weighed once on the first exposure of this exposure period, once on the third and once on the eight exposure. The last body weight determination was on the day of the gross necropsy.

FOOD CONSUMPTION:
- Food consumption was generally determined once a week for the male and female parental animals.
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-4.
• Food consumption of the females during the 9 exposure days after necropsy of the pups was determined over one week from study day 47 to day 54.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER:
- The parturition and lactation behavior of the dams was evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

- Functional observational battery (FOB): For the males the FOB was carried out at the end of the administration period, for the females at the end of the premating period.

Litter observations:

CLINICAL EXAMINATIONS OF PUPS:
- Pup status and litter size after birth: Status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were examined for gross-morphological changes.
- Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays and public holidays.
Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Clinical signs: All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weights: The pups were weighed on the day after birth (PND 1) and on PND 4. The body weight determined on PND 1 was also used to determine runts. Those pups whose body weight was - 25% below the mean body weight of the control group (separately according to male and female pups) was defined as runts.
- Postmortem examinations of pups: On PND 4, the pups were sacrificed under isoflurane anesthesia with C02. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. Pups that die or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death. All pups without any notable findings were discarded after their macroscopic evaluation.

Postmortem examinations (parental animals):

SACRIFICE
All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava.
- Male animals: All surviving animals were sacrificed after the end of the administration period (at least 28 days)
- Maternal animals: The parental females were allowed to deliver and rear their pups until PND 4. After PND 4 of the female, which delivers last, all parental females were exposed to the test substance on 9 consecutive days. They were sacrificed on the day after and were examined.

GROSS NECROPSY
The animals were necropsied and assessed by gross pathology.
- The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCI) and further processed pathologically.

HISTOPATHOLOGY / ORGAN WEIGHTS
- 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).

Postmortem examinations (offspring):

On PND 4, all pups were sacrificed and examined

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: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.
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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male reproduction data: For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One high-concentration male (150 mg/m³) did not generate implants in the mated female. This male did not show any relevant histological findings impair fertility. Even though, the male fertility index ranged between 90% and 100% without showing any relation to exposure concentration. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
Female reproduction and delivery data: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.0 and 2.4 days without any relation to exposure concentrations. All sperm positive rats delivered pups or had implants in utero with the following exception: One high-concentration female did not become pregnant. The fertility index varied between 90% in test group 3, and 100% in test groups 1, 2 and in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant female did not have any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 21.9 and 22.0 days). The gestation index was 88.9 % in test group 3 and was 100 % in test groups 1 and 2 as well as in control. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (11.9 / 13.0 / 12.1 and 11.8 implants/dam in test groups 0-3 (0, 15, 50 and 150 mg/m³). There were no statistically significant differences in postimplantation loss between the groups (3.4% / 6.8% / 5.7% / 20.7%), and the mean number of F1 pups delivered per dam remained unaffected (11.5 / 12.1 / 11.4 and 11.1 pups/dam at 0, 15, 50 and 150 mg/m³). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (control, test group 1, and test group 3 ) and 99.1% (test group 2). One stillborn pup (0.9 %) was only in test group 2 animals. However, this is within the normal range of biological variation inherent in the strain of rats used for this study.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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. The female animal, which was not pregnant as well as its male mating partner did not show relevant gross lesions.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in in the nasal cavity (levels I and II) in males and females. 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.
The female animal, which was not pregnant as well as its male mating partner did not show relevant histopathological findings.

Effect levels (P0)

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:

reproductive toxicity

Effect level:

150 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Results: F1 generation

Details on results (F1)

PUP NUMBER AND STATUS AT DELIVERY
The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The low rate of stillborn pups in test group 2 was within the biological variation of this stain and was considered to be not related to the test substance.

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 86.5% (test group 3), 99.1 % (control and test group 1) and 100% (test group 2). The low viability index in test group 3 was derived by one female animal with only one live born, which was found dead on PND 0. The viability index for this litter was 0 %. This value decreased the mean viability of 8 litters in this group to 86.5 % with standard deviation of 35.1 %. With exception of this one litter, the viability of other litters was within biological variations. Therefore, this finding was considered not to be substance-related.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups. One female runt was seen in the test group 1 (15 mg/m³), and one female runt in test group 2 (50 mg/m³). As there was no relation to the exposure concentration, this finding was considered to be incidental and not substance-related.

GROSS PATHOLOGY (OFFSPRING)
No findings were observed at gross necropsy in any male pups of test groups 1 to 3. One male pup of control group showed large heart and discolored liver (pale), another one showed discolored testes. This finding is considered to be incidental, because they did not occur in animals exposed to the test substance. One female pup in test group 1 could not be assessed because it had been cannibalized. As no pup was cannibalized in high concentration groups, this finding is considered as incidental.

OTHER FINDINGS (OFFSPRING)
Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Effect levels (F1)

Dose descriptor:

NOAEC

Remarks:

developmental effects

Generation:

F1

Effect level:

150 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Sex ratio of live F1 pups:

PND 0	Test group 0 (clean air)	Test group 1 /15 mg/m³)	Test group 2 (50 mg/m³)	Test group 3 (150 mg/m³)
Live males [%]	56.5	46.3	54.0	52.8
Live females [%]	43.5	53.7	46.0	47.2
PND 4
Live males [%]	57.0	46.7	54.0	54.0
Live females [%]	43.0	53.3	46.0	46.0

Sex ratio data were calculated using the individual values concerning pup status

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 in5 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	11.1	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.

Generally, no toxicologically relevant reproductive or developmental difference was observed between animals exposed to measured concentrations of 11.7, 48.4 and 156.8 mg/m³ and controls. These concentrations of pyrrolindine did not adversely impact the reproduction of these rats, nor did treatment impact delivery and pup viability. Furthermore, none of the F1 generation pups showed any evidence of developmental toxicity in response to pyrrolidine.

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.