1-ethylpyrrolidin-2-one

Endpoint summary

Description of key information

Oral administration:

In a subchronic oral study, administration of N-Ethyl-2 -pyrrolidone caused substance-related adverse effects at 1000 and 300 mg/kg bw/day in both sexes of rats and 100 mg/kg bw/day or higher in males (BASF, 2006).

Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Afunctional observational battery (FOB) and measurement of motor activity was carried out. Vaginal smears for estrus cycle determination of all female animals were prepared and evaluated each day during the last 4 weeks of the study. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. Ophthalmological examinations were performed before and towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations. Additionally, sperm parameters were determined immediately after necropsy and organ weight determination. 

No effects were observed in females at 100 mg/kg bw/day. Food consumption, body weight, grip strength was reduced at 1000 and 300 mg/kg bw/day. Target organs were the liver in both sexes (centrilobular hypertrophy of hepatocyctes liver weight increase) and the kidney in males (alpha 2u globuline which was confirmed immunohistochemically). Histomorphologically, a centrilobular hypertrophy of hepatocytes could be correlated to the increased liver weights in both sexes. In males, a centrilobular hypertrophy was noted for the mid (300 mg/kg bw/day) and low (100 mg/kg bw/day) dose groups, although dose-dependently less pronounced. In females, no centrilobular hypertrophy was noted in the mid and low dose groups. The centrilobular hypertrophy of hepatocytes is indicative for an enzyme induction of the cytochrome P450 system and is considered to be a detoxifying, adaptive effect (no signs for cytotoxicity recorded). Kidney effects in male rats at 100 mg/kg bw/day or higher are considered not relevant to humans as they are due to a rat specific mechanism.

Sperm analysis revealed an increased number of sperms with abnormal heads in males at 1000 mg/kg bw/day. The most common sperm head anomalies were abnormal hooks and amorphous heads. These results are indicative of disrupted sperm maturation, when the test compound is administered at high dosages. This effect was not associated with any histopathological changes in the testis and did not affect the number of homogenization resistant spermatids, epididymal sperm count and sperm motility. The reproductive organs in females were not affected. Beyond these findings, there were some effects on clinical chemistry and hematology parameters observed at 1000 mg/kg/day.

Therefore, the overall no observed adverse effect level (NOAEL) under the conditions of this study was 100 mg/kg bw/day for females and the lowest observed adverse effect level (LOAEL 300 mg/kg bw/day) and below 100 mg/kg bw/day for males (critical effect: alpha 2u nephropathy, which is considered not to be relevant to humans).

Inhalative administration:

In a 28 -day study according to OECD 412, 10 male and 5 female rats per dose group received doses of 0, 80, 200, and 400 mg/m3 as a liquid aerosol with vapor fraction via nose/head-only inhalation exposure for 4 weeks on 5 consecutive days per week and 6 h per day (BASF, 2011). A concurrent control group was exposed to air. On each exposure day a clinical examination was performed before, during and after exposure. Body weights and food consumption of the animals were determined weekly. 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). Selected tissues were processed histopathologically and were evaluated by light microscopy. Histological examinations were performed in respiratory tract, liver and testes according to standardized methods.

During the exposure period the animals of the control group and low concentration (80 mg/m³) showed no clinical signs and findings different from normal. During the exposure period several animals of the intermediate and high concentration (200 mg/m³) showed abnormal clinical signs including salivation, red encrusted nose, nose discharge, lacrimation of eyes. These signs were only observed directly after the daily exposure period and fully reversible before the start of next exposure. On the post-exposure observation day all animals were free of clinical signs. No treatment-related changes among clinical chemistry parameters, body weight, food consumption, or hematology were measured. The degeneration/ regeneration of the olfactory epithelium in animals of the mid and high dose group was regarded to be treatment-related. Furthermore most animals of the high dose group showed minimal focal epithelial alteration of cuboidal cells in the larynx at the base of epiglottis (level I). In females, a treatment-related effect cannot be ruled out. All other findings were considered to be incidental or spontaneous in origin and without any relation to treatment. Thus, under the test conditions of this 28 day-study, the NOAEC for local effect in nasal cavity was 80 mg/m3 while the NOAEC for systemic effects was 400 mg/m3.

In an inhalation study over 90 days (65 exposures) according to OECD TG 413, 10 male and 10 female rats per dose group were exposed to vapor for 6 hours per day on 5 consecutive days at concentrations of 0, 30, 60, and 200 mg/m3 of the test item via nose/head-only inhalation exposure (BASF, 2013). 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 was 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.

The inhalation exposure did not lead to any exposure-related clinical signs of toxicity nor were there any effect in clinical chemistry, hematology, urine and sperm parameters. Regarding pathology the target organ was the nasal cavity. Histological examination revealed signs of degeneration and regeneration of the olfactory epithelium at the highest tested concentration of 200 mg/m3. 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 (see table 5). Comparable findings in testes and in the epididymides have been observed frequently in head-nose exposed control animals in past inhalation studies in the same laboratory. Therefore, the occurrence of tubular degeneration as well as of the resulting debris and oligospermia in the epididymides in this inhalative subchronic study was considered to be incidental. All other pathological findings were considered to be incidental or spontaneous in origin and without relation to treatment. Therefore, under the current test conditions, the NOAEC for local effect in nasal cavity was at the mid concentration 60 mg/m3 and for systemic toxicity at the high concentration 200 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch-Id. 29151288P0
Purity 99.8%
Expiry date: Feb 22, 2007

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

35 +/- 1 day males/females were used. The rats were identified clearly by ear tattoo. The rats were housed singly in type DK III stainless steel wire mesh cages. Underneath the cages, waste trays were fixed containing absorbent material. Motor activity measurements were conducted in Polycarbonate cages with wire covers and small amounts of absorbent material. The animals were housed in a fully air-conditioned room. Central airconditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity. The day/night cycle was 12 hours light and 12 hours dark. Deviations from these ranges did not occur. The animal room was completely disinfected prior to the study. The floor and the walls were cleaned once a week. The food used was ground Kliba maintenance diet mouse/rat and drinking water (from water bottles) were available ad libitum.

Route of administration:

oral: feed

Details on route of administration:

For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dietary concentrations of N-Ethyl-2-pyrrolidon for each group and sex were adjusted weekly, based on body weight and food consumption measurements from the previous week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out. The stability of the test substance in the diet over a period of 49 days was proven prior to the study. Homogeneity and concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day (groups 0, 1, 2, 3)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and measurement of motor activity was carried out after 85 resp. 89 days of treatment in males and 86 resp. 90 days of treatment in females. Vaginal smears for estrus cycle determination of all female animals were prepared and evaluated each day during the last 4 weeks of the study. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. Ophthalmological examinations were performed before and towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations. Additionally, sperm parameters were determined immediately after necropsy and organ weight determination.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
FOOD EFFICIENCY: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes
URINALYSIS: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes

Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy
The test animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Weight parameters
Weight assessment was carried out on all animals sacrificed at scheduled dates. The weights of the following organs were determined:
Anesthetised animals, Liver, Kidneys, Adrenals, Testes, Epididymides, Ovaries, Uterus, Spleen, Brain, Heart, Thymus, Thyroids,,

Organ / Tissue preservation list
The left testis, left epididymis and both ovaries of all animals sacrificed at scheduled dates were fixed in Bouin’s solution. The right testis and right epididymis were used for sperm and spermatide examination. The following organs or tissues were preserved in neutral buffered 4% formaldehyde:
All gross lesions, Salivary glands (mandibular and sublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum and ileum, Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenals, Thyroids, Parathyroids, Trachea, Lungs, Pharynx, Larynx, Nose (nasal cavity), Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and mandibular lymph nodes, Spleen, Thymus, Kidneys, Urinary bladder, Oviducts, uterus and vagina, Prostate and seminal vesicle, Female mammary gland, Skin, Skeletal muscle, Sternum with marrow, Femur with knee joint, Extraorbital lacrimal glands

The following organ samples were processed histotechnically
All gross lesions, Salivary glands (mandibular andsublingual glands), Esophagus, Stomach (forestomach and glandular stomach), Duodenum and ileum, Jejunum (with Peyer’s patches), Cecum, colon and rectum, Liver, Pancreas, Brain, Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic and lumbar cords), Eyes, Adrenals (cortex and medulla), Thyroids, Parathyroids, Trachea, Lungs, Pharynx, Larynx, Nasal cavity (level III), Aorta, Heart, Bone marrow (femur), Lymph nodes (mesenteric and mandibular lymph nodes), Spleen, Thymus

Other examinations:

A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Ophthalmoscopy
Prior to the start of the administration period the eyes of all animals, and on day 91 the eyes of the control and high dose animals were examined for any changes using an ophthalmoscope after administration of a mydriaticum.

Vaginal smears for cycle determination were prepared in the morning and evaluated.

Sperm parameters
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
The following parameters were determined:
• Sperm motility (%, microscopic evaluation)
• Sperm morphology (%, vital staining with eosin, microscopic evaluation)
• Sperm head count (sperm heads x 10E6/ g cauda epididymis) (microscopic evaluaion with MAKLER chamber after homogenization)
• Sperm head count (sperm heads x 10E6 g testis, microscopic evaluaion with MAKLER chamber after homogenization)

Statistics:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

“Anogenital region, stained, slight” was observed in 7/10 males and 4/10 females of group 3 and 3/10 males of group 2 from day 63 till the end of the study; “anogenital region, stained, moderate” was observed in 1/10 males of group 3 from day 63 till day 92. “Urine discolored, orange” was observed in 10/10 males and females of groups 3 and 2 and 10/10 females of group 1 from day 2 till the end of the study. “Urine, discolored, dark yellow” was observed in 10/10 males of group 1 from day 7 till day 92. These findings were assessed as substance-related but caused by the physical property of the test article (yellow) and do not represent real toxicity. “High stepping gait, slight” was observed in 1 female of group 3 as well as in 1 female of dose group 1, from day 70 till day 83 and 76, respectively. This finding was assessed as not related to the test article due to the absence of a dose-response relationship. Findings like “alopecia, body, flank, left” in 1 female of group 3 from day 84 till day 93 and “aggressiveness, moderate” in 1 male of group 3 from day 35 till day 55 were assessed as spontaneous in nature and therefore not substance-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight in both sexes of group 3 was statistically significantly decreased from day 7 till day 91, up to -17.6% in males and -18.3% in females on day 91. Body weight was also decreased in both sexes of group 2, statistically significantly in males on days 28 and 91 and in females on day 21 and from day 49 till day 91, up to -8.7% in males on day 91 and in females on day 70. Body weight change in both sexes of group 3 and 2 was statistically significantly decreased during the whole study period in group 3, resp. the main part of the study in group 2, up to -39.1% in males and -52.6% in females of group 3 and -18.8% in males and -23.5% in females of group 2 on day 7 of the study, each. Due to a clear dose-response relationship, these findings were clearly substance-related. The increase in body weight change of both sexes in group 1 animals on day 7 of the study with +8.9% in males and +6.9% in females was assessed as being incidental, biologically not relevant as well as not substance-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in both sexes of group 3 was statistically significantly decreased during the whole study period, up to -21.1% in males and -23.3% in females on day 7. Food consumption in both sexes of group 2 was decreased during the study, statistically significantly on days 7, 21, 28, 63, 70 and 91 in males, up to -13.2% on day 7 and on days 7, not statistically significantly, and 70 in females, up to -7.5% on day 7, also. Due to a clear dose-response relationship, the influence on food consumption was assessed to be substance-related.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency was decreased on days 7 and 14 in males and on days 7 and 70 in females of group 3. This finding was assessed as being incidental and biologically not relevant.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

All findings were incidental in nature, due to the occurrence in single animals, only, and/or the
lack of a dose-response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period significantly increased platelet counts were found in the peripheral blood of males at 1000 mg/kg bw and in all treatment groups of females. Moreover, clotting analysis revealed significantly shortened prothrombin times in rats of either sex at 1000 mg/kg. No treatment-related changes were noted in the other hematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Marginally, but significantly decreased alanine aminotransferase and aspartate aminotransferase activities were found in the serum of male and female rats given 1000 mg/kg bw and reduced alanine aminotransferase activities were also seen in female animals at 100 mg/kg and 300 mg/kg. No treatment-related changes were observed in the other serum enzymes examined. Blood chemistry investigations revealed significantly increased inorganic phosphate, calcium, triglyceride and cholesterol concentrations and decreased creatinine levels in the serum of both sexes at 1000 mg/kg. The increase in triglycerides in males and the decrease in creatinine in females, however, were not significantly different to the respective control, but were seen only as a trend toward higher concentrations. In addition, decreased chloride, glucose and total bilirubin values were noted in males at 1000 mg/kg and total bilirubin was also reduced in males at 300 mg/kg. In the serum of females given 300 mg/kg bw and 1000 mg/kg bw significantly lower total protein and albumin concentrations were noted. No treatment-related changes were found in the other blood chemistry parameters.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males given 100 mg/kg bw and animals of either sex receiving 300 mg/kg bw and 1000 mg/kg bw of the test compound excreted urine, which was discolored from dark yellow to orange. Furthermore, microscopic examination of the urine sediments revealed increased numbers of transitional epithelial cells, granular casts and epithelial cell casts in all treated males at the end of the administration period. Most of the transitional epithelial cells found in the sediments were degenerated. No test article-related changes were observed in the other urine parameters of both sexes.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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.
Home cage observations: No substance related findings were observed.

Open field observations:
“Discolored fur” was observed in 7 males and 4 females of group 3 and 2 males of group 2, caused by discolored urine. “Discoloration of urine, orange” was observed in 4 males and females of group 3, 2 males and 8 females of group 2 and 4 females of group 1; “discoloration of urine, dark yellow” was observed in 8 males of group 1. These findings were assessed as substance-related but caused by the physical property of the test article (yellow) and do not represent real toxicity. “Alopecia” was observed in 1 female of group 3. This was assessed as spontaneous in nature and therefore not substance-related.

Sensorimotor tests/reflexes:
“Very frequent vocalizations when touched” was observed in 2 males and 1 female of group 3, 2 males and females of group 2, 3 males and 2 females of group 1 and 1 male and females of group 0. This was assessed as spontaneous in nature and therefore not substance-related. The value of the “grip strength forelimbs” was statistically significantly decreased in male animals of group 3 (-40.1%) and 2 (-33.0%). These findings were assessed as being related to the test article caused by systemic toxicity and reduced body weight, respectively.

Regarding the overall motor activity a statistically significantly decreased deviation was seen in females of group 3. This finding was assessed as being related to the test article caused by systemic toxicity and reduced body weight, respectively. Comparing the single intervals with the control groups, no substance-related findings were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-dependent decrease of the terminal body weights in dose groups 2 and 3. The significant increase of liver weights in dose group 3 is considered to be a treatmentrelated effect. The significant decrease of brain, heart, spleen, and thymus weights in dose groups 2 and 3 is regarded to be secondary to the decreased terminal body weights.
There was a dose-dependent decrease of the terminal body weights in dose groups 2 and 3. The significant decrease of adrenal glands, brain, heart, spleen, and thymus weights in dose group 3 is regarded to be secondary to the decreased terminal body weights. The significant weight increase of the thymus weights in dose group 1 is considered to be incidental and rather represents the wide, physiological biological range of thymus weights of animals of this age than a treatment-related effect.

The significant weight increase of the adrenal glands, brain, epididymides, and testes is considered to be secondary to the significant decrease of terminal body weights in the dose groups 2 and 3. In parts this is also true for the significant increase of the kidney and liver weights but histological examination also detected a direct compound-related effect that may be additionally responsible for the significant weight increase in dose groups 1 to 3.

All significant weight changes in dose groups 2 and 3 were regarded to be secondary to the significant weight decrease of the terminal body weights and do not represent a direct compound-related effect. The liver weight increase of dose group 3 animals is considered to be partly due to direct effect as treatment-related histological findings are present. The significant increase of the thymus weights in dose groups 1 and 2 represent rather the wide biological range of thymus weights of animals in this age than a treatment-related effect.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All gross lesions are considered to be spontaneous lesions in origin and are not related to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Regarding pathology, the liver and the kidneys of male test animals are found to be target organs. In the liver, a centrilobular hypertrophy of hepatocytes is found in all dose groups. In males, centrilobular hypertrophy of hepatocytes is found in all dose groups. The gradings
indicate a dose-dependent increase after treatment. In females, only the 1000 mg/kg bw/day dose group animals show centrilobular hypertrophy of hepatocytes. In the kidneys of male test animals, morphological indications for the development of an alpha 2u globulin nephropathy are found in all dose groups.
For each of the dose groups, one male kidney was immunhistochemically stained for the proof of alpha 2u globulin. In all cases, the hyaline droplets that showed positive staining in the Mallory Heidenhain stain, were shown to represent specifically alpha 2u globulin. All groups show an increased severity of basophilic tubules and accumulation of hyaline droplets in the proximal tubules, when compared to the control. All other findings noted are considered to be spontaneous or incidental in origin and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm examination showed an increased number of sperms with abnormal heads in males receiving 1000 mg/kg bw of the test compound (2.0, 2.2, 2.8 and 11.4% in controls, low, mid and high dose, respectively; males with >4% abnormal sperm: 0, 1, 2, 8 in controls, low, mid and high dose, respectively).. The most common sperm head anomalies were abnormal hooks and amorphous heads. Test substance administration did not affect number of homogenization resistant spermatids, epididymal sperm count and sperm motility.

Estrous cycle determination:
No substance-related findings were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

1000 mg/kg bw/day

- Food consumption in both sexes was statistically significantly decreased during the whole study period, up to -21.1% in males and -23.3% in females

- Body weight in both sexes was statistically significantly decreased from day 7 till day 91, up to -17.6% in males and -18.3% in females

- Body weight change in both sexes was statistically significantly decreased during the whole study period, up to -39.1% in males and -52.6% in females

- Grip strength forelimbs in males was statistically significantly decreased of -40.1% during functional observational battery

- Motor activity (summation of intervals) in females was statistically significantly decreased

- Platelets, inorganic phosphate, calcium, cholesterol and triglycerides were increased in both sexes

- Number of sperms with abnormal heads was increased in males

- Prothrombin time was shortened and creatinine was decreased in both sexes

- Chloride, glucose and total bilirubin were decreased in males

- Total protein and albumin were decreased in females

- Terminal body weight decrease in both sexes

- Liver weight increase in both sexes (+53% in males, + 29% in females)

- Centrilobular hypertrophy of hepatocytes in both sexes

- rel. kidney weight increase in males and females (+32% and + 22% respectively)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

300 mg/kg bw/day

- Food consumption in both sexes was decreased during the whole study period, statistically significantly on several days

- Body weight in both sexes was decreased from day 7 till day 91, statistically significantly in males on days 28 and 91 and in females on day 21 and from day 49 till day 91, up to -8.7%, each

- Body weight change in both sexes was statistically significantly decreased during the main part of the study, up to -18.8% in males and -23.5% in females

- Grip strength forelimbs in males was statistically significantly decreased of -33.0% during functional observational battery

- Total bilirubin in males and total protein and albumin in females was decreased

- Terminal body weight decrease in both sexes

- rel. liver weight increase in both sexes (+13% in males, + 9% in females)

- Centrilobular hypertrophy of hepatocytes in males

- rel. kidney weight increase in males (+14%)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

100 mg/kg bw/day

- rel. Liver weight increase in males (+7%)

- Centrilobular hypertrophy of hepatocytes in males

- rel. kidney weight increase in males (+9%)

- Increase of basophilic tubules and accumulation of hyaline droplets (alpha 2u globulin) in males

- No effects in females at 100 mg/kw/bw

Additional comments to be considered

The kidney effects observed in males at 100 mg/kg bw/day onwards are not relevant to humans (alpha-2 µ-globulin).

The centrilobular hypertrophy of hepatocytes is indicative for an enzyme induction of the cytochrome P450 system and is considered to be a detoxifying, adaptive effect (no signs for cytotoxicity recorded). In males, a centrilobular hypertrophy was also noted for the mid (300 mg/kg bw/day) and low (100 mg/kg bw/day) dose groups, although dose-dependently less pronounced. In females, no centrilobular hypertrophy was noted in the mid and low dose groups.

Sperm analysis revealed an increased number of sperms with abnormal heads in males at 1000 mg/kg/day abnormal sperm: (2.0, 2.2 , 2.8 and 11.4% in controls, low, mid and high dose, respectively; males with >4% abnormal sperm: 0, 1, 2, 8 for

in controls, low, mid and high dose, respectively). The summary of sperm evaluations can be found in the attached table. The most common sperm head anomalies were abnormal hooks and amorphous heads. These results are indicative of disrupted sperm maturation, when the test compound is administered at extremely high dosages. It should be noted that this effect was not associated with any weight changes or (histo)pathological changes in the testis. Beyond this, administration did not affect the number of homogenization resistant spermatids, epididymal sperm count and sperm motility (see table). The functional relevance of this effect is unclear as fertility was not examined in this study.

Conclusions:

The target organs were liver and kidneys.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality. (Klimisch score = 1)

System:

other:

Organ:

other: body weight

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/2013- 12/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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

Specific details on test material used for the study:

Batch-no. 52195875L0
Purity: 99.8% corr. area %

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.

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)

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

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

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.

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

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

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/m^3)	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/m^3)	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/m^3)	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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality. (Klimisch score = 1)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/2013- 12/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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

Specific details on test material used for the study:

Batch-no. 52195875L0
Purity: 99.8% corr. area %

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.

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)

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

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

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.

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

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

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/m^3)	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/m^3)	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/m^3)	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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

60 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality. (Klimisch score = 1)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Supportive studies:

Oral administration

In addition, N-ethyl-2 -pyrrolidone (NEP) was evaluated in a 4-week repeated dose study in rats. NEP diluted in distilled water was orally administered by gavage to male and female Sprague-Dawley rats at doses of 0 (vehicle control), 5, 50, and 250 mg/kg/day for 28 consecutive days (Saillenfait, 2017). Transient decreases in the body weight and in the body weight gain of the males was observed during the first days of treatment at the 50 and 250 mg/kg/day doses. There was a marked increase in urine volume at the beginning of treatment in males and female rats at doses of 50 and 250 mg/kg/day. No biologically significant differences were observed in hematological and clinical chemistry values in males and females at necropsy. Histological examination revealed an increase in hyaline droplets in the renal tubules of the kidneys and hepatocellular centrilobular hypertrophy in the liver of males at 250 mg/kg/day. Cytochrome P450 concentration in liver microsomes was slightly increased at 250 mg/kg/day in males. The results of this study demonstrate that NEP has mild to no effects at doses up to 250 mg/kg/day when administered orally to rats for 28 days with males being more susceptible than females. According to the authors, the NOAEL is 250 mg/kg bw/d for males and females based on abof permanent adverse effects at 250 mtg/kg bw/day and the NOEL in males is 5 mg/kg bw/day based on body weight effects at 50 mg/kg bw/day, whereas the NOEL for females is 50 mg/kg bw/day based on increased urine volume at 250 mg/kg bw/day).

Justification for classification or non-classification

According to the EU CLP Regulation (EC) No. 1272/2008) substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure shall be classified for specific organ toxicity after repeated exposure. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. The adverse effects include consistent and identifiable toxic effects in humans, or, in animals toxicologically significant changes which have affected the function or morphology of a tissue/organ, or have produced serious changes to the biochemistry or hematology of the organism and these changes are relevant for human health. 

Substances are classified in category 2 for specific target organ toxicity (repeated exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentration. Guidance dose/concentration values according to CLP (table 3.9.3) to assist in Category 2 classification are > 0.2 <= 1.0 mg/l/6h/day for inhalation (vapour; rat), > 0.02 <= 0.2 (dust, mist, fume) and > 10 <= 100 mg/kg bw/day for oral exposure (rat) for a 90 -day repeated dose study. In case of a subacute study, the guidance values are increased by a factor of three.

NEP caused localized findings in the nasal cavity in repeated dose inhalation studies with rats. Minimal to moderate signs of local irritation were observed in the nasal cavity at 200 mg/m3 (vapour) in the 90d study and more pronounced effects at a high concentration of 400 mg/m3 (aerosol/vapour mix) in the 28d study. Additionally, in the subacute inhalation study, most animals of the high dose group at 400 mg/m3 (aerosol/vapor mix) showed minimal focal epithelial alteration of cuboidal cells in the larynx at the base of epiglottis (level I) for which a treatment related effect cannot be completely ruled out.  No other signs of specific organ toxicity were observed in the available inhalation studies. NEP is known to be a locally irritating substance to mucous membranes (classified accord. to CLP with Eye damage Cat 1). Thus, considering the mild effects of irritant concentrations of NEP vapours or aerosols in the nasal tract, which will most likely be reversible after a sufficient period of time, and the corresponding effect levels, a classification for specific target organ toxicity after repeated exposure is not warranted according to the criteria of the CLP.

Regarding oral exposure, the observations in the 90 -day toxicity study (alpha 2u nephropathy in male rats being not relevant for humans and the adaptive liver hypertrophy in male and female rats without signs of cytotoxicity) are not considered relevant for a classification regarding specific target organ toxicity arising from a repeated exposure.