Registration Dossier

Administrative data

Description of key information

A LOAEL for subchronic oral toxicity of 130 mg/kg bw/day (BASF AG, 1991) was derived from a 90-day toxicity study in rats after oral (gavage) administration (OECD408).
A NOAEL for subacute oral toxicity of 50 mg/kg bw/day (Safepharm, 1989) was derived from a 28-day toxicity study in rats after oral (gavage) administration (OECD407).
A NOAEC for subchronic inhalation toxicity of 58 mg/m³ was derived from a 90 day toxicity study in rats exposed to the test item as vapor (OECD413).
A NOAEC systemic of 58 mg/m³ and NOAEC local of 1 mg/m³ for a subacute inhalation toxicity was derived from a 28-day inhalation study in rats exposed to the test item via vapor (OECD 412).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-Mar-03 to 1989-Apr-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study with GLP, acceptable with restriction (analytical purity of the test substance not reported).
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
analytical purity of the test substance not reported
GLP compliance:
yes
Remarks:
(Safepharm Laboratories Limited, UK)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Interfauna (U.K.) Limited, Cambridgeshire
- Weight at study initiation: males (147-188 g) and females (152-184 g)
- Housing: 5 per sex per cage
- Diet (e.g. ad libitum): A pelleted diet (SQC Rat and Mouse Diet No. 1 Expanded, Special Diet Services
Limited, Witham, Essex, U.K.)
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 35-62
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was liquified by warming in a water bath at 37°C, added to arachis oil at the appropriate concentrations and shaken thoroughly. Stability of the test material formulations was determined by Safepharm Analytical Laboratory prior to the start of the study and found to be stable for at least nine days. Formulations were therefore prepared weekly and stored at 4°C in the dark.


VEHICLE
- Concentration in vehicle: 0, 25, 125 and 250 mg/mL
- Amount of vehicle: 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The N-Vinylcaprolactam concentration of the test material formulations was determined by gas chromatography incorporating a flame ionisation detector using an external standard technique .The analytical measurements of test material formulation concentrations of 25, 125 and 250 mg/mL were 26.5, 130.0 and 253.3 mg/mL, respectively. Homogenicity and stability of the test item were analytically determined.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50, 250, 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose in the test groups, in addition 5 animals per sex per dose were included in 2 satellite groups (0 and 500 mg/kg bw)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose was selected based on a range-finding study where the test substance was administered by gavage to groups of 3 male and 3 female Sprague-Dawley CFY rats at dose levels of 50, 250, 500, 750, and 1000 mg/kg bw for 14 days. A control group of 3 rats/sex was administered the vehicle. Clinical observations and body weights were monitored. All surviving animals were killed on study day 15 and subjected to full macroscopic (internal and external) examination. Tissues were not retained.
Animals treated with 50 and 250 mg/kg bw/day showed no clinically observable signs of toxicity during the 14-day study period. At 500 mg/kg bw/day moderate signs of toxicity were noted from day 3 until the end of treatment including hunched posture, pilo-erection, lethargy, red/brown staining around the snout, mouth and of the fur. Severe signs of toxicity were noted in both the 750 and 1000 mg/kg bw/day dose groups from the start of the study. Observations included those seen at 500 mg/kg bw/day with further incidents of increased salivation, ataxia, ptosis, diuresis, pallor of the extremities, emaciation, vocalisation, noisy respiration and loss of righting reflex with two females treated with 1000 mg/kg bw/ day appearing comatose on day 3. This dose group was consequently killed in extremis the same day, with the 750 mg/kg bw/day dose group being terminated on day 5 of the study as one female of this group was found dead on day 5. No effect on bodyweight was seen in the 50 and 250 mg/kg bw/day dose groups in comparison with controls. At 500 mg/kg bw/day, a reduction in bodyweight gain was noted by day 4 with one female showing an actual loss. Body weights recovered by day 8 and animals seemed to show normal gains during the remainder of the study. Animals treated with 750 mg/kg/day, the females in particular, showed losses in bodyweight during the first four days of treatment. One female rat treated with 500 mg/kg bw/day showed congested lungs. Macroscopic abnormalities noted in the 750 mg/kg bw/day dose group included pale livers, with incidents of accentuated lobular pattern, and pale kidneys. The decedent from this dose group showed congested lungs, pale liver with a small green area noted adjacent to the stomach, plus a small dark depression on the glandular region and multiple raised white foci on the non-glandular region of the stomach. Abnormalities at 1000 mg/kg bw/day included extremely pale liver and kidneys with severe -ulceration and epithelial sloughing of the glandular region of the stomach. Hydronephrosis was seen in the 50, 250 and 500 mg/kg bw/day dose groups. This finding is commonly encountered and consistent with normally expected findings in laboratory maintained rats and, as such was considered not to be treatment-related. Based on these results, dose levels of 50, 250, and 500 mg/kg bw/day were selected for the main study.

- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: on day before treatment and on days 7, 14, 21 and 28; and in case of satellite group, also on days 35 and 42. Body weights were also recorded at necropsy.


FOOD CONSUMPTION. yes
- Time schedule for examinations: weekly


WATER CONSUMPTION: Yes by visual inspection of the water bottles
- Time schedule for examinations: daily


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 28 in test groups and on day 42 in satellite groups.
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: No
- How many animals: all
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, total leucocyte count, erythrocyte indices, plate count, differential leucocyte counts and clotting potential.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 28 in test groups and on day 42 in satellite groups.
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: No
- How many animals: all
- Parameters checked: blood urea, total protein, sodium, potassium, chloride, calcium, inorganic phosphorus, creatinine, alkaline phosphatase, alanine aminotransferase, glucose, gamma glutamyl transpeptidase, triglycerides and total cholesterol


URINALYSIS: Yes (only in test groups)
- Time schedule for collection of urine: during week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes but normal hydration
- Parameters checked: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances and blood.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes for the following organs:
Adrenals, Muscle (skeletal), Aorta (thoracic), Pancreas, Bone & Bone Marrow (femur), Pituitary, Brain, Rectum, Caecum, Sciatic nerve ,Colon, Skin (hind limb), Duodenum, Spleen, Eyes, Stomach, Gross lesions, Testes, Heart, Thymus, Ileum, Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Urinary bladder, Liver, Oesophagus, Lungs, Ovaries, Lymph nodes (cervical and mesenteric )

All animals of test and satellite groups were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. Adrenals, brain gonads, heart, kidneys, liver, pituitary and spleen were weighed.
Statistics:
Relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance.
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortality was observed. Animals treated with 500 mg/kg bw/day showed signs of increased salivation, red/brown staining around the snout and mouth and pilo-erection. Slight signs of staining around the mouth and increased salivation were noted in animals treated with 250 mg/kg bw/day. Satellite high dose animals showed immediate recovery after termination of treatment. No clinically observable signs of toxicity were observed in animals treated with 50 mg/kg bw/day.


BODY WEIGHT AND WEIGHT GAIN: A slight reduction in bodyweight gain was noted during the first week only, in animals treated with 500 mg/kg bw/day.


FOOD CONSUMPTION: A slight reduction was noted during the first week only in animals treated with 500 mg/kg bw/day.


WATER CONSUMPTION: Visual inspection of water bottles throughout the study period revealed no overt intergroup differences.


HAEMATOLOGY: Clotting time was significantly increased in females from the 500 mg/kg bw/day dose group but no effects were seen in the other dose groups.


CLINICAL CHEMISTRY: A slight increase in creatinine and urea was noted for high dose males. Such a small increase in one of these parameters would not be considered toxicologically significant but an increase in both is worthy of note since they are dual indicators of renal obstruction. Similar increases were not apparent in satellite group animals after the treatment-free period.


URINALYSIS: no abnormalities were detected.


ORGAN WEIGHTS: High dose males showed a slight reduction in kidney weight compared with controls. High dose females showed significantly increased liver weight and intermediate dose females also showed an increase but less marked. Liver weights were still slightly increased in satellite high-dose females after the fourteen day treatment-free period but the severity of this was substantially reduced.


GROSS PATHOLOGY: No treatment-related macroscopic abnormalities were detected.


HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related changes were observed in the liver of animals treated with 500 mg/kg bw/day. Changes were characterised by an increased staining intensity and tendency towards basophilia in the cytoplasm of centrilobular hepatocytes. There was no evidence of associated degenerative or inflammatory changes. No histopathological evidence of renal changes were observed.


Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver weight
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guideline study with GLP, acceptable with restriction (analytical purity of the test substance not reported).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
58 mg/m³
Species:
rat
Quality of whole database:
GLP and guideline study.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
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:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 9 weeks old
- Fasting period before study:
- Housing: up to 5 animals per cage
- Diet: Kliba laboratory diet, mouse/rat maintenance “GLP”, 10 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes: 15 air changes per hour
- Photoperiod: 06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark
- Acclimation period: 13/14 days: (2 days pre-exposure)

IN-LIFE DATES: From: 2012-10-09 To: 2012-11-22
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not relevant, animals were exposed to vapour.
Details on inhalation exposure:
Generation of the inhalation atmospheres (vapour)
For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the metering pump. The vapor / air mixture was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system. To achieve the low target concentration in test group 1, the so generated atmosphere was introduced into a mixing tube, where part of the atmosphere was replaced by conditioned air. Afterwards, the atmosphere was introduced into the inhalation chamber

Exposure systems
The following exposure systems with the specific technical conditions were used:
Whole-body inhalation systems:
During exposure, the animals were housed in wire cages (DK III) that were located in a glass-steel inhalation chambers, V approx. 200 L (control animals V approx. 1400 L). The inhalation atmospheres was passed into the inhalation chambers with the supply air and was removed by an exhaust air system.
a) Control group
The exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the supply air (positive pressure). This was to ensure that no laboratory roomair reached the control animals.
b) Test groups 1 – 4
The exhaust air system was adjusted in such a way that the amount of exhaust air was higher than the supply air (negative pressure). This was to ensure that no contamination of the laboratory occurred was the result of leakages from the inhalation chambers.

Measurement and recording of technical conditions in the exposure systems
In general, the technical parameters were measured and recorded as follows:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also recorded by means of this system. All these parameters were recorded continuously by a computerized data acquisition and control system BaseLab (BASF SE, Ludwigshafen, Germany) as analog signals (between 0 or 4 and 20 mA), converted into digital data every 10 seconds, transferred to a personal computer and displayed on the screen. The computer (Baselab-Software, BASF SE, Ludwigshafen, Germany) checked the incoming values against the preset threshold values, gave warnings if violations of these values occurred and recorded the start and the end of the violation. Daily protocols were prepared from the values measured every 10 seconds using Microsoft Excel. If values above or below the preset limits occurred for longer than 10 minutes, values were printed and documented in the printed daily records. The digital data and the printed daily records were considered as raw data. The systems and software were validated in house by professionals of the electronic data processing.
The pump rate of the dosing pumps were read and recorded once per exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the inhalation atmosphere
The analytical determination of the atmospheric concentrations in the inhalation chambers was performed at the Laboratory of Inhalation Toxicity and at the Laboratory of Analytical Chemistry, Experimental Toxicology and Ecology of BASF SE.

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

Analytical methods of determination
The atmospheric concentration of the test substance was measured either on-line by calibrated total hydrocarbon analyzer (FID, abbreviation for flame ionization detector, which is anonym of total hydrocarbon analyzer) or by gas chromatography (GC) of absorption samples obtained by off-line sampling (sampling method see the following section).
The FID will be calibrated with certificated test gas propane using specific response factor provided by the manufacture of the FID, matched against GC analyses during the technical trial. The measured values over a study day gives information about the constancy of N-Vinylcaprolactam concentration over the exposure time.
As the measurements with FID present the sum of the hydrocarbon in the air, to confirm the composition of the test atmosphere, the test atmospheres was analyzed once a week by gas chromatography.
At low concentrations, the sensitivity of FID may be not sufficient to determine such low atmospheric concentration of N-Vinylcaprolactam. In this case, absorption samples will be taken offline and analyzed by GC.

Sampling
Absorption samples were taken adjacent to the animals noses in order to determine the analytical concentration in the inhalation apparatus (sampling probe, appropriate absorption vessels connected in series, filled with appropriate solvent, gas sampling station). The sampling velocity in the sampling probe was 1.25 m/sec.
The control atmosphere was sampled on one day during the exposure period.
Duration of treatment / exposure:
20 treatments for 6 hours (28-days exposure period)
Frequency of treatment:
On each workday (5 times every week, Monday to Friday)
Remarks:
Doses / Concentrations:
1, 6, 58 or 173 mg/m³ (0.2, 1.0, 10 or 30 ppm)
Basis:
nominal conc.
No. of animals per sex per dose:
8 rats (m/f)
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on available data, four concentrations were selected ranging from the low concentration of 1 mg/m³ (0.2 ppm) representing the expected NOAEC, to the high concentration of 173 mg/m³ (30 ppm) expected to cause toxic effects.
Observations and examinations performed and frequency:
MORTALITY: Yes
- Time schedule: A check for moribund or dead animals was carried out twice per day on working days and once per day on weekends and holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: A clinical inspection was performed on each animal at least three times on exposure days and once a day during pre-exposure and post exposure observation days. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until the day of gross necropsy. The body weight change of the respective week was calculated as the difference of Friday to the previous Monday.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was determined weekly (e.g. Monday-Friday) and calculated as mean food consumption in grams per animal and day.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS
- Organ weights (all animals): anesthetized animals, epididymides, testes, adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, thyroid glands.

HISTOPATHOLOGY: Yes, nasal cavity (4 levels, Hematoxylin and Eosin stain/all animals per test group, BrdU stain/all male animals per test group), liver (Hematoxylin and Eosin stain/all animals per test group, BrdU stain/all female animals per test group, TUNEL stain/all female animals per test group), all gross lesions (all animals affected/test group)

PRESERVATION OF TISSUES
The following organs or tissues of all animals were fixed in 4% neutral-buffered formaldehyde or in modified Davidson’s solution:
All gross lesions, adrenal glands, brain with olfactory bulb, bone marrow (femur), esophagus, eyes with optic nerve (preserved with the head), heart, jejunum, kidneys, larynx/pharynx, liver, lungs, lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), nose (nasal cavity), ovaries, seminal vesicle, spinal cord (cervical, thoracic and lumbar cords), stomach (forestomach and glandular stomach), spleen, testes (modified Davidson’s solution), thyroid glands, thymus, trachea, urinary bladder, uterus.


Other examinations:
Implantation of minipumps
At approx. 3 days (males) or 7 days (females) before necropsy, Alzet osmotic minipumps (ALZET osmotic minipumps, type 2ML1, Alzet Corporation, Palo Alto, USA; supplied by SAVO, Kißlegg, Germany) were implanted subcutaneously to the dorsal region under isoflurane anesthesia (Essex GmbH, Munich, Germany). Additionally, in order to reduce pain during and after the implantation, the animals received an s.c. injection of an appropriate analgetic compound (e.g. Buprenorphine, 0.05 mg/kg bw) 10 to 30 minutes prior to surgery.

The pumps were filled with approx. 2 mL of a solution of 5´-bromo-2-deoxyuridine (BrdU (Sigma Chem., 20 mg/mL physiological saline)) approx. 2-4 hours before implantation. The filled pumps were stored in physiological saline at 37°C until use. The minipumps were implanted 3 days prior to necropsy for male animals and 7 days prior to necropsy for female animals.

Immunohistology
Staining procedure: After deparaffination and rehydration, the slides for S-phase response assessment were immunostained with a monoclonal antibody (anti BrdU) with the streptavidine method, the apoptosis was determined using a TUNEL-kit.

Thymus and jejunum of all test group animals served as positive control for the TUNEL and the BrdU cell proliferation assay, respectively. No histopathological examination of these organs was performed.

Quantitative assessment of S-phase response and apoptosis
Liver (female animals)
Positively stained cells (BrdU) were identified by red/brown pigment over their nuclei using an image analysis system (Quantimet 500). Hepatocytes were discriminated from non-hepatocytes on the base of their size and shape. Labeled and unlabeled cells were counted by genuine color detection.

Cell proliferation in the liver can be induced diffusely in all hepatocytes, or it can be localized in specific regions of the lobule. The liver lobule is, therefore, subdivided into three zones, comprising the portal zone (zone 1), the zone of the central vein (zone 3) and the intermediate zone in between (zone 2). According to BAHNEMANN et al. (1997), more than 1,000 cells per zone (> 3,000 cells per liver) were assessed.

The Labeling index (LI) in BrdU immunostained sections were calculated as follows:
LI (%) = labeled cells/unlabeled + labeled cells

Apoptotic bodies that showed a positive reaction (red/brown pigment over their nuclei) were discriminated from false positive staining due to their morphology and localization by light microscopy and counted per liver lobe. The total number of apoptotic cells was determined in the liver of control and all test group female animals.

Nasal cavity (male animals)
Positively stained cells are characterized by a red reaction product covering the nuclei.
Light microscopy was performed at an original magnification of 200 x using an image analysis system (Leica).

Cells of epithelium were differentiated and detected on the base of their shape and size. The positive nuclei were then related to the length of the epithelium within the measurement. The measurement was performed in the areas detected in H&E routine diagnostic to be the target areas (area 1, 2, 3, 4, 5, 6). In level I area 1, 2, and 3 (respiratory epithelium) and area 4 (transitional epithelium) were evaluated. In level II and III area 1, 2, 3, and 4 (olfactory epithelium) were evaluated). In level IV area 1, 2, 3, 4, 5, and 6 (olfactory epithelium) were evaluated. In the two cell layers next to the basal membrane the BrdU positive cells were counted and the ULLI was calculated.

The unit length labeling index (ULLI) in BrdU-immunostained slides was calculated as follows:
ULLI (%) = labeled cells/length of epithelium
Statistics:
Mean values and standard deviations were calculated.
The following statistical analyses were carried out, additionally:
Parameter: Body weights and body weight change.
Statistical test: DUNNET`s.

Statistics of pathology
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
Parameter: Weight of the anesthetized animals and absolute and relative organ weights.
Statistical test: KRUSKAL-WALLIS-H and WILCOXON test

Parameter: Weight parameters, results of cell proliferation and apoptosis
Statistical test: WILCOXON test, one sided
Details on results:
CLINICAL SIGNS AND MORTALITY
In all male animals frequent masticatory movements were observed on day 1 of the study in test group 4 (173 mg/m³). In females of the same test group 4 animals showed salivation on day 1 and 1 animal an encrusted nose on day 1, respectively 2 animals on day 8 of the study.

No mortality occured in males and females during the course of the study.

BODY WEIGHT
A statistically significant reduction in the body weight gain was observed in test group 4 males (173 mg/m³) compared to test group 0 males (0 mg/m³) on day 8. In the summary bodyweight development, a relevant statistically significant decrease was observed in test group 4 for days 0 - 1, and study days -9 - 28. The statistically significant differences observed in test group 1 and 2 (1 and 6 mg/m³) for days -2 - 0 and day 0 - 1, as compared to controls, were regarded as incidental and without biological relevance.
In females, a statistically significant reduction of the body weight gain compared to test group 0 was observed in test group 4 (173 mg/m³) on days 2, 12, 16, 19 and 26. No statistically significant differences in the summary body weight development were observed in females for the different time intervals investigated.

FOOD CONSUMPTION
The food consumption in male and female animals in test group 4 (173 mg/m³) was slightly reduced during the course of the study as compared to controls. These findings were considered as treatment related. The reduction in the food consumption in male animals of test group 3 (58 mg/m³) on days 25 - 27 and in female animals in test group 2 (6 mg/m³) observed for days 0 - 2 and 5 - 9 as compared to controls, were regarded as incidental and without biological relevance.

ORGAN WEIGHTS
- Absolute organ weights: when compared to control group 0 (set to 100%), the mean absolute organ weights of following organs were statistically significant changed: heart (male animals in test group 3 and 4 (58 and 173 mg/m³)), thyroid glands (male animals in test group 1, 2 and 3 (1, 6 and 58 mg/m³)), thymus (female animals in test group 2 and 4 (6 and 173 mg/m³)).

- Relative organ weights: when compared to control group 0 (set to 100%), the mean relative organ weights of following organs were statistically significant changed: liver (male animals in test group 4 (173 mg/m³)), female animals in test group 3 and 4 (58 and 173 mg/m³)), kidneys (female animals in test group 3 (58 mg/m³) ), thymus (female animals in test group 2 (6 mg/m³)), thyroid glands (male animals in test group 1 - 4 81 - 173 mg/m³)), female animals in test group 1 and 3 (1 and 58 mg/m³).

The increased relative liver weights in males and females of test group 4 (173 mg/m³) were regarded to be treatment related.

For the other changed organ weights a substance-related effect cannot be ruled out as no histopathologic examination of these organs was performed. For the thymus and the heart it is not regarded to be very likely that the changes were treatment related, as no concentration-response relationship was present.

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS LESIONS
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY
Treatment-related findings were observed in the nasal cavity in all levels and in the liver of males and females.

The incidences and grading are shown for females for the liver and for males for the nasal cavity in the tables listed under "Any other information on results incl. tables". Only the evaluation in accordance with the cell proliferation examinations is listed.

All female animals of test group 4 (173 mg/m³) revealed a minimal hypertrophy of centrilobular liver cells. This finding was regarded to be treatment related.

In the nasal cavity level I there was minimal to slight hyperplasia of the respiratory epithelium in test group 2 (6 mg/m³) increasing in a concentration-response manner up to severe in test group 4 (173 mg/m³). Mainly the respiratory epithelium in the middle part of the nasal septum was affected. The transitional epithelium was minimal to slight diffusely hyperplastic in males from test group 2-4 (6 – 173 mg/m³). At the very basal region of level I where the nasal entrance is located, there was a minimal to slight focal squamous metaplasia of the respiratory epithelium observed just at the transition from squamous to respiratory epithelium. Again, animals from test group 2-4 (6 – 173 mg/m³) were affected with an increase in number and severity with concentration of the test substance. In addition, there were minimal to slight inflammatory cell infiltrates in these three test groups corresponding the above mentioned findings.

In the nasal cavity level II there was degeneration/regeneration of the olfactory epithelium. Mainly the dorsal meatus was affected. The findings were minimal to moderate and occurred in males of test group 3 and 4 (58 and 173 mg/m³). The term degeneration/regeneration was used when there was a decrease in height of and vacuoles within the epithelium (degeneration) as well as large, mostly basally located intensive blue staining cells (regeneration). One male of the test group 4 (173 mg/m³) revealed a synechia, means an adhesion of the olfactory epithelium of two opposite located turbinates.

In the nasal cavity level III there was degeneration/regeneration of the olfactory epithelium of test group 3 and 4 animals (58 and 173 mg/m³), almost exclusively in the dorsal meatus. The olfactory epithelium covering the nasal septum and the turbinates revealed minimal to severe hyperplasia in test groups 3 and 4 (58 and 173 mg/m³) with increase in severity and number of affected animals with concentration. In contrast to degeneration/regeneration the diagnosis hyperplasia was used when there was an increase in epithelium height without any signs of cell loss. Again two animals of test group 4 (173 mg/m³) revealed an adhesion (synechia) of the olfactory epithelium.

In the nasal cavity level IV there was exclusively hyperplasia observed in males of test groups 3 and 4 (58 and 173 mg/m³). Mainly the dorsal areas of the turbinates were affected. Again one male of test group 4 (173 mg/m³) revealed an adhesion (synechia) of the olfactory epithelium.

Females revealed similar findings. 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.

S-Phase response and apoptosis (tables are listed under "Any other information on results incl. tables")
Liver
S-Phase (only investigated in females): A slight increase in the labeling index (LI) in zone 1 and 2 and the corresponding increase in all zone LI of treated animals was regarded to be not related to treatment as no concentration-response relationship was present. The 6-fold increase in LI of zone 3 in the test group 4 females (173 mg/m³) was regarded to be treatment related.
Apoptosis (only investigated in females): The total number of apoptotic cells was determined in the liver of control and all treated females. There was no clear concentration-response relationship in the number of apoptotic cells. The significant numbers mainly in zone 2 were therefore regarded to be incidental and not related to treatment.

Nasal cavity (tables are listed under "Remarks on results including tables and figures")
S-Phase (only investigated in males): In the nasal cavity level I the respiratory and transitional epithelium revealed a 3-19-fold increase in (Unit Length Labelling Index (ULLI) in test group 3 and 4 (58 and 173 mg/m³). The respiratory epithelium covering the middle region of the nasal septum revealed also in test group 2 (6 mg/m³) an 11-fold increase in the ULLI.
The olfactory epithelium in levels II-IV showed a clear effect on cell proliferation in test group 4 (173 mg/m³). The significant increases in ULLI in the remaining test groups were questionable. The increase compared to control was in test group 1-3 very low (below 2-fold) and occasionally without a concentration-response relationship. The subtle increase of cell proliferation (< 2-fold) was not regarded to be biological relevant.
The 2.3-fold increase in level 4 area 2 of test group 1 (1 mg/m³) was regarded to be incidental as all other areas were below control values and most of the control values were 0 (single animal data).
Therefore, a clear effect on S-phase was observed in the olfactory epithelium of males of test group 3 and 4 (58 and 173 mg/m³), in the transitional epithelium of males of test group 3 and 4 (58 and 173 mg/m³) and in the respiratory epithelium of males of test group 2-4 (6-173 mg/m³).

Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
58 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on increased relative liver weights in males and females and hepatocellular hyperthropy in females at 173 mg/m³.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on histopathological findings in the nasal cavity and corresponding increases in the S-phase at 6 mg/m³ and above.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified

Histopathology

Table 1: Histopathological examination of the liver (females only)

 

Female animals

Test group

(mg/m³)

0

(0)

1

(1)

2

(6)

3

(58)

4

(173)

No. of animals

8

8

8

8

8

Liver

 

 

 

 

 

Hypertrophy centrilobular

0

0

0

0

8

·        Grade 1

 

 

 

 

8

Table 2: Histopathological examination of the nasal cavity (males only)

 

Male animals

Test group

(mg/m³)

0

(0)

1

(1)

2

(6)

3

(58)

4

(173)

No. of animals

8

8

8

8

8

Nasal cavity, level I

 

 

 

 

 

Hyperplasia respiratory epithelium, (m)f

0

0

5

8

8

·        Grade 1

 

 

4

2

 

·        Grade 2

 

 

1

5

4

·        Grade 3

 

 

 

1

3

·        Grade 4

 

 

 

 

1

Hyperplasia transitional epithelium, d

0

0

8

8

8

·        Grade 1

 

 

5

5

1

·        Grade 2

 

 

3

3

7

Metaplasia, squamous

0

0

3

8

7

·        Grade 1

 

 

3

4

 

·        Grade 2

 

 

 

4

7

Inflammatory cell infiltrates, (m)f

0

0

2

2

7

·        Grade 1

 

 

2

1

2

·        Grade 2

 

 

 

1

5

Nasal cavity, level II

 

 

 

 

 

Degeneration/regeneration olfactory epithelium

0

0

0

7

8

·        Grade 1

 

 

 

4

 

·        Grade 2

 

 

 

3

1

·        Grade 3

 

 

 

 

7

Synechia

 

 

 

 

1

Nasal cavity, level III

 

 

 

 

 

Hyperplasia olfactory epithelium, (m)f

0

0

0

6

8

·        Grade 1

 

 

 

3

 

·        Grade 2

 

 

 

3

 

·        Grade 3

 

 

 

 

4

·        Grade 4

 

 

 

 

4

Degeneration/regeneration olfactory epithelium

0

0

0

2

7

·        Grade 1

 

 

 

2

 

·        Grade 2

 

 

 

 

2

·        Grade 3

 

 

 

 

5

Synechia

 

 

 

 

2

Nasal cavity, level IV

 

 

 

 

 

Hyperplasia olfactory epithelium, (m)f

0

0

0

4

8

·        Grade 1

 

 

 

2

 

·        Grade 2

 

 

 

2

 

·        Grade 3

 

 

 

 

2

·        Grade 4

 

 

 

 

6

Synechia

 

 

 

 

1

S-Phase response and apoptosis

Liver

Table 3: S-Phase (females only)

 

Females

Test group (mg/m³)

 

Zone 1

Zone 2

Zone 3

All zones

LI

%

LI

%

LI

%

LI

%

0

M

0.20

100

0.98

100

0.86

100

0.68

100

(0)

SD

0.20

 

0.31

 

0.50

 

0.28

 

 

n

8

8

8

8

1

M

0.44

220

1.94**

192

0.90

105

1.10*

162

(1)

SD

0.55

 

0.84

 

0.52

 

0.48

 

 

n

8

8

8

8

2

M

0.73**

365

2.44**

249

1.22

142

1.48**

218

(6)

SD

0.67

 

0.79

 

0.62

 

0.56

 

 

n

8

8

8

8

3

M

0.71**

355

2.51**

256

1.51

176

1.59**

234

(58)

SD

0.29

 

0.58

 

0.86

 

0.37

 

 

n

8

8

8

8

4

M

0.25

125

1.71**

174

5.37**

624

2.48**

365

(173)

SD

0.33

 

0.38

 

1.70

 

0.72

 

 

n

8

8

8

8

* p<= 0.05, ** p<= 0.01

LI = labeling index; M = mean; SD = standard deviation; n = number of animals

Table 4: Apoptosis (females only)

 

Females

Test group (mg/m³)

 

Zone 1

Zone 2

Zone 3

All zones

0

N

6

19

10

32

(0)

n

8

8

8

8

1

N

13*

39**

24*

76**

(1)

n

8

8

8

8

2

N

5

34**

16

55*

(6)

n

8

8

8

8

3

N

4

14

13

31

(58)

n

8

8

8

8

4

N

5

33**

10

48*

(173)

n

8

8

8

8

* p<= 0.05, ** p<= 0.01

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

An oral study was conducted according to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) with deviations (only 2 dose levels; no haematology, urinalysis and opthalmological examination) and is reliable with restrictions. Ten animals per sex per dose group were administered with 130 and 260 mg/kg bw of the test substance daily for 3 months. One high dose male died after 80 days of treatment; however, the cause of death was unclear. In the high dose group, salivation was observed in males and females after dosing. Body weights of males were significantly reduced from day 77 until the end of the study. Gamma-glutamyl transferase activity in the liver was increased in both sexes. Absolute liver weights were increased in females and relative liver weights were increased in both sexes. Other findings occurred incidentially and infrequently and were not of statistical significance. According to the authors, based on these results, repeated oral administration of the test substance to male and female rats for 3 months produced changes in liver weight and an increase in the gamma-glutamyl transferase activity. However, histopathology revealed no hepatocellular alterations. A correlation between biochemical determination of gamma-glutamyl transferase activity and histopathological findings could not be detected in this study. The no observed adverse effect level (NOAEL) was 130 mg/kg bw/d for males and below 130 mg/kg bw/d for females under the conditions of this test due to increase in the liver gamma-glutamyl transferase activity. Under the conditions of this test, the lowest observed adverse effect level (LOAEL) for systemic toxicity was 260 mg/kg bw/d for males and 130 mg/kg bw/d for females. (BASF AG, 1991)

 

A second oral study was conducted according to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) and is reliable with restrictions as analytical purity of the test substance is not reported. Five animals per sex per dose group were administered with 0, 50, 250 and 500 mg/kg bw of the test substance daily for 28 days. In addition, 5 animals per sex per dose were included in 2 satellite groups (0 and 500 mg/kg bw) for observation of reversibility, persistence, or delayed occurrence of toxic effects, for 14 days post treatment. During the dosing period, animals treated with 500 mg/kg bw/day showed signs of increased salivation whilst bodyweight gain and food consumption were adversely affected during the first week. Hematological examination revealed prolonged blood coagulation time noted in females from this group. Histopathological examination of liver sections from animals in this dose group showed centrilobular hepatocellular cytoplasmic changes. Animals treated with 250 mg/kg bw/day also showed increased salivation and liver weights were slightly increased in females, although-there were no associated histopathological changes. Effects of treatment with this test material were apparently reversible since satellite high dose animals, after two weeks without treatment, showed no histopathological liver changes and blood coagulation time was normal. Slightly elevated liver weights persisted but the severity of this was reduced. Oral administration of the test material, N-Vinylcaprolactam, to rats for a period of twenty-eight consecutive days resulted in treatment-related changes at dose levels of 250 and 500 mg/kg bw/day. No such effects were demonstrated in animals treated with 50 mg/kg bw/day and therefore the no adverse effect level (NOAEL) was 50 mg/kg bw/day. Under the conditions of this test, lowest observed adverse effect level (LOAEL) for systemic toxicity was considered to be 250 mg/kg bw/day. (Safepharm, 1989)

 

Inhalation:

An inhalation study was conducted according to OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day) and is reliable without restrictions. Ten rats per sex per dose group were exposed to 0.0059, 0.058, 0.181 mg/L (1, 10, 32 ppm) of test substance 5 hours/day, 5 days/week for 3 months. Exposure to the test substance at 32 ppm for 3 months resulted in irritation of the nasal cavity and in slight systemic toxicity characterized by depression of body weight development. The systemic effects were substantiated by clinico-pathological findings of mild anemia, adaptive response of the liver and probably slight disturbance of renal tubular function. Pathomorphological correlates for nasal irritation and liver changes could be found. No systemic toxicity was observed in rats exposed to 10 or 1 ppm, but nasal irritation was detectable by histopathology in a dose-dependent manner at both dose levels. The NOAEC for systemic toxicity was 0.058 mg/L (10 ppm) under the conditions of this test. No NOAEC for nasal irritation was reached, based on the histopathologic finding of hyperplasia of the transitional epithelium in the nasal cavity which was observed at all dose levels. Low observed adverse effect concentration (LOAEC) based on slight systemic toxicity was 0.181 mg/L (32 ppm) under the conditions of this test. (BASF AG, 1995)

 

An inhalation study was conducted according to OECD Guideline 412 (Subacute inhalation Toxicity: 28 Day) and is reliable without restrictions. Eight rats per sex per dose group were exposed to 1, 6, 58 and 173 mg/m³ of test substance vapour on 6 hours/day, 5 days/week for 28 days (20 treatments). A treatment related increase in the relative liver weights was observed in males and females of test group 4 (173 mg/m³). Regarding pathology, the nasal cavity in all four levels was the target organ. The transitional and respiratory epithelium in level I showed hyperplasia in test groups 2, 3, and 4 (6, 58, and 173 mg/m³). This corresponds to an increase in S-phase measured in these epithelia. At the nasal entrance at the transition from squamous to respiratory epithelium, there was a focal squamous metaplasia of the respiratory epithelium. This occurs when lesser resistant tissue is damaged and then replaced by a more resistant tissue. Furthermore inflammatory cell infiltrates were found in the submucosa in affected animals. In this level animals of test group 2-4 (6 - 173 mg/m³) showed these findings with a concentration-response related manner. In the levels II - IV mainly the olfactory epithelium was affected. In the dorsal meatus were degeneration and regeneration present; in the epithelium covering the turbinates mainly on the dorsal surface there was hyperplasia observed. Only animals of test group 3 - 4 (58-173 mg/m³) showed these findings. This also corresponds to the S-phase evaluation, were only in these two test groups ULLI higher than 2-fold compared to control were observed. All these histopathologic findings observed in the different levels of the nasal cavity were regarded to be treatment related and adverse in nature.The fact that hyperplastic and degenerative lesions were seen together might indicate a primary cell loss caused by the test substance and a secondary regenerative hyperplasia. The liver showed a minimal centrilobular hypertrophy in female animals of test group 4 (173 mg/m³). Furthermore, a 6-fold increase in S-phase in female animals was observed in the zone 3 (centrilobular). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The NOAEC for systemic toxicity was 58 mg/m³ under the conditions of this test, based on increased relative liver weights in males and females at 173 mg/m³ and the hepatocellular hypertrophy noted in females at the same concentration. The NOAEC for local toxicity was 1 mg/m³, based on the histopathological findings in the nasal cavity and corresponding increases in the S-phase at 6 mg/m³ and above. (BASF, 2013)

Dermal:

No data available


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
From the two available key studies, the study with the lowest NOAEL was selected.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The same systemic NOAEC was established for both reliable key studies (subacute and subchronic). Both studies provided the basis for the DNEL derivation.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
The most reliable study was selected.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

N-Vinylcaprolactam is classified according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 with R48/23 (danger of serious damage to health by prolonged exposure through inhalation) and STOT RE cat. 1 (H372: causes damage to liver and respiratory tract through prolonged or repeated exposure).