1-vinylhexahydro-2H-azepin-2-one

Repeated dose toxicity: inhalation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-vinylhexahydro-2H-azepin-2-one

EC Number:

218-787-6

EC Name:

1-vinylhexahydro-2H-azepin-2-one

Cas Number:

2235-00-9

Molecular formula:

C8H13NO

IUPAC Name:

1-vinylazepan-2-one

Test material form:

other: liquid, yellowish

Details on test material:

- Name of test material: N-Vinylcaprolactam

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 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

Administration / exposure

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)

Doses / concentrations

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.

Examinations

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

Results and discussion

Results of examinations

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).

Effect levels

1

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.

2

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.

3

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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