4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to birds: acute oral toxicity test (LD50-only)

Type of information:

experimental study

Adequacy of study:

other information

Study period:

18 Oct - 2 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-1 (Avian Acute Oral Toxicity Test)

Version / remarks:

version: October 1982

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2100 (Avian Acute Oral Toxicity Test)

Version / remarks:

version: April 1996

Qualifier:

according to guideline

Guideline:

OECD Guideline 223 (Avian Acute Oral Toxicity Test)

Version / remarks:

Draft-OECD guideline for testing of chemicals "Avian Acute Toxicity Test - Oral Toxicity" (November, 1992)

Qualifier:

according to guideline

Guideline:

other: MAFF-Working Document No. 7/5: "Evaluating the acute oral and short-term cumulative oral toxicity of pesticides to birds."

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Dose method:

capsule

Remarks:

Gelatin capsula, Size No. 00 (one per bird); disintegration time: 5 Minutes (US-federal standard No. 285A-0,5% HCL); Source: Shionogi Qualicaps, Inc., Whitsett, N.C. 27377-9215 USA; Carrier: Deionized water

Analytical monitoring:

no

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: The birds were individually weighed and dosed as given in Table 1 under "Any other information on materials and methods incl. tables" below

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite Quail
- Source: Pen-reared, sexual mature Bobwhite Quails were purchased from Morris Quail Farm, 18370 SW 232 Street Goulds, Florida 33170. All birds were from the same hatch (June 01,1999) and appeared phenotypically similar to birds from wild populations.
- Age at test initiation (mean and range, SD): adult, 20 weeks; hatch from June 01,1999; Bobwhite Quails were approaching their first breeding season and had not been used in previous testing.
- Weight at test initiation (mean and range, SD): mean 211.6 g; range 186 - 262 g (for details on individual weigths see Table 1 under "Any other information on materials and methods incl. tables" below
- Sexes used / mixed or single sex: mixed sexes
- Disease free: yes
- Keeping: After arrival, all birds were placed in a large-size aviary for acclimatization to the new climatic conditions. 15 days prior to test 50 well developed birds were selected for randomized assignment to the exposure groups.

Limit test:

no

Remarks:

Single oral administration in gelatine capsules.

Post exposure observation period:

Dosing was followed by a subsequent observation period of 14 days.

No. of animals per sex per dose and/or stage:

5 males, 5 females

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

control, 432, 720, 1200, and 2000 mg ai/kg bw (nominal)

Details on test conditions:

ACCLIMATION
- Acclimation conditions (same as test or not): same
- Feeding: A standard rearing diet for quails (type: "0729-Extrudat", batch No. 181299 / 0755 of the company "Altromin GmbH", D-32791 Lage (BRD)) was supplied ad libitum prior and throughout the study, except a 15 hours starving period immediately before application.
- Water: Drinking water (Monheim City municipal water), analytically controled according to the Federal German Quality Standard (TVO) was available ad libitum prior and throughout the study.
- Health (any disease or mortality observed): no
- Fasting period before study: 15 hours starving period immediately before application

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: 15 hrs

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The birds were housed in stainless steel wire cages which were placed indoors in four superposed rows. The front of each cage was equipped with a polyethylene-feeder. The cages measured 38 x 25 cm with a height of 23 cm and were provided with tap water via an open waterpipe along the back side of each cage. The larger cage size as well as the modified water supply posed no impairment on performance and result of the study.
- Suitable to avoid crowding stress: yes
- Caging: individual

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 1
- For treated: 1

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: 10
- For treated: 10

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 13 - 25 °C
- Relative humidity (%): 35 - 85%
- Photoperiod: The photoperiod was maintained by fluorescent tubes which were arranged in a line above the cages; 8 hour light / 16 hour dark cycle. Natural daylight reached the hall through windows in the roof and in one wall. The photoperiod followed seasonal daylengths.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Observations on mortality and signs or intoxication were made approximately hourly on the day of dosing and at least once workdaily throughout the 14 days observation period.

BODY WEIGHT
- Time schedule for examinations: Individual body weights of animals for each dose and control group at the day before dosing, one week after dosing, at test termination, and of the birds which died prematurely.

FOOD CONSUMPTION
- Time schedule for examinations: On study days 3, 7 and 14, all remaining feed was replaced by fresh feed after cleaning. The removed feed was weighed for determination of the feed consumption.

WATER CONSUMPTION (if drinking water study)
- Time schedule for examinations: To secure a faultless supply with water, waterpipe nozzles were checked work-daily for proper function.

PATHOLOGY
- Dose groups that were examined: At the end of the study all surviving birds were sacrificed by CO2 asphyxiation. Gross necropsies were carried out on all premature deads and on all survivors at the end of the study.

REGURGITATION
- Test material was regurgitated: no

Reference substance (positive control):

no

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Dose descriptor:

other: NOAEL

Effect level:

720 mg/kg bw

Conc. / dose based on:

act. ingr.

Basis for effect:

other: behaviour, feed consumption, body weight

Mortality and sub-lethal effects:

For details on sub-lethal effects see Tables under "Any other information on results incl. tables" below

MORTALITY
Only one male died in the concentration group of 1200 mg ai./kg. One female of the treatment group 2000 mg ai. was sacrificed at day +1 because its leg was broken.

CLINICAL SIGNS
- Results: In the treatment group of 432 mg/kg diarrhea was the only symptom, which was observed on two females (one female on day +2, the other one from day +6 to 10). These effects were reversible and had no influence on the body weight development of these birds. At 1200 mg/kg all birds showed diarrhea from day +1 on, some of them up to day +4. In the highest concentration group (2000 mg ai./kg) half of the birds suffered on diarrhea on the days after the application.

ABNORMAL BEHAVIOUR
- Results: At 1200 mg/kg discoordinated movements were found on one female. Further effects were only observed with the male, which died later: discoordinated movements, apathy, narcotic effects. In the highest concentration group (2000 mg ai./kg) two birds showed discoordinated movements. In one case additionally other symptoms were observed: Ptosis, fluffed feathers, reduced vigilance, apathy. All these birds recovered and survived the test without any symptoms.

BODY WEIGHT
- Results: Body weight decreased in the highest test concentration (2000 mg ai./kg). Since the standard deviation was quite high, these individual body weight data revealed no statistical significance at p=0.05 compared to the control. A dose-response relationship between test concentration and body weight development could not be evaluated. In some cases statistical differences (for p=0.05) between the body weight development of treatment groups and control were found:
a) for males of the treatment groups 432 mg ai./kg and 1200 mg ai./kg for the period from day 0 to day +7 and day 0 to day 14,
b) for females of the treatment group of 1200 mg ai./kg for the period day 0 to day +14.
In the other two concentrations (720 mg ai./kg, 2000 mg ai./kg) no statistically significant differences (p=0.05) could be found.

FOOD CONSUMPTION
- Results: At the highest concentration (2000 mg ai./kg) a decrease in food consumption was determined for the first three days after administration.

PATHOLOGY
- Results: Observations in post-mortem necropsies of surviving birds showed no dosage related effects. Bleeding intestines were observed at several males (not at females) in all treatment group, inclusive the control. Enlarged spleens were found on single individuals of the control. No dosage depending effect on testicles / ovaries were detectable (inhomogeneous pattern of sexual maturity at control). The male, which died during the post exposure period, showed discoloured intestines, reduced liver and a discoloured pancreas.

Reported statistics and error estimates:

Initially all data for the testing endpoints were analysed on variance equality on a 1% level of significance (Bartletts test) using the treatment levels as co-variates.
This statistic displayed, showed no statistically significant difference amongst the standard deviations of the compared datagroups.
In case of equal variances, subsequent analyses were conducted using parametric techniques; performed at p < 0.05.

Parametric procedures involved body weight data to a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, a two-sample comparisons (single treatment-groups versus control) was used on a 5% level (p < 0.05), to indicate which treatment groups differed significantly from the control.
For non-parametric procedures, the Mann-Whitney / Wilcoxon test for independent samples was used, which allows a comparison of the medians from unpaired groups of different sizes.
All described statistical procedures were carried out by using the STATGRAPHICS-Plus® Software.
Summarizing of raw data as well as pre-calculations (mean and standard deviation) were performed by using " Excel 97 for Windows©" of the Microsoft Corporation / USA.

Table 1: Summarized Signs of Intoxication

Dose Level (mg ai/kg bw)	# Toxic signs / # Dead / # Dosed			Observed effects
	Males	Females	Combined
0	0 / 0 / 5	0 / 0 / 5	0 / 0 / 10	--
432	0 / 0 / 5	2 / 0 / 5	2 / 0 / 10	DR
720	0 / 0 / 5	0 / 0 / 5	0 / 0 / 10	--
1200	5 / 1 / 5	5 / 0 / 5	10 / 1 / 10	DR/DM//AP/NA
2000	3 / 0 / 5	2 / 0 / 4*	5 / 0 / 9*	DR/DM/PT/FF/RV/AP

DR = diarrhea, AP = apathy, DM = discoordinated movements, NA = narcotic effects, PT = ptosis, FF = fluffed feathers, RV = reduced vigilance, * = out of valuation, this female was sacrificed because of a broken leg

 

Table 2: Gross Necropsy Observations (male)

	# animals with observations / # dosed
Dose (mg ai/kg bw)	0	432	720	1200	2000
Found dead	0 / 5	0 / 5	0 / 5	1 / 5	0 / 5
survivors	5 / 5	5 / 5	5 / 5	4 / 5	5 / 5
Observed effects	# animals with observations / # dosed
no pathological changes	2 / 5	2 / 5	0 / 5	3 / 5	4 / 5
Intestinal haemorrhages	2 / 5	3 / 5	5 / 5	1 / 5	1 / 5
dark discoloured pancreas	0 / 5	0 / 5	0 / 5	1 / 5	0 / 5
dark discoloured intestine	0 / 5	0 / 5	0 / 5	1 / 5	0 / 5
enlarged spleen	1 / 5	0 / 5	0 / 5	0 / 5	0 / 5
enlarged gall bladder	0 / 5	0 / 5	0 / 5	0 / 5	0 / 5
liver reduced	0 / 5	0 / 5	0 / 5	1 / 5	0 / 5
testicles reduced	1 / 5	0 / 5	1 / 5	0 / 5	1 / 5
testicles discoloured	0 / 5	0 / 5	0 / 5	0 / 5	0 / 5

 

Table 3: Gross Necropsy Observations (female)

	# animals with observations / # dosed
Dose (mg ai/kg bw)	0	432	720	1200	2000
Found dead	0 / 5	0 / 5	0 / 5	0 / 5	0 / 4*
survivors	5 / 5	5 / 5	5 / 5	5 / 5	4 / 4
Observed effects	# animals with observations / # dosed
no pathological changes	3 / 5	5 / 5	5 / 5	5 / 5	4 / 4
Intestinal haemorrhages	0 / 5	0 / 5	0 / 5	0 / 5	0 / 4*
enlarged spleen	1 / 5	0 / 5	0 / 5	0 / 5	0 / 4*
enlarged gall bladder	1 / 5	0 / 5	0 / 5	0 / 5	0 / 4*
ovaries reduced	2 / 5	3 / 5	2 / 5	2 / 5	2 / 4*
ovaries discoloured	0 / 5	0 / 5	0 / 5	0 / 5	0 / 4*

* =one bird excluded from valuation (sacrificed because of a broken leg)

 

Table 4: Average Body Weight Data (Male Survivors)

Dose level (mg ai/kg bw)		Body weights (g)
		day -1		day 7		day 14
0	Mean	207.7	(n=5)	213.0	(n=5)	214.0	(n=5)
	SD	9.9		11.4		12.8
432	Mean	222.8	(n=5)	220.8	(n=5)	209.8	(n=5)
	SD	9.9		24.3		10.5
720	Mean	209.0	(n=5)	211.2	(n=5)	213.4	(n=5)
	SD	7.6		9.7		12.0
1200	Mean	218.4	(n=5)	217.8	(n=4)	220.3	(n=4)
	SD	11.8		11.0		10.3
2000	Mean	204.8	(n=5)	186.0	(n=5)	191.2	(n=5)
	SD	7.3		34.0		28.8

 

Table 5: Average Body Weight Data (Female Survivors)

Dose level (mg ai/kg bw)		Body weights (g)
		day -1		day 7		day 14
0	Mean	203.4	(n=5)	210.0	(n=5)	220.8	(n=5)
	SD	16.6		19.9		23.9
432	Mean	205.8	(n=5)	210.8	(n=5)	215.6	(n=5)
	SD	16.2		14.0		17.3
720	Mean	220.0	(n=5)	228.8	(n=5)	238.8	(n=5)
	SD	12.5		14.5		15.2
1200	Mean	211.2	(n=5)	213.2	(n=4)	214.8	(n=4)
	SD	13.6		17.8		13.6
2000	Mean	213.0	(n=5)	196.5	(n=5)	205.5	(n=5)
	SD	6.2		37.0		28.6

 

Table 6: Summarized Food Consumption

Dose level (mg ai/kg bw)	Feed Consumed per Treatment Group (g)
	days 0-3 (3 x 24 h)	days 3-7 (4 x 24 h)	days 7-14 (7 x 24 h)
0	510	825	1175
432	450	731	1122
720	431	814	1168
1200	598	504	1084
2000	209	588	1023

 

Table 7: Estimated Food Consumption per Bird per Day

Dose level (mg ai/kg bw)	Feed Consumed per Treatment Group (g)
	days 0-3 (3 x 24 h)		days 3-7 (4 x 24 h)		days 7-14 (7 x 24 h)
0	17.0	(n=30)	18.3	(n=40)	16.8	(n=70)
432	15.0	(n=30)	20.4	(n=40)	16.0	(n=70)
720	14.4	(n=30)	13.6	(n=40)	16.7	(n=70)
1200	19.9	(n=30)	16.3	(n=37)	17.2	(n=63)
2000	7.5	(n=28)	18.3	(n=36)	16.2	(n=63)

n = number of “bird-living-days”

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

short-term toxicity to birds: acute oral toxicity test (LD50-only)

Type of information:

experimental study

Adequacy of study:

other information

Study period:

15 - 29 Nov 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 223 (Avian Acute Oral Toxicity Test)

GLP compliance:

no

Dose method:

gavage

Analytical monitoring:

no

Vehicle:

yes

Remarks:

2% Cremophor solution

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: no
- Preparation of doses: The dosing volume was 1 mL/100 g body weight
- Type, identity and function of solvent/vehicle: 2% Cremopher solution

Test organisms (species):

Coturnix coturnix japonica

Details on test organisms:

TEST ORGANISM
- Common name: Japanese quail (reported as Coturnix japonica)
- Source: Saitama Laboratory Animals Co., Japan
- Age at test initiation (mean and range, SD): 7 week-old
- Weight at test initiation (mean and range, SD): 80 - 100 g (70 - 90 g at receipt)
- Sexes used / mixed or single sex: only males used

Limit test:

no

Remarks:

Single oral administration via stomach sonde

Post exposure observation period:

Dosing was followed by a subsequent observation period of 14 days.

No. of animals per sex per dose and/or stage:

10 males per group

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

0 (vehicle control), 500, 1000, and 2000 mg/kg bw (nominal)

Details on test conditions:

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions (same as test or not): same
- Feeding: Throughout the study period, the diet (quail mush, Nihon Nosan Industry Inc.) and tap water (Hino-city Water Works) were given ad libitum

FEED WITHHOLDING PERIOD BEFORE DOSING
- No. of hours: one overnight fasting before administration (Feeding was resumed 30 minutes after administration of the test substance)

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: stainless steel cage
- Caging: group (5 birds)

NO. OF BIRDS PER STAGE OR REPLICATE
- For vehicle control: 5
- For treated: 5

NO. OF STAGES OR REPLICATES PER GROUP
- For vehicle control: 2
- For treated: 2

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 20 - 24 °C
- Relative humidity (%):
- Photoperiod: 12 hours light/dark cycle (light on 7:00 a.m. to 7:00 p.m.)

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: The birds were examined carefully for clinical signs frequently on the day of administration and thereafter once or more daily for 14 days.

BODY WEIGHT
- Time schedule for examinations: The birds were weighed before application, and 7 and 14 days after administration.

Reference substance (positive control):

no

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Mortality and sub-lethal effects:

For details on cumulative mortality and sub-lethal effects see Table 1 under "Any other information on results incl. tables" below

No symptoms and deaths were observed at all dose groups. And there was no significant changes in body weight gain at all dose groups.

Table 1: Results on mortality, symptoms and body weight

Dose mg/kg	Results**	Symptoms		Death		Mean Body Weight (g)
	A/B/C	Start	End	Start	End	Administration	7-day	14-day
2000	0/0/10	--	--	--	--	86	111	113
1000	0/0/10	--	--	--	--	88	114	115
500	0/0/10	--	--	--	--	95	117	119
0*	0/0/10	--	--	--	--	82	109	112

*: Vehicle control

**: A: The number of dead animals, B: The number of animals showing symptoms, C: The number of animals used

Validity criteria fulfilled:

not applicable

Description of key information

LD50 (14 d) > 2000 mg a.i./kg (OECD 223)

Additional information

Two studies are available (1999, 1995) investigating the toxicity of the test substance to birds. The studies were conducted according (1999) or similar (1995) to OECD guideline 223. GLP principles were followed in the key study (1999). The test substance was applied via capsule to Bobwhite Quail (Colinus virgianus) (1999) or via gavage to Japanese Quail (Coturnix coturnix japonica) (1995).

In the study with Bobwhite Quail (1999) 10 birds per treatment level (5 per sex) were dosed at 432 mg a.i./kg, 720 mg a.i./kg, 1200 mg a.i./kg, and 2000 mg a.i./kg b.w. In addition a control was run. Dosing was followed by a subsequent observation period of 14 days. Adverse effects on behaviour were recorded as well as effects on feed consumption and body weight. Additionally, gross pathological changes were determined by necropsies on premature deaths and on all survivors at study termination. Only one male died in the concentration group of 1200 mg a.i./kg. One female of the treatment group of 2000 mg a.i./kg had to be sacrificed because of a broken leg. A clear relationship between effects and dose was not observable: At 432 mg a.i./kg two birds showed diarrhoea, no effects at 720 mg a.i./kg; all birds at 1200 mg a.i./kg and half of the birds at 2000 mg a.i./kg suffered from diarrhoea. Other single birds in the higher treatment groups showed additional effects: one female at 1200 mg a.i./kg discoordinated movements; the male which died at 1200 mg a.i./kg discoordinated movements, apathy, narcotic effects; one female at 2000 mg a.i./kg: discoordinated movements; one male at 2000 mg a.i./kg: discoordinated movements, ptosis, fluffed feathers, apathy, reduced vigilance. In addition, a dose-response relationship between test concentration and body weight could not be observed. At the highest concentration (2000 mg a.i./kg) a decrease in food consumption was determined for the first three days after the administration. Observations in post-mortem necropsies of surviving birds revealed no difference between control and treatment groups. Thus an LD50 of >2000 mg a.i./kg and a NOAEL of 720 mg a.i./kg was determined.

The second study the with Japanese Quail (1995) was conducted with single doses of 0 (vehicle control), 500, 1000, and 2000 mg/kg. 10 males per treatment level were dosed. The birds were examined carefully for clinical signs frequently on the day of administration and thereafter once or more daily for 14 days. The birds were weighed before application, and 7 and 14 days after administration. The LD50 value of the test substance was determined to be >2000 mg/kg. Even in the highest dose group (2000 mg/kg), no toxic signs and deaths were observed throughout the observation period. In measurement of body weight, there was no significant changes in weight gain at all dose groups.