N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to birds: acute oral toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2000 - 2 May 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Dose method:

capsule

Analytical monitoring:

no

Vehicle:

yes

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern Bobwhite
- Age at test initiation: 27 weeks
- Weight at test initiation: Birds weighed between 175.2 and 224.4 g
- Sexes used: Male and female
- Kept according to standard practices: Yes

Limit test:

no

Remarks:

The birds were exposed to a single oral dose

Post exposure observation period:

The birds were exposed to a single oral dose and observed for 14 days.

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

5 animals per sex per dose in the treatment groups; 10 animals per sex per dose in the control group.

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

- Nominal concentrations: 0, 125, 250, 500, 1000 and 2000 mg/kg bw

Details on test conditions:

ACCLIMATION
- Acclimation period: 15 days
- Acclimation conditions (same as test or not): Yes. Birds were acclimated to the test cages and rooms for 15 days prior to administration of the test material.
- Feeding: Feed and water was provided ad libitum
- Fasting period before study: Yes

FEED WITHHOLDING PERIOD BEFORE DOSING
The birds were fasted for 15 hours prior to dosing

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The cage dimensions were 51 cm deep, 25 cm wide and 20.5- 25 cm in maximum height. The sides, floor and top of the cages were of epoxy-coated wire mesh.
- Caging: Birds were paired and kept in cages for acclimation, dosing, and post-dosing periods.

NO. OF BIRDS PER REPLICATE
- For vehicle and capsule control: 20 (10 per sex)
- For treated: 10 (5 per sex)

NO. OF REPLICATES PER GROUP
- For vehicle and capsule control: 1
- For treated: 1

TEST CONDITIONS
- Temperature: Temperature was maintained between 19 and 24 °C, with an average temperature of 21 °C and a standard deviation of 1 °C.
- Relative humidity: Relative humidity was maintained between 50 and 78 %, with an average of 63 % and a standard deviation of 7 %.
- Photoperiod: Light was provided by fluorescent lights that closely approximate natural sunlight spectrum.

DOSE PREPARATION AND METHODOLOGY
The test material was mixed into a solution using acetone as a vehicle and the requisite amount, per bird, placed into a capsule. The 2000 mg/kg treatment group birds received the test material in two capsules due to the quantity required for the dose. The acetone was evaporated and the bird dosed one time with the capsule(s). Control birds were dosed with one capsule having evaporated acetone. The solution was magnetically stirred to provide even dissolution.

RANGE FINDING STUDY
- Test concentrations: 0, 125, 250, 500, 1000 and 2000 mg/kg bw
- Test conditions: The range-finder was conducted on the same manner as the definitive study, with the exception that 3 animals per sex were exposed to each dose level (6 animals per sex in the control group).
- Results used to determine the conditions for the definitive study: Yes

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Study animals were observed for signs of toxicity and mortality approximately every two hours during the first six hours post dosing and one time just before the end of the first 24 hour interval. They were observed twice daily thereafter except during weekends within the observation period when they were observed once daily.

BODY WEIGHT
- Time schedule for examinations: All test birds were weighed at the start of acclimation, on Day 0 just before dosing, Day 7 and on Day 14.
- Remarks: Body weights for each weighing event were statistically compared among groups for differences when possible.

FOOD CONSUMPTION
- Time schedule for examinations: Feed consumption was measured in each dose group and the control group for four intervals during the study, which consisted of Days: 0 through 1; 1 through 3; 3 through 7; and 7 through 14.
- Remarks: Feed spillage was controlled and visually monitored but was not calculated. The group feed consumption for each period was divided by the number of live birds in the dose group per day to calculate mean daily feed consumption per bird.

PATHOLOGY
- Dose groups that were examined: For the definitive phase, test birds were euthanised by carbon dioxide asphyxiation at the end of day 14 post-dosing. Post-mortem examinations were conducted immediately after euthanasia on four birds (2 male and 2 female) from each dosing group, and four birds from the control group (2 male and 2 female) determined by computerized random number generation of the surviving birds.

REGURGITATION
- Test material was regurgitated: No

Reference substance (positive control):

no

Key result

Duration (if not single dose):

14 d

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Mortality and sub-lethal effects:

MORTALITY / CLINICAL SIGNS
No mortality occurred in the range finder or in the definitive phase of the test.
There were no notations of abnormal behaviour, toxicity or morbidity in the behavioural observations during the range finder or during the definitive phase of the test.

BODY WEIGHT
There were no significant differences detected.

FEED CONSUMPTION
Consumption was fairly even across the groups for the four feeding intervals measured.

POST-MORTEM OBSERVATIONS
There were no abnormal findings in the post-mortem examinations.

Reported statistics and error estimates:

Statistical tests for this study were conducted using TOXSTAT Version 3.4 (West Inc. Cheyenne, WY).
For the range finder, individual body weights of male and female bobwhite were separately tested during three periods for normality, homogeneity and mean differences between treatment groups and the control group.
For the definitive phase, mean body weights of male and female bobwhite were separately tested during four time periods for normality, homogeneity, and mean differences between treatment groups and the control group.
There were no mortalities. An LD50 was, therefore, not calculated statistically.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the LD50 was determined to be >2000 mg/kg bw.

Executive summary:

A study was conducted to assess the acute oral toxicity of the test material to the Northern Bobwhite in accordance with the standardised guideline EPA OPP 71-1 under GLP conditions.

Colinus virginianus were exposed to dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg bw, administered as a single oral dose via capsule. The study involved dosing five groups (one group per dose level) of ten Northern Bobwhite, each containing five males and five females, and a control group (dosed with an empty capsule) of twenty Northern Bobwhite, containing ten males and ten females.

The birds were monitored for survival and behavioural signs of intoxication. The birds' feed consumption was measured over four intervals during the study, consisting of Days: 0 through 1; 1 through 3; 3 through 7; and 7 through 14. Their body weights were measured pre-dosing (Day 0) and on Days 7 and 14 post-dosing. These data were analysed to test for effects induced by exposure to the test material.

No mortality occurred and there were no notations of abnormal behaviour, toxicity or morbidity in the behavioural observations. No significant differences in body weight were detected and feed consumption was fairly even across the groups for the four feeding intervals measured. There were no abnormal findings in the post-mortem examinations.

Under the conditions of this study the LD50 was determined to be >2000 mg/kg bw.

Reference 2

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2001 - 10 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 205 (Avian Dietary Toxicity Test)

Deviations:

yes

Remarks:

- see below

Qualifier:

according to guideline

Guideline:

EPA OPP 71-2 (Avian Dietary Toxicity Test)

Deviations:

yes

Remarks:

- see below

Principles of method if other than guideline:

The guidelines recommend that each individual is given 300 cm² of floor space. During this study, 195 cm² floor space was provided per individual. It was the opinion of the study director that this deviation did not affect the results or interpretation of the test.

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: Yes
- Preparation of doses: Test diets were prepared by mixing the test material (dissolved in acetone) directly into the feed using a Hobart mixer. A predetermined amount of basal diet was measured out for each dosing level. Calculations were made to determine the amount of test material to be added to each dosing level mix in order to obtain the required test concentration. Calculated weights of the test material were increased by approximately 3 % to compensate for anticipated loss during mixing. For each level, the test material was first dissolved in approximately 50 mL acetone. The container holding the pre- weighed test material was rinsed twice with approximately 5 mL of acetone (10 mL total). This slurry was then added directly to the basal diet and mixed thoroughly for approximately 20 minutes. The solvent was completely evaporated prior to feeding. Acetone (50 mL) was added to the control diet and subsequently evaporated completely prior to feeding. The control diet contained no test material. In order to prepare a sufficient volume of feed, the control diet was prepared three times during testing.
- Storage of test diet: Test diets were stored throughout the exposure in a freezer at a temperature of below -10 °C to prevent degradation.

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
Homogeneity and stability (animal room and freezer) of the test diet were determined prior to initiation of the definitive test (presentation of the test diet to the birds). A homogeneity and stability determination was performed for the low and high dietary concentration.
Samples for homogeneity verification were removed at the time of mixing from the low and high dietary treatment levels. One sample was removed from each treatment at the top, middle and bottom of each batch of food.
A subsample of the mixed feed (low and high treatment level) to be used for the freezer stability study was sent for analysis along with the homogeneity samples. These subsamples were stored in a freezer (<-10 °C) and sampled (two samples per treatment level) and analysed on days 6 and 14. Animal room diet stability samples were placed in uniquely identified animal feeders and exposed in the animal room. They were placed in such a location as to avoid cross contamination. There were no animals in the room during the stability test. Samples (two) were collected on day 5 (at the end of day) for chemical analysis to determine the stability of the test material in the avian diet at ambient animal room temperature.

TEST DIET CONCENTRATION VERIFICATION
Test diets were analysed to confirm proper dietary concentrations of the test material prior to presentation to the birds. Analysis was performed for all dietary levels and the control. Test diets were used only if the recoveries were within 20 % of the stated target concentration. Two individual samples were collected for each treatment level, consisting of a subsample from the top, middle and bottom of the batch of feed, which was then homogenised.

ANALYTICAL METHODS
All homogeneity, stability and verification samples, including QC samples, were analysed using a high performance liquid chromatography (HPLC) procedure based on methodology validated at the testing facility. The method validation study was conducted prior to the initiation of the definitive test and established an average recovery of 100 ± 5.00 % for the test material from avian feed. Conditions and procedures used throughout the analysis of feed and QC samples during this study were similar to those used in the method validation study.

RESULTS
CONFIRMATION OF TEST MATERIAL HOMOGENEITY AND STABILITY
Samples removed from the 5200 and 325 mg a.i./kg dietary concentrations prior to test initiation ranged from 95 to 110 % of the nominal dietary concentrations, demonstrating the homogeneity of the diet. Analysis of the three quality control samples resulted in measured concentrations which ranged from 87.5 to 100 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed).
Analysis of the samples of treated frozen feed removed on day 6 and day 14 resulted in recoveries (based on nominal concentrations) ranging from 101 to 113 % and demonstrated stability over 14 days. When stored at ambient temperature (animal room) analyses demonstrated stability for 5 days, based on nominal concentrations. Recoveries were 95.5 and 110 % of nominal. Based on these results, the dietary concentrations were determined to be stable through the test period. Analysis of the three QC samples resulted in measured concentrations which ranged from 92.2 to 96.7 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed).

TEST MATERIAL VERIFICATION
The measured concentrations ranged from 89 to 101 % of nominal concentrations and defined the treatment levels tested as 320, 580, 1200, 2500 and 5300 mg a.i./kg feed.
Analysis of the quality control samples resulted in measured concentrations which ranged from 90.3 to 101 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed). Based on the results of these analyses, it was established that the appropriate quality control was maintained during the analyses of the exposure solutions.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern Bobwhite Quail
- Age at test initiation: Quail were 13 days old at experimental start
- Sexes used: Test animals were of indeterminate sex, since they lack sexual dimorphism at 11 to 13 days of age.
- Cultural background: Birds were phenotypically indistinguishable from wild stock and were hatched at the testing facility
- Disease free: Yes
- Kept according to standard practices: Yes

Limit test:

no

Total exposure duration (if not single dose):

5 d

Post exposure observation period:

5 days

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

16 animals per dose group; sex was not determined

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

- Nominal concentrations: 0 (Control), 325, 650, 1300, 2600 and 5200 mg a.i./kg feed
- Mean measured concentrations: 0 (Control), 320, 580, 1200, 2500 and 5300 mg a.i./kg feed

Details on test conditions:

ACCLIMATION
- Acclimation period: 13 days
- Acclimation conditions (same as test or not): Yes. Prior to testing, the quail were housed in brooder units constructed of epoxy-coated, galvanized, welded-wire cages under a photoperiod of 14 hours light, 10 hours dark per 24 hour interval. Radiant heaters were used on a 24 hour basis and maintained brooder temperatures ranging from 79 to 104 °F (26 to 40 °C).
- Feeding: The quail were fed a basal diet and provided water ad libitum, daily.
- Health (any disease or mortality observed): During the last three days of acclimation (i.e., 72 hours prior to the initiation of the test), the test animals were observed for signs of illness, disease or mortality. Birds used in the test were in apparent good health. There were no mortalities in the 72 hour period preceding the start of treated feed.
- Fasting period before study: No

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The toxicity test was conducted in brooder units consisting of cages that were adjoining, but not stacked, in two sets of a 4-square pattern. The cages were constructed of epoxy-coated wire and were approximately 40 cm width x 82 cm length x 26 cm height, and were arranged to eliminate the chance of cross-contamination among groups, dose levels and controls.
- Compliant to good husbandry practices: Yes
- Suitable to avoid crowding stress: Yes
- Caging: individual / group: Group

NO. OF BIRDS PER REPLICATE
- For vehicle control: 16
- For treated: 16

NO. OF REPLICATES PER GROUP
- For vehicle control: 3
- For treated: 1

TEST CONDITIONS
- Brooder temperature: The brooder units were designed to maintain test temperatures ranging from 95 to 100 °F (35 to 38 °C). These temperatures cover the entire brooder, thus include areas directly under the heating element and areas away from the heating elements. The test birds move about the brooder cage to seek their desired level of comfort.
- Room temperature: 80 to 99 °F
- Humidity: 47 to 86 %
- Photoperiod: Exposure cages were maintained throughout the test in an area illuminated by GE fluorescent bulbs at an average light intensity of not less than 6 footcandles at pen level. The photoperiod was 14 hours light and 10 hours of darkness. Sudden transitions from light to dark and vice versa were avoided. Light intensity of the test area ranged from 11 to 23 footcandles.

RANGE FINDING STUDY: No

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Each animal was observed for mortality, morbidity and intoxication three times on the initial day of exposure and twice daily (early in the light cycle and late in the light cycle) thereafter until test termination.
- Remarks: Observations were made to note mortality and any signs of morbidity or other symptoms of intoxication. Typical signs of intoxication are considered those behaviours apparently due to ingestions of the test material and may include a wide array of behaviours, such as wing droop, ataxia, laboured respiration, leg weakness, convulsions, and pilo-erection. All signs of intoxication and any other abnormal behaviour, such as excessive aggression, toe-picking, etc. that may or may not have been attributed to the test material were also recorded, if observed. Following the removal of treated feed, remission of signs of intoxication and cessation of abnormal behaviour were recorded, if observed, for surviving organisms for dose level and observation day. The time at which each animal was found dead was recorded if observed as well as the apparent cause of death, if it appeared that the cause of death was unrelated to test material exposure.

BODY WEIGHT
- Time schedule for examinations: Body weight was measured for each test animal within 24 hours prior to the start of the test (start of treated feed) to ensure a homogeneous distribution of mean cage weights. Body weight was also measured on test days 5 and 10.
- Remarks: Prior to the start of treated feed, statistical comparisons were made among groups to ensure that all groups exhibited similar mean weights and that body weights had similar variances among groups.

FOOD CONSUMPTION (if feeding study)
- Time schedule for examinations: Feed consumption was measured approximately every 24 hour period. Spillage was not measured, but was visually monitored.
- Remarks: Feed consumption per bird per day was calculated for each of the ten days. Feed consumption/bird-day was calculated for the exposure and post-exposure periods by dividing the grams of feed removed from a feed container by the total number of bird-days for the cage.

PATHOLOGY
- Dose groups that were examined: A post-mortem examination was performed as soon as possible on all birds found dead during the test. In addition, four birds that survived the duration of the test were indiscriminately selected from each cage and received post-mortem examinations at test termination.
- Remarks: Gross examinations included examinations of the general physical condition, digestive tract, pancreas, lungs, liver, kidneys, heart, spleen, testes and ovaries.

REGURGITATION
- Test material was regurgitated: Not observed

Reference substance (positive control):

no

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LC50

Effect level:

4 100 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 3200 to 5000 mg a.i./kg feed

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

320 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

other: mortality and weight gain

Duration (if not single dose):

5 d

Dose descriptor:

other: Lowest Lethal Concentration (LLC)

Effect level:

580 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Mortality and sub-lethal effects:

MORTALITY AND CLINICAL SIGNS
The survival data and sub-lethal effects are presented in Table 1. No abnormal behaviour or mortality was observed among birds in the control or the 320 mg a.i./kg feed groups. In the 580 mg a.i./kg feed group, one bird died on day 5. Two birds demonstrated wing droop and diminished mobility on day 5. Both birds recovered by day 7. Total mortality in the 580 mg a.i./kg feed group was one bird. For the 1200 mg a.i./kg feed group, wing droop in one bird was first noted on day 4 and was noted for a second bird on day 5. One mortality was recorded on day 6 and another on day 7. Total mortality in the 1200 mg a.i./kg feed group was two birds. In the 2500 mg a.i./kg feed group, wing droop and diminished mobility in two birds was observed on days 3 and 4, and one was recorded on day 4. Two birds demonstrated wing droop and/or diminished mobility again on day 5 and 6 with three additional mortalities recorded by day 6. Total mortality in the 2500 mg a.i./kg feed group was four birds. For the 5300 mg a.i./kg feed group, on day 3 observations were made for six birds demonstrating wing droop and sluggish movement, and on day 4, four birds died. Mortality, wing droop and diminished mobility continued to be observed in this group through day 7. Total mortality in the 5300 mg a.i./kg feed group was 11 birds.

In summary, mortalities observed in the control, 320, 580, 1200, 2500 and 5300 mg a.i./kg feed groups were 0, 0, 1, 2, 4 and 11 birds, respectively. Mortality first occurred in the 5300 and 2500 mg a.i./kg feed group on the 4th day from the start of treated feed (day 0). Mortality was at its maximum on day 5 with birds succumbing in the 580, 2500 and 5300 mg a.i./kg feed groups.
On day 6, mortality occurred in the 1200, 2500 and 5300 mg a.i./kg feed groups and on day 7, mortality occurred in the 1200 and 5300 mg a.i./kg feed groups. The mortality pattern demonstrated the lower concentration groups to be the least affected, affected later in time and for a briefer period. The only exception to this pattern was the one bird in the 580 mg a.i./kg feed group that died on day 5 having had no abnormal behavioural observations noted.

BODY WEIGHT
Body weight summaries are presented in Table 2. Mean group body weights did increase during the treated feed portion of the study for all groups with the exception of the 5300 mg group which demonstrated significant weight loss. All treatment groups increased in weight over the 10 day test period. Recovery was made by the surviving birds in the 5200 mg a.i./kg feed group between day 5 and day 10.
Prior to the start of treated feed (day 0), the body weights of all groups of test birds were measured. All data sets were normally distributed and had homogeneous variances. No significant differences were observed between any of the treatment or control group of birds prior to the start of treated feed.

FEED CONSUMPTION
Daily feed consumption data is presented in Table 3. Feed consumption for the higher treatment groups was diminished compared to the controls and lower treatment groups at the onset of start of treated feed. By the end of the five-day feeding period, the 5300 mg a.i./kg feed group was barely eating. Their water consumption was also at a minimum.
At the end of treated feed on day 5 and at the start of clean feed, consumption picked up noticeably. The 5300 mg a.i./kg feed group was the slowest to recover but did markedly increase consumption as well as demonstrate a noticeable weight gain.

PATHOLOGY
Post-mortem findings (Table 4) on all birds that succumbed during the test included abnormal findings for the intestinal tract. In addition, most birds that succumbed were emaciated and dehydrated. The intestinal wall disintegration was evidenced more severely in birds within the higher treatment levels. One bird in the 5300 mg a.i./kg feed treatment group had intestines which were not discernible, having no definition. Many birds were noted to have black to brownish bile in their intestinal tract. Birds that survived the test were noted to be within normal limits with the following exceptions. A few birds were noted to have gaseous intestines, including one control bird.

Reported statistics and error estimates:

Body weight data taken prior to the start of treated feed were analysed using Bartlett’s test for homogeneity of variance among cages. The mean cage body weights were compared using two-way analysis of variance to test for differences between groups.
The measured diet concentrations tested and the corresponding mortality data derived from the toxicity test were used to estimate the median lethal concentrations (LC50) and 95 % confidence intervals. If at least one test concentration caused mortality of 50 % of the test population, then a computer program (TOXSTAT, 1994) was used to calculate the LC50 values and 95 % confidence intervals. Probit analysis was used to calculate the LC50 value.
Statistical analyses (TOXSTAT, 1994) were conducted to determine whether significant (p<0.05) differences existed between the control group mean and any of the treatment group means for body weights (percent change). Data sets (days 0 to 5 and days 0 to 8) were first tested for normality using Chi-square Test (Weber, 1989) and for homogeneity of variance using Bartlett’s Test (Weber, 1989). If the data sets passed the test for homogeneity and normality, Bonferroni’s Test (Weber, 1989) was used to determine the NOEC. If the data sets did not pass the test for homogeneity and normality, Kruskal-Wallis Test (Sokal and Rohlf, 1981) was used to determine the NOEC. These tests were selected in place of Dunnett’s Test (Dunnett, 1965) because there were unequal sample sizes (i.e., at start of treated feed study, control N = 48, treatment N = 16 in each group). The results of these analyses, in combination with the mortality data were used to define the No-Observable-Effect Concentration (NOEC) for percent change in body weight (weight gain).

Table 1: Summary of Survival data

Dose group (mg a.i./kg feed)	Cumulative Percent Survival
	Day 0	Day 1	Day2	Day 3	Day 4	Day 5
Control	100	100	100	100	100	100
320	100	100	100	100	100	100
580	100	100	100	100	100	94¹
1200	100	100	100	100	100²	100³
2500	100	100	100	100¹	94¹	88⁴
5300	100	100	100	100⁵	75⁶	50⁷

¹ Two birds exhibited wing droop and were lethargic.

² One bird exhibited wing droop.

³ One bird exhibited wing droop. One bird exhibited wing droop and was not walking.

⁴ Several birds exhibited wing droop. One bird was lethargic.

⁵ Several birds exhibited wing droop and were lethargic.

⁶ One bird exhibited wing droop. All birds were not drinking water. Several birds were lethargic.

⁷ Several birds exhibited wing droop. Several birds were not walking. One bird was lying still.

 

Table 2: Summary of Mean Body Weight Data (g)

Dose group (mg a.i./kg feed)	Day 0 (Start of Treated Feed)	Day 5 (End of Treated Feed)	Change (g)	% Change	Day 10 (Termination)	Total Change (g)	% Change
Control	26.5	44.8	18.4	69	58.3	31.8	120
320	25.4	43.4	18.1	71	56.8	31.4	124
580	26.0	36.0	10.1	39*	50.6	24.7	95**
1200	25.4	36.1	10.7	42*	56.0	30.5	120
2500	24.7	28.5	3.7	15*	45.5	20.8	84**
5300	24.8	20.0	-4.8	-19*	36.4	11.6	47**

* Significantly reduced compared to the control, based on Kruskal-Wallis' Test.

** Significantly reduced compared to the control, based on Bonferroni's t-Test.

 

Table 3: Feed Consumption by Cage per Bird (g)

Dose group (mg a.i./kg feed)	Cage Number	Day 1	Day 2	Day 3	Day 4	Day 5*	Day 6	Day 7	Day 8	Day 9	Day 10
Control	2	4.1	5.7	6.8	6.0	9.4	5.3	8.8	7.3	8.8	9.8
Control	6	4.4	6.3	6.6	5.6	10.5	6.1	7.6	7.0	9.8	10.0
Control	8	4.0	6.1	6.5	5.3	9.9	5.7	6.6	7.4	9.6	9.7
320	4	4.6	5.8	6.5	6.7	10.3	6.5	8.2	7.6	9.6	9.4
580	5	4.1	5.2	5.1	4.3	7.4	5.4	7.1	7.3	9.4	9.7
1200	1	4.2	5.3	5.5	5.3	9.4	5.5	8.7	8.8	8.8	10.0
2500	3	4.2	4.4	3.3	3.2	7.3	3.7	7.5	9.7	8.0	7.8
5300	7	3.2	2.5	3.1	2.5	3.3	2.8	5.0	7.5	6.8	8.0

* The Day 5 feed measurement was taken later in the day than on other days and therefore reflects additional hours of consumption for Day 5 and fewer than 24 hours of consumption for Day 6.

 

Table 4: Summary of Post-mortem Observations for Animals That Died

Dose group (mg a.i./kg feed)	ID Number	Observation
650	2893	White crusty material on rear end and sternum area. Some food in crop and mucous present. Deteriorated intestines filled with brownish bile.
1300	2921	Emaciated and dehydrated. Blood haemorrhage around beak top and bottom. Dark blackish bile in crop. Deteriorated intestines, black in colour.
	2825	Emaciated and dehydrated. Appears that intestinal tract is backed-up.
2600	2891	Emaciated and dehydrated. Blood in nostrils. Blood/haemorrhage apparent on lower beak. Deteriorated intestines. Intestines mucous coated and brownish in colour. Enlarged gall bladder.
	2835	Emaciated and dehydrated. Crop empty. Deteriorated intestines, greyish black in colour. Gall bladder filled with green liquid.
	2907	Emaciated and dehydrated. Severely deteriorated intestines and intestinal tract. Brownish bile present.
	2827	Emaciated and dehydrated. Severely deteriorated intestinal tract.
5200	2903	Emaciated, dehydrated, deteriorated intestines
	2879	Emaciated, dehydrated, deteriorated intestines
	2935	Emaciated, dehydrated, deteriorated intestines
	2823	Emaciated, dehydrated, deteriorated intestines - black
	2863	Crop filled with mucous. Severely deteriorated intestines.
	2887	Emaciated and dehydrated. Severely deteriorated intestines - black in colour.
	2959	Emaciated and dehydrated. Dark brown bile in crop. Deteriorated black intestines.
	2839	Emaciated and dehydrated. Deteriorated intestines.
	2855	Emaciated and dehydrated. Lungs dark. Severely deteriorated intestines and deteriorated intestinal system. Not intact. Black bile present. Organs not discernable. Liver dark in colour.
	2807	Severely deteriorated intestinal tract.
	2943	Emaciated and dehydrated. Gaseous intestines/appears to be backing up intestinal tract.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the NOECs for mortality and percent of weight change (weight gain) was determined to be 320 mg a.i./kg feed, the lowest measured dietary concentration tested. The LC50 was calculated to be 4100 mg a.i./kg feed (95 % CI 3200 to 5000 mg a.i./kg feed). The lowest lethal concentration (LLC) was determined to be 580 mg a.i./kg feed.

Executive summary:

The acute dietary toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 205 and EPA OPP 71-2 under GLP conditions.

Northern Bobwhite Quail (Colinus virginianus) were exposed to the test material in the feed at five dosing levels: 325, 650, 1300, 2600 and 5200 mg a.i./kg feed, along with a control. Each treatment group contained 16 birds and the control group contained 48 birds (16 per cage). The birds were fed treated feed for 5 days (5 twenty-four hour intervals). Non-treated feed was then provided for five additional days (total test duration: 10 days). The animals were observed for mortality, morbidity and intoxication, along with body weight changes and the amount of feed consumed. A post-mortem examination was performed on all birds found dead during the test. In addition, four birds that survived the duration of the test were indiscriminately selected from each cage and received post-mortem examinations at test termination.

Mortalities were observed in the four highest treatment levels during the study. Percent survival at test termination (day 10) in the 320, 580, 1200, 2500 and 5300 mg a.i./kg feed treatment levels was 100, 94, 88, 75 and 31 %, respectively. Mean group body weights did increase during the treated feed portion of the study for all groups with the exception of the 5300 mg a.i./kg feed group which demonstrated significant weight loss. Surviving animals in all treatment groups increased in weight over the 10-day test period. Feed consumption for the higher treatment groups was diminished compared to the controls and lower treatment groups at the onset of the treated feed. By the end of the five-day exposure period, the 5300 mg a.i./kg feed group was noticeably reduced. Their water consumption was also at a minimum. Following the exposure period on day 5, at the presentation of non-treated feed, consumption picked up noticeably. The 2500 and 5300 mg a.i./kg feed groups were the slowest to recover but markedly increased their consumption and demonstrated a noticeable weight gain.

Under the conditions of this study, the NOECs for mortality and percent of weight change (weight gain) was determined to be 320 mg a.i./kg feed, the lowest measured dietary concentration tested. The LC50 was calculated to be 4100 mg a.i./kg feed (95 % CI 3200 to 5000 mg a.i./kg feed). The lowest lethal concentration (LLC) was determined to be 580 mg a.i./kg feed.

Reference 3

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2001 - 10 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 205 (Avian Dietary Toxicity Test)

Deviations:

yes

Remarks:

- see below

Qualifier:

according to guideline

Guideline:

EPA OPP 71-2 (Avian Dietary Toxicity Test)

Deviations:

yes

Remarks:

- see below

Principles of method if other than guideline:

The guidelines recommend that each individual is given 600 cm² of floor space. During this study, 426 cm² floor space was provided per individual. It was the opinion of the study director that this deviation did not affect the results or interpretation of the test.

GLP compliance:

yes

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: Yes
- Preparation of doses: Test diets were prepared by mixing the test material (dissolved in acetone) directly into the feed using a Hobart mixer. A predetermined amount of basal diet was measured out for each dosing level. Calculations were made to determine the amount of test material to be added to each dosing level mix in order to obtain the required test concentration. Calculated weights of the test material were increased by approximately 3 % to compensate for anticipated loss during mixing. For each level, the test material was first dissolved in approximately 50 mL acetone. The container holding the pre- weighed test material was rinsed twice with approximately 5 mL of acetone (10 mL total). This slurry was then added directly to the basal diet and mixed thoroughly for approximately 20 minutes. The solvent was completely evaporated prior to feeding. Acetone (50 mL) was added to the control diet and subsequently evaporated completely prior to feeding. The control diet contained no test material. In order to prepare a sufficient volume of feed, the control diet was prepared three times during testing.
- Storage of test diet: Test diets were stored throughout the exposure in a freezer at a temperature of below -10 °C to prevent degradation.

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
Homogeneity and stability (animal room and freezer) of the test diet were determined prior to initiation of the definitive test (presentation of the test diet to the birds). A homogeneity and stability determination was performed for the low and high dietary concentration.
Samples for homogeneity verification were removed at the time of mixing from the low and high dietary treatment levels. One sample was removed from each treatment at the top, middle and bottom of each batch of food.
A subsample of the mixed feed (low and high treatment level) to be used for the freezer stability study was sent for analysis along with the homogeneity samples. These subsamples were stored in a freezer (<-10 °C) and sampled (two samples per treatment level) and analysed on days 6 and 14. Animal room diet stability samples were placed in uniquely identified animal feeders and exposed in the animal room. They were placed in such a location as to avoid cross contamination. There were no animals in the room during the stability test. Samples (two) were collected on day 5 (at the end of day) for chemical analysis to determine the stability of the test material in the avian diet at ambient animal room temperature.

TEST DIET CONCENTRATION VERIFICATION
Test diets were analysed to confirm proper dietary concentrations of the test material prior to presentation to the birds. Analysis was performed for all dietary levels and the control. Test diets were used only if the recoveries were within 20 % of the stated target concentration. Two individual samples were collected for each treatment level, consisting of a subsample from the top, middle and bottom of the batch of feed, which was then homogenised.

ANALYTICAL METHODS
All homogeneity, stability and verification samples, including QC samples, were analysed using a high performance liquid chromatography (HPLC) procedure based on a methodology validated at the testing facility. The method validation study was conducted prior to the initiation of the definitive test and established an average recovery of 100 ± 5.00 % for the test material from avian feed. Conditions and procedures used throughout the analysis of feed and QC samples during this study were similar to those used in the method validation study.

RESULTS
CONFIRMATION OF TEST MATERIAL HOMOGENEITY AND STABILITY
Samples removed from the 5200 and 325 mg a.i./kg dietary concentrations prior to test initiation ranged from 95 to 110 % of the nominal dietary concentrations, demonstrating the homogeneity of the diet. Analysis of the three quality control samples resulted in measured concentrations which ranged from 87.5 to 100 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed).
Analysis of the samples of treated frozen feed removed on day 6 and day 14 resulted in recoveries (based on nominal concentrations) ranging from 101 to 113 % and demonstrated stability over 14 days. When stored at ambient temperature (animal room) analyses demonstrated stability for 5 days, based on nominal concentrations. Recoveries were 95.5 and 110 % of nominal. Based on these results, the dietary concentrations were determined to be stable through the test period. Analysis of the three QC samples resulted in measured concentrations which ranged from 92.2 to 96.7 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed).

TEST MATERIAL VERIFICATION
The measured concentrations ranged from 89 to 101 % of nominal concentrations and defined the treatment levels tested as 320, 580, 1200, 2500 and 5300 mg a.i./kg feed.
Analysis of the quality control samples resulted in measured concentrations which ranged from 90.3 to 101 % of the nominal fortified levels (211 to 6320 mg a.i./kg feed). Based on the results of these analyses, it was established that the appropriate quality control was maintained during the analyses of the exposure solutions.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard duck
- Age at test initiation: Ducks were 9 days old at experimental start
- Sexes used: Test animals were of indeterminate sex, since they lack sexual dimorphism at 7 to 9 days of age.
- Cultural background: Birds were phenotypically indistinguishable from wild stock, and were from a single hatch one day of age when received at the testing facility
- Disease free: Yes
- Kept according to standard practices: Yes

Limit test:

no

Total exposure duration (if not single dose):

5 d

Post exposure observation period:

3 days

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

12 animals per dose group; sex was not determined

Control animals:

yes, concurrent vehicle

Nominal and measured doses / concentrations:

- Nominal concentrations: 0 (Control), 325, 650, 1300, 2600 and 5200 mg a.i./kg feed
- Mean measured concentrations: 0 (Control), 320, 580, 1200, 2500 and 5300 mg a.i./kg feed

Details on test conditions:

ACCLIMATION
- Acclimation period: 8 days
- Acclimation conditions (same as test or not): Yes. Prior to testing, the ducks were housed in brooder units constructed of epoxy-coated, galvanized, welded-wire cages under a photoperiod of 14 hours light, 10 hours dark per 24 hour interval. Radiant heaters were used on a 24 hour basis and maintained brooder temperatures ranging from 73 to 100 °F (23 to 38 °C).
- Feeding: The ducks were fed a basal diet and provided water ad libitum, daily.
- Health (any disease or mortality observed): During the last three days of acclimation (i.e., 72 hours prior to the initiation of the test), the test animals were observed for signs of illness, disease or mortality. Birds used in the test were in apparent good health. There were no mortalities in the 72 hour period preceding the start of treated feed.
- Fasting period before study: No

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The toxicity test was conducted in brooder units consisting of adjoining cages aligned in a straight line, with a heating element overhead. The cages were constructed of polycarbonate-coated wire and were approximately 36 cm width x 142 cm length x 45 cm height, and were arranged to eliminate the chance of cross-contamination among groups, dose levels and controls.
- Compliant to good husbandry practices: Yes
- Suitable to avoid crowding stress: Yes
- Caging: individual / group: Group

NO. OF BIRDS PER REPLICATE
- For vehicle control: 12
- For treated: 12

NO. OF REPLICATES PER GROUP
- For vehicle control: 3
- For treated: 1

TEST CONDITIONS
- Brooder temperature: The brooder units were designed to maintain test temperatures ranging from 89.6 to 95 °F (32 to 35 °C). These temperatures cover the entire brooder, thus include areas directly under the heating element and areas away from the heating elements. The test birds move about the brooder cage to seek their desired level of comfort.
- Room temperature: 76 to 89 °F (24 to 32 °C)
- Humidity: 53 to 69 %
- Photoperiod: Exposure cages were maintained throughout the test in an area illuminated by full sunlight-spectrum fluorescent bulbs at an average light intensity of not less than 6 footcandles at pen level. The photoperiod was 14 hours light and 10 hours of darkness. Sudden transitions from light to dark and vice versa were avoided. Light intensity of the test area ranged from 6.9 to 23 footcandles.

RANGE FINDING STUDY: No

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Each animal was observed for mortality, morbidity and intoxication three times on the initial day of exposure and twice daily (early in the light cycle and late in the light cycle) thereafter until the deaths of all animals in the group or test termination.
- Remarks: Observations were made to note mortality and any signs of morbidity or other symptoms of intoxication. Typical signs of intoxication are considered those behaviours apparently due to ingestions of the test material and may include a wide array of behaviours, such as ataxia, laboured respiration, leg weakness, convulsions, and pilo-erection. All signs of intoxication and any other abnormal behaviour, such as excessive aggression, toe-picking, etc. that may or may not have been attributed to the test material were also recorded, if observed. Following the removal of treated feed, remission of signs of intoxication and cessation of abnormal behaviour were recorded, if observed, for surviving organisms for dose level and observation day. The time at which each animal was found dead was recorded if observed as well as the apparent cause of death, if it appeared that the cause of death was unrelated to test material exposure.

BODY WEIGHT
- Time schedule for examinations: Body weight was measured for each test animal within 24 hours prior to the start of the test (start of treated feed) to ensure a homogeneous distribution of mean cage weights. Body weight was also measured on test days 5 and 8.
- Remarks: Prior to the start of treated feed, statistical comparisons were made among groups to ensure that all groups exhibited similar mean weights and that body weights had similar variances among groups.

FOOD CONSUMPTION (if feeding study)
- Time schedule for examinations: Feed consumption was measured approximately every 24 hour period. Spillage was not measured, but was visually monitored.
- Remarks: Feed consumption was determined for days 0 to 5 (exposure period), and days 5 to 8 (post-exposure period). In addition, feed consumption per bird per day was calculated for each of the eight days. Feed consumption/bird-day was calculated for the exposure and post-exposure periods by dividing the grams of feed removed from a feed container by the total number of bird-days for the cage.

PATHOLOGY
- Dose groups that were examined: Four birds of those that survived the duration of the test were indiscriminately selected from each cage and received post-mortem examinations at test termination.
- Remarks: Gross examinations included examinations of the digestive tract, pancreas, lungs, liver, kidneys, heart, spleen, testes and ovaries.

REGURGITATION
- Test material was regurgitated: Not observed

Reference substance (positive control):

no

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LC50

Effect level:

> 5 300 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

5 300 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

320 mg/kg bw/day (actual dose received)

Conc. / dose based on:

act. ingr.

Basis for effect:

body weight

Duration (if not single dose):

5 d

Dose descriptor:

other: Lowest Lethal Concentration (LLC)

Effect level:

> 5 300 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Mortality and sub-lethal effects:

MORTALITY AND CLINICAL SIGNS
No mortalities were observed throughout the test, and therefore statistical analysis of mortality data was not performed. There were no observations made of any abnormal behaviour or signs of intoxication.

BODY WEIGHT
Body weight summaries are presented in Table 1. Body weights were measured after 5 days of exposure to treated feed. The percentage of weight gainfor all treatment groups except the 320 mg a.i./kg feed group, was significantly lower than that of the control group.
Final body weights were measured again 3 days following the end of the treated feed portion of the study (day 8). Statistical differences were detected between two treatment groups (1200 and 2500 mg a.i./kg feed) and the control group for the percentage of weight gained between day 0 (start of treated feed) and day 8 (end of test). The 1200 and 2500 mg a.i./kg feed treatment groups demonstrated significantly less weight gain (percent change) than the controls.
Prior to the start of treated feed (day 0), the body weights of all groups of test birds were measured. All data sets were normally distributed and had homogeneous variances. No significant differences were observed between the control body weights and body weights of any treatment group of birds prior to the start of treated feed.

FEED CONSUMPTION
Visual analysis of feed consumption data demonstrated a fairly even pattern of consumption between groups (Table 2). There was no clear evidence of any diminished feed consumption until day 5 of treated feed when the 2500 and 5300 mg a.i./kg feed groups demonstrated slightly decreased consumption compared to the controls. The 5300 mg a.i./kg feed group resumed feed consumption comparable to the controls after the presentation of clean feed; however, the individuals in the 2600 mg a.i./kg feed group were more erratic in their recovery. There was no evidence of feed aversion.

PATHOLOGY
During post-mortem examinations, all birds were noted to be within normal levels with no exceptions.

Reported statistics and error estimates:

Body weight data taken prior to the start of treated feed, were analysed using Bartlett’s test (or similar test) for homogeneity of variance among cages. The mean cage body weights were compared using one-way analysis of variance to test for differences among cages.
The measured diet concentrations tested and the corresponding mortality data derived from the toxicity test were used to estimate the median lethal concentrations (LC50) and 95 % confidence intervals. If at least one test concentration caused mortality of 50 % of the test population, then a computer program (Toxstat, 1994) was used to calculate the LC50 values and 95 % confidence intervals. Since no treatment level tested resulted in mortality, LC50 values were not calculated.
Statistical analyses (TOXSTAT, 1994) were conducted to determine whether significant (p<0.05) differences exist between the control group mean and any of the treatment group means for body weights (percent change). Data sets (days 0 to 5 and days 0 to 8) were first checked for normality using Chi-square Test (Weber, 1989) and for homogeneity of variance using Bartlett’s Test (Weber, 1989). If the data sets passed the test for homogeneity and normality, Bonferroni’s Test (Weber, 1989) was used to determine the NOEC. This test was selected in place of Dunnett’s Test (Dunnett, 1965) because there were unequal sample sizes (control N = 36, treatment N = 12 in each group). The results of these analyses, in combination with the mortality data were used to define the No-Observable-Effect Concentration (NOEC) for percent change in body weight (weight gain).

Table 1: Summary of Mean Body Weight Data (g)

Dose group (mg a.i./kg feed)	Day 0 (Start of Treated Feed)	Day 5 (End of Treated Feed)	Change (g)	% Change	Day 8 (Termination)	Total Change (g)	% Change
Control	123.9	273.7	149.8	120	322.9	199.0	161
320	120.8	260.0	139.2	115	326.8	206.0	171
580	127.1	255.0	127.9	101*	320.5	193.4	152
1200	122.1	236.1	114.0	93*	292.0	169.8	139**
2500	127.8	240.7	112.9	88*	301.9	174.1	136**
5300	121.4	221.0	99.5	82*	302.5	181.1	149

* A statistical difference was detected in the 650, 1300, 2600 and 5200 mg a.i./kg feed groups compared to the controls for percent change between start of treated feed and end of treated feed.

** A statistical difference was detected in the 1300 and 2600 mg a.i/kg groups compared to the controls for percentage change between start of treated feed and test termination. 

 

Table 2: Average Feed Consumption by Bird (g)

Dose group (mg a.i./kg feed)	Day 1	Day 2	Day 3	Day 4	Day 5	Average Days 0 to 5	Day 6	Day 7	Day 8	Average Days 6 to 8
Control	34.3	47.7	48.7	49.7	55.1	47.1	64.0	69.9	66.5	66.8
320	36.4	47.6	50.8	51.4	53.8	48.0	63.4	68.0	755	68.9
580	32.5	48.7	48.6	51.1	53.6	46.9	62.2	65.4	73.0	66.9
1200	33.2	48.4	45.3	50.1	50.8	45.5	61.6	65.5	74.2	67.1
2500	36.5	47.7	50.7	50.7	47.6	46.7	65.5	58.5	66.7	63.6
5300	32.9	46.7	51.0	48.8	47.5	45.4	63.3	73.5	71.8	69.5

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the NOEC for mortality was determined to be 5300 mg a.i./kg feed, the highest measured dietary concentration tested. The NOEC for percent of weight change (weight gain) was 320 mg a.i./kg feed. The LC50 was determined to be >5300 mg a.i./kg feed and the lowest lethal concentration (LLC) was also determined to be >5300 mg a.i./kg feed.

Executive summary:

The acute dietary toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 205 and EPA OPP 71-2 under GLP conditions.

Mallard ducks (Anas platyrhynchos) were exposed to the test material in the feed at five dosing levels: 325, 650, 1300, 2600 and 5200 mg a.i./kg feed, along with a control. Each treatment group contained 12 birds and the control group contained 36 birds (12 per cage). The birds were fed treated feed for 5 days (5 twenty-four hour intervals). Non-treated feed was then provided for three additional days (total test duration: 8 days).

The animals were observed for mortality, morbidity and intoxication, along with body weight changes and the amount of feed consumed. A post-mortem examination was performed at test termination on four birds that survived the duration of the test and were indiscriminately selected from each cage.

No mortalities were observed during the study.

After 5 days of exposure to treated feed, the weight gain (percent change) for all treatment groups except the 320 mg a.i./kg feed group, was significantly lower than that of the control group.

Between day 0 (start of treated feed) and day 8 (end of test), the 1200 and 2500 mg a.i./kg feed treatment groups demonstrated significantly less weight gain (percent change) than the controls.

Under the conditions of this study, the NOEC for mortality was determined to be 5300 mg a.i./kg feed, the highest measured dietary concentration tested. The NOEC for percent of weight change (weight gain) was 320 mg a.i./kg feed. The LC50 was determined to be >5300 mg a.i./kg feed and the lowest lethal concentration (LLC) was also determined to be >5300 mg a.i./kg feed.

Description of key information

In the Northern bobwhite quail, the LD50 was determined to be >2000 mg/kg bw  after a single exposure via capsule and the LC50 was calculated to be 4100 mg a.i./kg feed (95 % CI 3200 to 5000 mg a.i./kg feed) in a 5 day toxicity study in which the test material was administered in the diet.

In a further 5 day toxicity study in which the test material was administered via the diet to the mallard duck, the LC50 was determined to be >5300 mg a.i./kg feed.

Key value for chemical safety assessment

Short-term EC50 or LC50 for birds:

4 100 mg/kg food

Additional information

Acute Toxicity (Single Dose, Capsule)

A study was conducted to assess the acute oral toxicity of the test material to the Northern Bobwhite in accordance with the standardised guideline EPA OPP 71-1 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Colinus virginianus were exposed to dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg bw, administered as a single oral dose via capsule. The study involved dosing five groups (one group per dose level) of ten Northern Bobwhite, each containing five males and five females, and a control group (dosed with an empty capsule) of twenty Northern Bobwhite, containing ten males and ten females. The birds were monitored for survival and behavioural signs of intoxication. The birds’ feed consumption was measured over four intervals during the study, consisting of Days: 0 through 1; 1 through 3; 3 through 7; and 7 through 14. Their body weights were measured pre-dosing (Day 0) and on Days 7 and 14 post-dosing. These data were analysed to test for effects induced by exposure to the test material.

No mortality occurred and there were no notations of abnormal behaviour, toxicity or morbidity in the behavioural observations. No significant differences in body weight were detected and feed consumption was fairly even across the groups for the four feeding intervals measured. There were no abnormal findings noted during the post-mortem examinations.

Under the conditions of this study the LD50 was determined to be >2000 mg/kg bw.

Acute Toxicity (5 Days, Dietary)

There are two 5 day toxicity studies available, conducted in the Northern bobwhite quail and the mallard duck. Both studies were awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute dietary toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 205 and EPA OPP 71-2 under GLP conditions.

Northern Bobwhite Quail (Colinus virginianus) were exposed to the test material in the feed at five dosing levels: 325, 650, 1300, 2600 and 5200 mg a.i./kg feed, along with a control. Each treatment group contained 16 birds and the control group contained 48 birds (16 per cage). The birds were fed treated feed for 5 days (5 twenty-four hour intervals). Non-treated feed was then provided for five additional days (total test duration: 10 days). The animals were observed for mortality, morbidity and intoxication, along with body weight changes and the amount of feed consumed. A post-mortem examination was performed on all birds found dead during the test. In addition, four birds that survived the duration of the test were indiscriminately selected from each cage and received post-mortem examinations at test termination.

Mortalities were observed in the four highest treatment levels during the study. Percent survival at test termination (day 10) in the 320, 580, 1200, 2500 and 5300 mg a.i./kg feed treatment levels was 100, 94, 88, 75 and 31 %, respectively. Mean group body weights did increase during the treated feed portion of the study for all groups with the exception of the 5300 mg a.i./kg feed group which demonstrated significant weight loss. Surviving animals in all treatment groups increased in weight over the 10-day test period. Feed consumption for the higher treatment groups was diminished compared to the controls and lower treatment groups at the onset of the treated feed. By the end of the five-day exposure period, the 5300 mg a.i./kg feed group was noticeably reduced. Their water consumption was also at a minimum. Following the exposure period on day 5, at the presentation of non-treated feed, consumption picked up noticeably. The 2500 and 5300 mg a.i./kg feed groups were the slowest to recover but markedly increased their consumption and demonstrated a noticeable weight gain.

Under the conditions of this study, the NOECs for mortality and percent of weight change (weight gain) was determined to be 320 mg a.i./kg feed, the lowest measured dietary concentration tested. The LC50 was calculated to be 4100 mg a.i./kg feed (95 % CI 3200 to 5000 mg a.i./kg feed). The LLC was determined to be 580 mg a.i./kg feed.

The acute dietary toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 205 and EPA OPP 71-2 under GLP conditions.

Mallard ducks (Anas platyrhynchos) were exposed to the test material in the feed at five dosing levels: 325, 650, 1300, 2600 and 5200 mg a.i./kg feed, along with a control. Each treatment group contained 12 birds and the control group contained 36 birds (12 per cage). The birds were fed treated feed for 5 days (5 twenty-four hour intervals). Non-treated feed was then provided for three additional days (total test duration: 8 days). The animals were observed for mortality, morbidity and intoxication, along with body weight changes and the amount of feed consumed. A post-mortem examination was performed at test termination on four birds that survived the duration of the test and were indiscriminately selected from each cage.

No mortalities were observed during the study. After 5 days of exposure to treated feed, the weight gain (percent change) for all treatment groups except the 320 mg a.i./kg feed group, was significantly lower than that of the control group. Between day 0 (start of treated feed) and day 8 (end of test), the 1200 and 2500 mg a.i./kg feed treatment groups demonstrated significantly less weight gain (percent change) than the controls.

Under the conditions of this study, the NOEC for mortality was determined to be 5300 mg a.i./kg feed, the highest measured dietary concentration tested. The NOEC for percent of weight change (weight gain) was 320 mg a.i./kg feed. The LC50 was determined to be >5300 mg a.i./kg feed and the LLC was also determined to be >5300 mg a.i./kg feed.