spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Toxicity to birds

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to birds

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life initiated/competed: 11-Jan-2000 to 11-July-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This experimental study report is reliable for use in the risk assessment as it has been conducted by a trusted laboratory, under GLP and following EPA Guidelines relevant at the time of the study initiation. There are no significant deviations from the current guideline therefore the data in this study been deemed appropriate to use.

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Version / remarks:

FIFRA

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM standard E 1062-86

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Dose method:

homogenously mixed into feed (accounts for technical substances)

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

Preparation of test diet: The spirodiclofen technical was mechanically mixed with the adult feed using a commercial Hobart mixer. No vehicles or solvents were needed. Hatchlings were given clean feed only.

Description of basal diet: Harlan Teklad gamebird feed for adults and Harlan Teklad starter ration for hatchlings. All feed analysis indicated appropriate diet.

Stability and homogeneity of test material: Stability was acceptable under ambient lab conditions and storage conditions. Homogeneity was determined and was acceptable. All analyses confirmed stability and homogeneity. All measured concentrations were within 85 to 111% of the nominal concentration.

Verification by chemical analysis: Measured concentration of spirodiclofen in feed was determined for week 1, 5, 10, 15 and week 20.

TEST CONCENTRATIONS:
Nominal: control (raw feed only), 80, 240 and 720 mg/kg feed.
Measured: control (< 3.4), 79.4, 239 and 734 mg/kg feed.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

Age at study initiation: 23 weeks
Body weight recording: Recorded in week 1 (study initiation), week 3, week 5, week 7, week 9 and at termination.
Body weight (mean and range): The mean initial female body weight was 966 g + 70 g and the mean initial male body weight was 1093 g + 90 g in the control group.
Number of organisms: 72 pairs of mallards were received.
Acclimation period: Six weeks.
Feeding: food and water were provided ad libitum.

Limit test:

no

Total exposure duration (if not single dose):

20 wk

Remarks:

Pre-laying exposure: Pre-laying exposure was from January 11 to March 7, 2000 (week 1 to week 8).
Egg-laying exposure: Egg laying exposure was from March 8 to May 30, 2000 (week 9 - week 20).
No withdrawal period was necessary.

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

15 adult pens at each treatment level; 1 male and 1 female adult mallard per pen. Randomly assigned by body weight to each treatment level. Pen location was also randomly assigned. 15 pens for each treatment; 15 pens for control (raw feed). No solvent control was needed in this study.

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

Nominal: 80, 240 and 720 mg/kg feed.
Mean measured: 79.4, 239 and 734 mg/kg feed.

Details on test conditions:

Pens (for parental and offspring) size and construction:
Adults - Britz-Heidbrink adult mallard cages; stainless steel cages with plastic coated flooring. Pen dimension: 79L x 61W x 55H cm. 15 adult pens at each treatment level. All procedures and equipment were appropriate to prevent cross contamination. Birds identified by a unique ID number (leg band). Pens marked for test level, study number, band numbers and pen number.
Hatchlings - G.Q.F. Poultry/Gamebird box brooders made of galvanized metal. Pen dimension: 91L X 81W X 25H cm. Gang housed by parental treatment level and age. Approx. 30 birds per pen. Pen labeled for parental treatment, study number and hatch date.

Feeding and husbandry: Food and water was available to adults and hatchlings ad libitum. All husbandry procedures were appropriate and humane.

Adults - Adult cages were exchanged and thoroughly cleaned every third week; bedding pans and pens were rinsed clean at least 3 times a week.

Hatchlings - Hatchlings were housed in a brooder to allow a temperature gradient from brooder temp. to room temp.; brooder temperature was 29 to 38°C and the room temperature was 22 ± 2°C. Hatchling room photoperiod was 14 hours light and 10 hours dark. Bedding changed at least once per week.

Environmental Conditions: Mean room temperature was 21°C and humidity was 51% for duration of study. The photoperiod was 7 hours light and 17 hours dark for acclimation through study week 8 (pre-laying phase). The lights were changed in week 9 to 17 hours light and 7 hours dark (laying phase). Light intensity was measured 3 times during the study. Light intensity mean was 7.4 foot candles; minimum was 4 and maximum was 12 foot candles.

Details on examinations and observations:

Parental parameters measured: Adult endpoints were mortality, behavior, body weight (male and female), growth, feed consumption per pen, and gross pathology.

Egg collection and subsequent development: Egg endpoints included: eggs laid/hen, eggs set/hen, eggs cracked, eggshell strength and thickness, eggs defective, egg fertility/hen, embryo viability/hen.

Other measurements: no. hatched, no. of 14-day survivors, average weight of 14-day old survivors, mortality, gross pathology, others. Hatchling endpoints: hatched/hen, hatched of fertile eggs, hatched of viable embryos, 14-day survivors/hen, 14-day survivors of viable embryos/hen, hatchling body weight upon hatch, hatchling growth, hatchling behavior, and hatchling 14-day old body weight.

Regurgitation: no regurgitation was observed throughout the study.

Observation intervals: Adult body weights were taken every other week until the onset of the laying phase and the final body weights were taken upon sacrifice. Food consumption was determined weekly throughout the adult exposure period.

Details on reproductive parameters:

Egg collection and incubation: Collection was done twice daily on weekdays and once per day on holidays and weekends. All eggs were labeled, cleaned, disinfected and stored in an egg cooler on the day of collection. Egg cooler average temperature was 14°C and humidity was 80%.

Were eggs candled for cracks prior to setting for incubation?: Yes. Eggs were candled to assess cracks prior to setting in incubator (day 0). The incubator settings were appropriate. Incubator was an average 55% humidity and average temperature of 36°C.

Were eggs set weekly?: Yes. Eggs were set weekly except for the first set (lot A) where two weeks of eggs were combined. All other sets (lot B to lot K) were done weekly.

Was candling done for fertility?: Yes. Incubated mallard eggs were candled for fertility on day 14 and for 3-week embryo viability on Day 21.

When were the eggs transferred to the hatcher?: Eggs were transferred to the hatcher after 23 days of incubation. The eggs were in the hatcher until day 27 or day 28.

Hatching conditions: Hatcher conditions were appropriate. Average temperature was 37°C and average humidity was 65%.

Day the hatched eggs were removed and counted: Hatchlings were collected on day 27 and day 28. Any unhatched eggs remaining after collection on day 28 were recorded and discarded. The hatchlings were banded, weighed and transferred to the appropriate brooder pen.

Were egg shells washed and dried for at least 48 hrs before measuring?: Yes

Additional details on measuring reproductive parameters: On one day of every other week, the newly laid eggs were collected for egg shell strength and thickness during the egg laying phase of the study. The shell plus membrane were measured for thickness in 3 locations ±0.01 mm with Mitutoyo micrometer. Egg shell strength was measured (kg) using the Chatillon force gauge.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

20 wk

Dose descriptor:

NOEC

Effect level:

734 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

signs of toxicity

Remarks on result:

other: No observed effects

Key result

Duration (if not single dose):

20 wk

Dose descriptor:

LOEC

Effect level:

> 734 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

signs of toxicity

Remarks on result:

not determinable

Mortality and sub-lethal effects:

No statistically significant mortality occurred in the adults or hatchlings at any treatment level as compared to the controls. There was no mortality (0%) in the control hatchlings and parents.

Effects on reproduction:

There were no adverse effects on adult birds at any spirodiclofen technical treatment level as compared to the control for any of the adult endpoints, including mortality, behavior, growth, food consumption and gross pathology. There were no compound related effects observed throughout the 20 week exposure period. There were no adverse reproductive effects at any treatment level as compared to the control. The hatchlings were observed for 14-days post-hatch and evaluated for body weight, growth, mortality and sublethal effects. A statistical evaluation of all reproductive endpoints indicated no adverse effects as compared to the controls. No reference chemical was included in this study.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

Statistical data analyzed using TOXSTAT version 3.4 (West and Gulley, 1994).
Adult data - male and female weekly body weight, body weight change and feed consumption
data from treatment groups will be compared to controls using Levene's test of equal variance
(Snedecor and Cochran, 1971) to determine if dose groups have unequal variances. If variances
are equal, then parametric analyses will be conducted using analysis of variance (ANOVA)
followed by Dunnett's test (Snedecor and Cochran, 1971). If unequal variances are detected and
nonparametric procedures are required, the ranks of the values over all cages will be determined,
followed by an analysis of variance and a one-tailed Dunnett’s test performed on these ranks
(Conover, 1980).
Reproductive Data - Number of eggs laid per hen, eggs per hen per day, percentage of cracked
eggs, percentage of defective eggs, percent fertile of eggs set, percent live 3-week embryos of
fertile eggs, number hatched per hen, percent hatched of fertile eggs and of 3-week embryos,
hatchling and 14-day survivor body weight, percent 14-day survivors of hatchlings, number of
14-day survivors per hen and eggshell strength/ thickness measurements of the treatment groups
will all be compared to controls using Levene's test of equal variance (Snedecor and Cochran,
1971) to determine if dose groups have unequal variances. If variances are not determined to be
unequal then analyses will be conducted using ANOVA followed by Dunnett's test (Snedecor
and Cochran, 1971). If variances are unequal then the data will be transformed using the
appropriate transformation and subjected to the Levene's test again. If variances are not unequal
then analyses will be conducted using ANOVA followed by Dunnett's test. If unequal variances
are detected and nonparametric procedures are required, the ranks of the values over all cages
will be determined, followed by an analysis of variance and a one-tailed Dunnett’s test performed
on these ranks (Conover, 1980).

Table 2: Effect of spirodiclofen technical on mortality of the mallard duck in different observation periods

Treatment (mg/kg diet)	Acclimation	Acclimation	Pre-laying phase	Pre-laying phase	Laying phase	Laying phase
	Dead males	Dead females	Dead males	Dead females	Dead males	Dead females
Control	1	1	0	0	0	0
80 nominal, 79.4 measured	0	0	0	0	0	0
240 nominal, 239 measured	0	0	0	0	1 sac	1
720 nominal, 734 measured	0	0	0	0	0	0

sac = sacrificed since pen mate was found dead.

 

Table 3a: Reproductive and other parameters - Results for each test group indicate the units of measurement

Parameter	Control	79.4 ppm	239 ppm	734 ppm	NOAEC/LOAEC
Eggs laid/pen	43.9	35.8	38.2	50.1	734
Eggs laid/hen/day	0.52	0.43	0.46	0.60	734
Eggs cracked	54	53	39	70	734
Eggs laid	658	537	573	752	734
Eggs set	495	408	407	543	734
Shell thickness (mm ± SD)	0.339 ± 0.023	0.325 ± 0.027	0.337 ± 0.020	0.329 ± 0.019	734
Viable embryos	413	362	320	446	734
Live 3-week embryos	398	359	310	432	734
No. of hatchling/hen	20.9	17.4	16.0	23.6	734
No. of normal hatchlings	313	261	240	354	734
Hatchling weight (g)	35.5	36.0	36.8	36.2	734
14-day old survivors	313	261	239	350	734

 

Table 3b: Reproductive and other parameters - Results for each test group indicate the units of measurement

Parameter	Control	79.4 ppm	239 ppm	734 ppm	NOAEC/LOAEC
14-day old survivors weight (g)	259.4	264.8	255.4	261.6	734
Mean food consumption g/bird/day	163	163	163	159	734
Weight of females (parent) at test initiation:	966	938	955	946	734
Weight of females (parent)at onset of egg laying (week 9):	997	1002	1004	1007	734
Weight of females (parent) at termination of parental phase:	1086	1076	1077	1072	734
Weight of males (parent)at test initiation:	1093	1089	1100	1087	734
Weight of males (parent)at onset of egg laying:	1095	1106	1104	1103	734
Weight of males (parent)at termination of parental phase:	1091	1069	1089	1071	734
Gross pathology	no abnormal findings	no compound related findings	no compound related findings	no compound related findings	734
Egg shell strength (kg)	2.5 ± 0.46	2.4 ± 0.44	2.4 ± 0.45	2.3 ± 0.31	734

 

Validity criteria fulfilled:

yes

Conclusions:

The findings of this study concluded that the NOAEC of the test material is 734 mg /kg feed. The LOAEC is therefore >734 mg/kg feed.

Executive summary:

The one generation reproductive toxicity of spirodiclofen tech. to groups of 15 pairs of approximately 23 week-old mallard ducks was assessed over 20 weeks. spirodiclofen technical was administered to the birds in the diet at 0 (control), 80, 240 and 720 mg/kg diet. The NOAEC was determined to be 720 mg/kg diet based on all measured parameters including adult body weight and feed consumption and all reproductive parameters including eggshell strength and thickness, eggs laid, eggs cracked, eggs defective, eggs set, egg fertility, egg viability, normal hatchlings, hatchling survival and hatchling growth. There were no statistically significant differences from the control at any treatment level tested. Therefore, the LOAEC was greater than 720 mg/kg diet when compared to the control. There were no parental generation behavioral abnormalities or other visible signs of toxicity during the exposure to spirodiclofen technical. Feed consumption and body weight at all treatment levels were normal as compared to the parental controls. At all treatment levels, for all reproductive endpoints including egg production, eggshell thickness, eggshell strength, egg quality, egg fertility, embryo survival, hatch success, hatchling number, hatchling survival and hatchling growth there were no statistically significant adverse effects as compared to the controls. This toxicity study is classified as scientifically sound and does satisfy the guideline requirement for a Mallard reproductive toxicity study.

Description of key information

The one generation reproductive toxicity of spirodiclofen to groups of 15 pairs of approximately 23 week-old mallard ducks was assessed over 20 weeks. Spirodiclofen was administered to the birds in the diet at 0 (control), 80, 240 and 720 mg/kg diet. The NOAEC was determined to be 720 mg/kg diet based on all measured parameters including adult body weight and feed consumption and all reproductive parameters including eggshell strength and thickness, eggs laid, eggs cracked, eggs defective, eggs set, egg fertility, egg viability, normal hatchlings, hatchling survival and hatchling growth. There were no statistically significant differences from the control at any treatment level tested. Therefore, the LOAEC was greater than 720 mg/kg diet when compared to the control. There were no parental generation behavioral abnormalities or other visible signs of toxicity during the exposure to spirodiclofen. Feed consumption and body weight at all treatment levels were normal as compared to the parental controls.

At all treatment levels, for all reproductive endpoints including egg production, eggshell thickness, eggshell strength, egg quality, egg fertility, embryo survival, hatch success, hatchling number, hatchling survival and hatchling growth there were no statistically significant adverse effects as compared to the controls. This toxicity study is classified as scientifically sound and does satisfy the guideline requirement for a Mallard reproductive toxicity study.

 

Two additional GLP-studies, testing Spirodiclofen to birds were not included as study entry in this IUCLID-dossier, but added to the key description here.

One of these studies, a 5-day-dietary study to Mallard ducks was performed by Barfknecht, 1998. The main guideline followed for this study was EPA Pesticide Assessment Guidelines-Subdivision E, §71-2 "Avian dietary LC₅₀ test" from October 1982. Based on the results of this study, the LC₅₀ was determined as being greater than the highest, tested dietary concentration of 5000 mg/kg food. The no observed effect level (NOEC) at this study corresponded to the highest tested concentration of 5000 mg/kg food.

The second study not included in this IUCLID-dossier was a GLP-study performed by Barfknecht, 1999 that investigated the acute oral LD₅₀ for Bobwhite quail. The main guideline followed for this study was EPA Pesticide Assessment Guidelines-Subdivision E, §71-1 "Avian single dose oral LD₅₀ test" from October 1982. The acute oral LD₅₀ for Bobwhite Quail orally dosed was >2000 mg/kg. The NOEL was 2000 mg/kg (highest tested dose). The final examinations of the birds from all groups revealed no treatment-related effects on body organs.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

720 mg/kg food