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

Toxicity to birds

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug 1993 to 4 Mar 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 71-4 (Avian Reproduction Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

feed

Details on preparation and analysis of diet:

It was not necessary to use a vehicle in the incorporation of test substance in the diet. A premix of suitable strength was prepared weekly by mixing the required quantity of test substance with untreated basal diet. Blending of the premix was achieved by mixing in a mixer for a minimum period of 5 minutes. The required concentrations were prepared by direct dilution of the prepared premix. Blending of the inclusion levels for feeding was achieved by mixing in a Gardner double-cone blender for a minimum period of 7 minutes.
Prior to the start of the main study, samples were taken from a trial mix to determine stability and homogeneity of the test substance in SDS layer diet at 200 and 600 ppm. Samples were taken from the top, middle and bottom of the mix for analysis of homogeneity. Additional samples were taken to determine the stability of the test substance in avian diet over 0, 8 and 16 days under animal room conditions. Results from the trial mix indicated that the test substance was stable in avian diet under normal animal room conditions for up to 16 days. A 200 g random sample was taken at the time of mixing from each dietary level prepared for Weeks 1, 12 and 22 for analysis of active ingredient content. All samples were sent for analysis.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Bobwhite quail
- Supplier culture condition: The Supplier had maintained the birds on deep litter under a photoperiod of 7 hours light : 17 hours darkness and reported no health problems. On arrival at the test facility the birds received a routine course of antibiotic.
- Age at test initiation: Approximately 11 months old (were approaching their first reproductive season.)
- Weight at test initiation: 169 - 228 g
- Sexes used / mixed or single sex: mix
- Disease free: Yes (All birds were in apparent good health at the start of the pre—treatment period)

Limit test:

no

Total exposure duration (if not single dose):

23 wk

Remarks:

11 weeks pre-egg laying period and 12 weeks egg production period

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

1 male and 1 female / replicate

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

- Norminal concentrations: 0 (control), 200, 400 and 600 mg/kg diet

Details on test conditions:

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The birds were housed in 5 batteries of cages, each battery consisting of 4 tiers of 4 cages. Each cage, was constructed of polythene coated steel wire and measured approximately 0.31 x 0.39 x 0.24 m.
- Floor covering: The cages had sloping floors with 0.1 m egg-catchers, and had externally attached food hoppers and automatic drinkers.
- Compliant to good husbandry practices: Yes
- Suitable to avoid crowding stress: Yes
- Caging: Group (1 male and 1 female)

NO. OF BIRDS PER REPLICATES
- For negative control: 1 male and 1 female
- For vehicle control: 1 male and 1 female
- For treated: 1 male and 1 female

NO. OF REPLICATES PER GROUP
- For negative control: 20
- For vehicle control: 20
- For treated: 20

TEST CONDITIONS
- Room temperature: 18 - 21˚C
- Relative humidity (%): 44
- Photoperiod: 7 hours light : 17 hours darkness (from arrival until the beginning of Week 6); The photoperiod was then increased to
16 hours and remained so until the end of egg production.
- Light intensity: 60 to 150 lux (6 to 14 foot candles)
- Feeding: The basal diet used was avian layer diet. The diet was known to contain no added antibiotics or other non-nutritional feed additives.was available ad libitum throughout the study. Domestic quality potable water (Anglian Water) was available ad libitum at all times.

RANGE FINDING STUDY
Dose levels were selected on the basis of known residue levels in crops and on a preliminary study conducted between May and June 1993.
- Test concentrations: 0, 50, 200 and 400 ppm
- Test conditions: Fifteen male and fifteen female adult Bobwhite quail were used, including spare birds for possible
replacements during the pre-treatment period. The birds were housed in in treatment replicate groups of one male and one female bird, in polythene coated steel wire tiered cages measuring approximately 0.31 x 0.39 x 0.24 m. Each cage had an externally attached food hopper and an automatic drinker. The room in which the birds were housed was designed to provide suitable environmental conditions for the species. Ventilation fans were adjusted as required. A photoperiod of 16 hours was adopted. The mean maximum and minimum temperatures of the animal room were 25°C and 21 °C respectively and the mean daily relative humidity value was 67 %. Birds were given basal (untreated) diet during the 14-day pre-treatment period, and basal diet (Control) or treated diet during the treatment period. The test substance was given via the diet for 4 weeks following a 14-day acclimatisation period.
- Results used to determine the conditions for the definitive study: There were no mortalities and all birds remained in good health throughout the study. Bodyweight changes were similar in all groups and there was no evidence of any treatment-related effect. Food consumption was similar in all groups and there was no evidence of any treatment-related effect. It was concluded that dietary administration of up to 400 ppm the test substance over a period of four weeks had no effect on health, bodyweights or food consumption of adult Bobwhite quail.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
Adult birds and chicks were observed daily for mortalities and clinical signs. Individual adult bodyweights were recorded in Weeks -2, 0 (immediately prior to the introduction of test diets), 2, 4, 6, 8 and at termination, Week 23.

BODY WEIGHT
Individual chick bodyweights were recorded within 24 hours of hatching and again after 14 days.

FOOD CONSUMPTION
Food consumption for each replicate was recorded weekly throughout the pre-treatment and treatment periods of the adult phase.

OTHER: All sporadic mortalities were subjected to a macroscopic external and internal examination. At termination all adult birds were examined following sacrifice by cervical dislocation. Chicks were not examined post mortem at termination of 14 days observation.

Details on reproductive parameters:

The following parameters were examined per week, except for egg shell thickness which was recorded on alternate weeks.
- Eggs laid : Eggs were collected and the number recorded daily for each pen over the 12-week egg production period from the start of Week 12 to the end of Week 23. Each egg was labelled with the study schedule number, replicate number and treatment group, together with the date of collection. The eggs were then stored on plastic egg trays in a refrigerator which was set to run at 16 ˚C. At the end of each 7-day period the eggs were removed from the refrigerator and allowed to reach room temperature for at least 12 hours prior to incubation.
- Eggs cracked; eggs broken : Each egg was candled and any broken or cracked eggs were recorded and discarded. The remaining eggs (except those for shell thickness measurement) were incubated.
- Eggshell thickness: Eggs laid on the first days of Weeks 12, 14, 16, 18, 20 and 22 in each replicate were removed for shell thickness measurement. The eggs were cracked open at the widest point and the contents washed out with tap water. The shells were then left to dry at room temperature for a minimum of 48 hours. The shell thickness of each egg was measured at 4 points around the circumference using a micrometer calibrated to 0.01 mm.
- Eggs incubation: Eggs were incubated in an incubator for 21 days prior to transferring to a hatcher. The incubator was set to run at 37.7°C and 55% relative humidity. The eggs were turned automatically once every hour through 90° each side of the vertical throughout the incubation period. Eggs were candled for cracks prior to incubation and again on Days 11 and 18 of the incubation period. At Day 11 all infertile eggs and eggs showing early embryonic death were identified, recorded and discarded. On Day 18 all late embryonic mortalities were similarly discarded. Early and late embryonic mortalities were recorded on the basis of candling only and internal examination was carried out only if the candling result was ambiguous.
- Normal hatchlings : After 21 days incubation, the eggs were transferred to a still air hatcher which was set to run at a temperature of 37.5 ˚C. The eggs were placed on wire mesh trays and separated according to replicate. All chicks which hatched were removed and transferred to floor pens within 24 hours of hatching. Any unhatched eggs remaining after 3 days were classed as dead in shell, with a record being made of whether or not the shells had been pipped.
- Chicks : On removal from the hatcher, chicks were individually weighed and identified by coloured plastic leg bands. Each was allocated a separate series of numbers with letter suffix, and each chick had a unique identification comprising colour code, number and letter suffix. The chicks were housed in wooden box floor pens according to the week of hatch. Each box contained a low level feeder and a drinking fount supplied with domestic quality tap water. Bedding consisted of wood shavings from an approved source. A heat lamp was suspended over each box to provide additional heat. Ventilation fans were adjusted as required and a 14-hour photoperiod was adopted. The mean maximum and minimum temperatures in the animal room were 30°C and 24°C respectively and the mean daily relative was 57%. All chicks were fed standard HRC chick diet in meal or crumb form. Food and water were available ad libitum. At the end of the l4-day observation period, chicks were killed by carbon dioxide asphyxiation.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

23 wk

Dose descriptor:

NOEC

Effect level:

200 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

reproductive parameters

Remarks on result:

other: equivalent to a daily dietary dose of 19.9 mg/kg bw/day

Mortality and sub-lethal effects:

An overview of the results is provided in Table 1 – Table 10 in ‘Any other information on results incl. tables’
- Adult clinical observations and mortalities: There was no evidence of any treatment-related signs of toxicity. A small number of mortalities occurred in each group, and there was no evidence of any treatment-related effect. Several birds were observed to have cut heads during the study; this, together with cut feet, was related to the long-term housing of birds in cages.
- Adult body weight: Bodyweights were similar in all groups throughout the study. Statistical analysis revealed no significant treatment-related effects.
- Adult food consumption: Food consumption was similar in all groups and there was no evidence of any treatment-related effect. Statistical analysis revealed no significant differences between control and treated groups.
- Microscopic post mortem examination: Some post mortem observations were related to long-term housing in cages. None of the observations was considered to be related to treatment.
- Chick body weight: Mean chick bodyweights at hatching were slightly reduced in Groups 3 and 4 (400 and 600 ppm test substance) compared with control values. Statistical analysis revealed these reductions to be significant (P <0.01). Mean 14-day bodyweights were similar in all groups and there was no evidence of any treatment- related effects. This was supported by statistical analysis.
- Chick health and mortality: Although a large number of treatment-related mortalities occurred, all chicks appeared normal at hatching and no clinical signs of toxicity were observed during the 14-day observation period. At post mortem examination, a small proportion of chicks was observed to have been peeked by other birds. In addition, two birds from the Week 18 hatch were observed to have swollen eyes. None of these observations was considered to be related to treatment.

Effects on reproduction:

An overview of the results is provided in Table 1 – Table 10 in ‘Any other information on results incl. tables’
- Number of eggs laid: The number of eggs laid was slightly reduced in Groups 3 and 4 (400 and 600 ppm the test substance) relative to control values. However, statistical analysis revealed no significant treatment-related effects.
- Number of cracked eggs: The proportion of cracked eggs of eggs laid was variable and there was no evidence of any treatment-related effect. This was supported by statistical analysis.
- Egg shell thickness: Egg shell thickness was similar in all groups and there was no evidence of any treatment-related effect. This was supported by statistical analysis.
- Candling: The proportion of viable embryos (fertile eggs) of eggs set initially was slightly reduced in Group 4 (600 ppm test substance) relative to the control. However, statistical analysis revealed no significant treatment-related effects. The proportions of live 3-week embryos of viable embryos were similar in all groups. Statistical analysis revealed no significant differences between control and treated group.
- Hatching: The proportion of normal hatchlings of viable embryos was slightly reduced in all groups relative to control values. The proportion of normal hatchlings of live 3-week embryos was slightly reduced in Groups 3 and 4 (400 and 600 ppm the test substance) relative to control values. However, statistical analysis of these parameters revealed no significant treatment-related effects.
- 14-day survivors: The proportion of 14-day survivors of eggs laid was slightly reduced in all treated groups as compared with the control. However, statistical analysis showed this decrease to be significant only in Group 4 (600 ppm test substance; P <0.01). The proportion of 14-day survivors of normal hatchlings also appeared slightly reduced in all treated groups. Statistical analysis revealed this decrease to be significant in Groups 3 and 4 (400 and 600 ppm test substance; P <0.01) The number of 14-day survivors per adult female also appeared slightly reduced in all treated groups. However, statistical analysis revealed no significant treatment-related effects.

Reported statistics and error estimates:

Analyses of variance were carried out on the data; factors in the analyses were: (i) treatment (the main component of interest); (ii) position (batteries, and rows by columns within batteries). Williams' test for contrasting increasing dose levels of a compound with a zero dose control was used to compare the treated groups with the control. Significant differences are indicated in the Tables using the conventional asterisk notation: * P < 0.05; ** P < 0.01.

Table 1. Mean bodyweights (g) adult birds

		Week of study
Dose (ppm)	Sex	-2	0	2	4	6	8	23
0 (control)	M	193	187	186	187	187	186	193
	F	189	184	184	186	189	201	207
200	M	188	185	187	189	192	192	197
	F	188	184	188	187	186	197	204
400	M	196	189	191	191	191	191	195
	F	190	187	186	188	186	190	202
600	M	192	184	186	187	186	187	187
	F	184	181	184	188	188	194	207

 

Table 2. Mean weekly food consumption (g/bird/day) 

Dose (ppm)	Week of study (Pre-egg production)
	-2	-1	1	2	3	4	5	6	7		9	10	11
0 (control)	10	13	13	14			14	16	16	17	19	19	20
200	10	13	13	14	13	13	13	15	16	16	18	18	19
400	11	12	13	14	13	14	14	15	16	16	17	18	18
600	11	14	14	15	15	15	15	15	16	16	18	19	19

 

Dose (ppm)	Week of study (egg production)													Mean weeks 1 - 23
		12	13	14	15	16	17	18	19	20	21	22	23
0 (control)	1	21	21	22	22	23	23	23	22	22	22	22	22	19
200	2	20	20	21	22	22	23	23	21	22	22	22	22	19
400	3	19	20	21	22	21	22	22	21	22	22	22	22	18
600	4	20	20	21	23	22	23	23	22	23	23	22	22	19

Table 3. Number of eggs laid

Parameter	Dose (ppm)	Week of study												Total
		12	13	14	15	16	17	18	19	20	21	22	23
Number of eggs laid	0 (control)	37	45	57	46	62	77	72	57	57	58	53	46	667
	200	27	45	57	53	68	77	87	79	73	64	52	45	727
	400	20	31	38	40	46	49	48	50	49	60	59	45	535
	600	16	31	33	39	45	53	58	62	58	60	51	46	552
Number of eggs laid per female	0 (control)	1.9	2.4	3.0	2.4	3.3	4.1	3.8	3.0	3.0	3.1	2.8	2.6	35.4
	200	1.4	2.3	2.9	2.7	3.4	3.9	4.4	4.0	3.8	3.4	2.7	2.4	37.3
	400	1.2	1.8	2.2	2.4	2.7	2.9	2.8	2.9	2.9	3.5	3.5	2.6	31.4
	600	0.8	1.6	1.7	2.1	2.4	2.8	3.1	3.3	3.1	3.2	2.8	2.7	29.6

 

Table 4. Cracked eggs

Parameter	Dose (ppm)	Week of study												Total
		12	13	14	15	16	17	18	19	20	21	22	23
Number of eggs cracked	0 (control)	1	0	3	2	2	4	1	2	1	0	0	2	18
	200	2	7	2	1	3	5	5	5	5	5	4	4	48
	400	1	1	1	0	1	0	1	1	1	1	1	1	10
	600	1	0	1	1	3	2	1	4	3	6	3	5	30
% cracked of eggs laid	0 (control)	2.7	0	5.3	4.3	3.2	5.2	1.4	3.5	1.8	0	0	4.3	2.7
	200	7.4	15.6	3.5	1.9	4.4	6.5	5.7	6.3	6.8	7.8	7.7	8.9	6.6
	400	5.0	3.2	2.6	0	2.2	0	2.1	2.0	2.0	1.7	1.7	2.2	1.9
	600	6.3	0	3.0	2.6	6.7	3.8	1.7	6.5	5.2	10.0	5.9	10.9	5.4

 

Table 5. Mean shell thickness (mm)

Dose (ppm)	Week of study						Mean
	12	14	16	18	20	22
0 (control)	0.20	0.22	0.21	0.21	0.21	0.21	0.21
200	0.20	0.21	0.21	0.20	0.20	0.20	0.20
400	0.20	0.22	0.22	0.21	0.21	0.21	0.21
600	(0.21)	0.21	0.21	0.21	0.20	0.20	0.21

() One value day

Table 6. Candling

Parameter	Dose (ppm)	Week of study												Total
		12	13	14	15	16	17	18	19	20	21	22	23
Number of eggs set initially	0 (control)	30	45	45	44	49	72	63	55	46	57	46	44	596
	200	22	38	49	52	55	71	70	74	56	59	39	40	625
	400	17	30	34	40	39	49	40	49	42	59	50	44	493
	600	14	31	29	38	36	51	49	58	52	54	39	41	492
Number of viable embryos	0 (control)	27	36	38	40	48	63	53	50	40	49	37	33	514
	200	12	22	33	42	45	56	58	60	43	46	31	32	480
	400	12	19	29	37	39	46	40	49	38	52	38	34	433
	600	9	18	19	24	18		38	45	37	42	29	29	344
Number of live 3-week embryos	0 (control)	26	34	37	40	48	60	52	47	38	49	35	33	499
	200	11	21	32	41	43	52	55	58	40	43	30	27	453
	400	12	19	28	37	39	40	36	49	37	50	33	34	414
	600	9	17	18	24	17	33	34	42	35	41	29	28	327
% viable embryos of eggs set initially	0 (control)	90	80	84	91	98	88	84	91	87	86	80	75	86
	200	55	58	67	81	79	83	81	77	78	79	80	77	55
	400	71	63	85	93	100	94	100	100	90	88	76	77	88
	600	64	58	66	63	50	71	78	78	71	78	74	71	70
% live 3-week embryos of viable embryos	0 (control)	96	94	97	100	100	95	98	94	95	100	95	100	97
	200	92	95	97	98	96	93	95	97	93	93	97	84	94
	400	100	100	97	100	100	87	90	100	97	96	87	100	96
	600	100	94	95	100	94	92	89	93	95	98	100	97	95

Table 7. Hatching results

Parameter	Dose (ppm)	Week of study												Total
		12	13	14	15	16	17	18	19	20	21	22	23
Number of normal hatching	0 (control)	22	29	35	36	46	53	48	42	37	48	33	32	461
	200	11	20	30	36	36	50	51	51	38	35	25	25	403
	400	10	19	25	26	35	39	29	42	31	41	27	30	354
	600	8	14	15	18	15	32	27	38	30	35	26	27	285
Number of dead in shell	0 (control)	4	5	2	4	2	7	4	5	1	1	2	1	38
	200	0	1	2	5	6	2	4	7	2	8	5	2	44
	400	2	0	3	10	4	1	7	7	6	9	6	4	59
	600	1	3	3	6	2	1	6	4	5	6	3	1	41
% normal hatchings of viable embryos	0 (control)	81	81	92	90	96	84	91	84	93	98	89	97	90
	200	92	91	91	86	80	89	88	85	88	76	81	78	85
	400	83	100	86	70	90	85	73	86	82	79	71	88	82
	600	89	78	79	75	83	89	71	84	81	83	90	93	83
% normal hatchings of live 3-week embryos	0 (control)	85	85	95	90	96	88	92	89	97	98	94	97	92
	200	100	95	94	88	84	96	93	88	95	81	83	93	90
	400	83	100	89	70	90	98	81	86	84	82	82	88	86
	600	89	82	83	75	88	97	79	90	86	85	90	96	87

 

Table 8. 14-day survivors

Parameter	Dose (ppm)	Week of study												Total
		12	13	14	15	16	17	18	19	20	21	22	23
Number of 14-day survivor	0 (control)	19	27	32	30	46	50	43	41	31	46	28	31	424
	200	9	19	27	23	30	37	43	45	33	27	16	22	331
	400	8	17	22	13	27	32	25	37	21	16	9	22	249
	600	4	9	9	5	10	21	23	27	14	16	10	9	157
% surviving of eggs laid	0 (control)	51	60	56	65	74	65	60	72	54	79	53	67	64
	200	33	42	47	43	44	48	49	57	45	42	31	49	46
	400	40	55	58	33	59	65	52	74	43	27	15	49	47
	600	25	29	27	13	22	40	40	44	24	27	20	20	28
% surviving of normal hatchings	0 (control)	86	93	91	83	100	94	90	98	84	96	85	97	92
	200	82	95	90	64	83	74	84	88	87	77	64	88	81
	400	80	89	88	50	77	82	86	88	68	39	33	73	70
	600	50	64	60	28	67	66	85	71	47	46	38	33	55
Number of 14-day survivor per female	0 (control)	1.0	1.4	1.7	1.6	2.4	2.6	2.3	2.2	1.6	2.4	1.5	1.7	22.4
	200	0.5	1.0	1.4	1.2	1.5	1,9	2.2	2.3	1.7	1.4	0.8	1.2	17.1
	400	0.5	1.0	1.3	0.8	1.6	1.9	1.5	2.2	1.2	0.9	0.5	1.3	14.7
	600	0.2	0.5	0.5	0.3	0.5	1.1	1.2	1.4	0.7	0.8	0.6	0.5	8.3

 

Table 9. Mean initial and 14-day body weight (g)

Parameter	Dose (ppm)	Week of study												Mean
		12	13	14	15	16	17	18	19	20	21	22	23
Mean initial bodyweight	0 (control)	6.8	7.1	6.8	6.8	7.2	7.1	6.6	7.3	7.2	6.8	7.0	6.9	7.0
	200	6.8	6.9	7.0	6.8	7.5	7.1	6.7	7.2	7.2	6.8	6.8	7.4	7.0
	400	6.9	6.9	6.8	6.3	6.9	6.6	6.4	6.5	6.7	6.6	6.5	6.7	6.6
	600	6.2	6.1	6.1	6.0	6.4	6.4	6.4	6.4	6.8	6.4	6.6	6.4	6.4
Mean 14-day bodyweight	0 (control)	20	24	23	23	25	24	26	25	24	24	22	25	24
	200	21	23	25	22	26	25	26	26	24	24	23	28	25
	400	24	24	24	23	26	26	26	23	25	26	21	25	24
	600	19	21	27	22	26	25	26	26	25	22	21	25	24

 

Table 10.Summary of reproductive data for effects of the test substance technical on bobwhite quail

Observation	Control	Test substance
		200 mg/kg	400 mg/kg	600 mg/kg
Adult mean daily food consumption over treatment period (g) Adult mean bodyweight over treatment period (g) Treatment as Daily Dietary Dose a (mg/kg bw/day)	19 190 0	19 191 19.9	18 191 37.7	19 188 60.6
Female mortality	1	1	2	1
Eggs laid per female	35.4	37.3	31.4	29.6
Eggs damaged	18	48	10	30
Eggs damaged of eggs laid (%)	2.7	6.6	1.9	5.4
Mean egg shell thickness (mm)	0.21	0.20	0.21	0.21
Eggs set	596	625	493	492
Viable embryos	514	480	433	344
Viable embryos of eggs set (%)	86	77	88	70
Live 3-week embryos	499	453	414	327
Live 3-week embryos of viable embryos (%)	97	94	96	95
Normal hatchlings	461	408	354	285
Normal hatchlings of viable embryos (%)	90	85	82	83
Normal hatchlings of live 3-week embryos (%)	92	90	86	87
14-day old survivors	424	331	249	157
14-day old survivors of eggs laid (%)	64	46	47	28 **
14-day old survivors of normal hatchlings (%)	92	81	70 **	55 **
14-day old survivors per female	22.4	17.1	14.7	8.3
Chick bodyweights at hatching (g)	7.0	7.0	6.6	6.4
Chick bodyweights at 14 days (g)	24	25	24	24

** Statistically significant difference from control (P<0.01; William’s test).

Validity criteria fulfilled:

yes

Conclusions:

In a reproduction toxicity study in birds, performed with Bobwhite quail and in accordance with EPA 71-4 guideline, the 23-wk NOEC was determined to be 200 mg/kg diet, equivalent to a daily dietary dose of 19.9 mg/kg bw/day.

Executive summary:

The effects of dietary administration of the test substance on reproduction in the Bobwhite quail (Colinus virginianus). The study was performed according to EPA guideline 71-4 and in compliance with GLP criteria. Three treated groups of 20 replicates, each of one male and one female, were offered test diet containing 200, 400 and 600 mg a.s./kg over a period of 23 weeks (for 11 weeks prior to the start of egg production and during 12 weeks of egg production). A similar sized control group was given untreated diet only. The birds (approximately 11 months old) were housed in 5 batteries of cages, each battery consisting of 4 tiers of 4 cages. Each cage had an externally attached food hopper and automatic drinker. The mean temperature and humidity (measured once daily) in the test room were 18 – 21 ˚C and 44%, respectively. An artificial light photoperiod of 7 hours daily was provided until the end of week 6, when the photoperiod was increased to 16 hours daily (light intensity range 60 to 150 lux). The test birds were fed ad libitum with a layer diet (treated or untreated) and water. Eggs were collected and recorded daily for each pen over the 12-week egg production period. The eggs were labelled and then stored on plastic egg trays at 16˚C. At the end of each 7-day period the eggs were removed and allowed to reach room temperature for at least 12 hours before being candled. Any broken or cracked eggs were recorded and discarded; the remaining eggs (except those removed for shell thickness measurement) were incubated. Eggs were incubated for 21 days at nominal 37.7°C and 55% RH. The eggs were automatically turned once every hour through 90˚each side of vertical. After 21 days the eggs were transferred to a hatcher. All eggs laid on the first day of weeks 12, 14, 16, 18, 20 and 22 in each replicate were removed for shell thickness examination. The eggs were cracked open at the widest point and the contents washed out. The shells were then left to dry at room temperature for at least 48 hours. The shell thickness of each egg was measured at four points around the circumference using a micrometre. Eggs were candled for cracks prior to incubation and then on days 11 and 18 of the incubation period. At day 11, all infertile eggs and eggs showing early embryonic death were recorded and removed. At day 18, late embryonic deaths were recorded and removed. After 21 days incubation, the eggs were transferred to a hatcher set at 37.5 ˚C. All chicks that hatched were removed within 24 hours of hatching and transferred to floor pens. Any eggs remaining unhatched after 3 days were classified as “dead in shell”. Adult birds and chicks were observed daily for mortalities and clinical signs. Individual adult bodyweights were recorded on weeks -2, 0 (immediately before introduction of the test diets), 2, 4, 6, 8 and 23 (termination of test). Food consumption for each replicate was recorded weekly during the adult phase. Individual chick bodyweights were recorded within 24 hours of hatching and again after 14 days. All data relating to food consumption, eggs and chicks were collated over weekly intervals, except for eggshell thickness, which was recorded on alternate weeks. All sporadic mortalities were subjected to a macroscopic external and internal examination. At termination, all adult birds were sacrificed and examined. Ducklings were not examined post-mortem.

Dietary administration of 200 mg/kg diet test substance to adult Bobwhite quail had no effect on adult birds or their reproductive performance. At 400 mg/kg diet, there was a reduction in the number of chicks surviving to 14 days as a proportion of normal hatchlings (P <0.01). Additionally, chicks were lighter in weight at hatching. At 600 mg/kg diet, there was a reduction in the number of chicks surviving to 14 days both as a proportion of normal hatchlings (P <0.01) and as a proportion of eggs laid (P <0.01). Additionally, chicks were lighter in weight at hatching. Based on this information, the NOEC was determined to be  200 mg/kg diet, equivalent to a daily dietary dose of 19.9 mg/kg bw/day.

Description of key information

All available data was assessed and the study representing the worst-case effect was included as key study. Other studies are included as supporting information. The effect value from the key study was selected for the CSA.

23-wk, NOEC = 200 mg/kg diet (equivalent to 19.9 mg/kg bw/day), Colinus virginianus, reproduction, EPA 71 -4, Johnson 1994

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:

200 mg/kg food

Additional information

Three acute and three chronic toxicity studies of the test substance are available. They all followed standard test guidelines and complied with GLP. One of the chronic studies (Johnson 1994; EPA 71 – 4; Reliability 1) on Bobwhite quail (Colinus virginianus) was selected as key study and its effect value was used as key value. This study was selected as the key study because a long-term toxicity test is preferred for this endpoint, according to ECHA guidance R7c (Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7c: Endpoint specific guidance).It represents the worst-case effect (i.e. shows the lowest NOEC value in daily dietary dose). Three treated groups of 20 replicates, each of one male and one female, were offered test diet containing 200, 400 and 600 mg a.s./kg over a period of 23 weeks (for 11 weeks prior to the start of egg production and during 12 weeks of egg production). A similar sized control group was given untreated diet only. The birds (approximately 11 months old) were housed in 5 batteries of cages, each battery consisting of 4 tiers of 4 cages. Each cage had an externally attached food hopper and automatic drinker. The mean temperature and humidity in the test room were 18 – 21 ˚C and 44%, respectively. An artificial light photoperiod of 7 hours daily was provided until the end of week 6, when the photoperiod was increased to 16 hours daily (light intensity range 60 to 150 lux). The test birds were fed ad libitum with a layer diet (treated or untreated) and water. The result show that the 200 mg/kg diet treatment had no adverse effect on adult birds or their reproductive performance. Higher than this dose, significant effects were observed. Therefore, the 23-week NOEC is 200 mg/kg diet (equivalent to 19.9 mg/kg bw/day) for reproduction parameters.

The other two chronic studies are a 23-wk reproduction test (Redgrave 1995; EPA guideline 71-4; Reliability 1) and a 90-day growth (body weight) test (Gallagher 2003; OECD TG 205 and EPA guideline 71-2; Reliability 1). They have been included as supporting information. In the study from Redgrave (1995), Mallard duck (Anas platyrhynchos) was offered the same dosages of the test substance in the diet as in the key study. The study concluded that the 23-wk NOEC for reproduction is 200 mg/kg diet, equivalent to a daily dietary dose of 32.2 mg/kg bw/day. In the 90 days growth test (Gallagher 2003), the test organisms were fed on 0, 2, 20, 200, 2000 or 20,000 mg/kg test substance in the diet. The NOEC was determined to be 20,000 ppm, the highest concentration tested.

Three supporting acute toxicity studies are available. Two EPA 71-1 guideline followed acute toxicity studies were carried out on mallard duck, Anas platyrhynchos. In one of the studies (Hakin 1990a; Reliability 1), the test organisms (approximately 5 months old) were exposed to control (corn oil), 500, 1000 or 2000 mg/kg feed treatment. Ten birds (5 male and 5 female) were tested for each treatment. After 14-d exposure, no mortalities occurred, no abnormalities were noted in any bird at any dose level. Thus, the LD50 was determined to be > 2000 mg/kg diet, the highest dose tested. In another study (Hakin 1990c ; Reliability 1), higher dosages were used: control, 163, 325, 650, 1300, 2600 and 5200 mg/kg diet. Ten mallard duck (8 days old; unsexed) were tested for each treatment (3 groups of 10 in the control). After 8-d exposure, the results indicate that the LC50 was higher than the highest dose tested (> 5200 mg/kg). The third acute toxicity study was performed on bobwhite quail (Colinus virginianus). The test organisms (12 days old) were exposed to control, 163, 325, 650, 1300, 2600 or 5200 mg/kg diet. Similar result to the other acute studies was found. The LC50 was higher than the highest dose tested (> 5200 mg/kg).