2,6-di-tert-butyl-p-cresol

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to birds

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2013 - 21 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

The aim of the study was to evaluate the tolerance of chickens for fattening to Butylated Hydroxytol uene (BHT) as technological feed additive (E 321). The study was carried out with 540 chickens at the start of the study. The treatments were: 1) Control group T1: animals fed with basal diet; 2) T2: animals fed basal (T1) diet supplemented with BHT at 150 g/tonne feed (recommended dose); 3) T3: animals fed basal (T1) diet supplemented with BHT at 1000 g/tonne feed (6.7 x recommended dose) and 4) T4: animals fed basal (T1) diet supplemented with BHT at 1500 g/tonne feed (10x recomm ended dose,). The feeds were issued to the appropriate pens for 35 consecutive days. Each feeding treatment was replicated in 9 pens. The data recorded during the feeding phase were live weight (LW) at 0, 10, 28 and 35 days from the start of the study, average daily gains (ADG), daily feed intake and feed:gain ratio (F:G) during the periods 0-10, 10-28, 28-35 and 0-35 days from the start of the study. At 35 days, blood samples were taken from one animal per pen (36 birds in total, 9 per treatment) a nd analysed for routine haematology and biochemistry parameters. The same 36 animals were then sacrificed and necropsied by the veterinary surgeon responsible for animal welfare.

GLP compliance:

yes (incl. QA statement)

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: The test product was supplemented to the basal diets using the vertical and horizontal mixers with a capacity of 0.15 and
0.60 tonnes respectively. With the exception of the test product, the animals did not receive any other product for the total study period. The control feed was free from any zootechnical feed additive as defined in (EC) No 1831/2003. Animals were fed by hand out of coded bags.

NUTRIENT DETERMINATION IN FEED
- When and at what dose levels were samples of treated food analyzed: Nine representative samples of at least 250 g were taken from each test diet: 3 in the beginning, 3 in the middle and 3 in the end of the production process (during the bagging-of the feeds). These 9 samples were pooled to 3 samples in such a way that 1 sample from the beginning, middle and end are pooled together and thoroughly mixed.
- Parameters analysed: Dry matter, crude ash, starch, crude protein content, crude fat content, crude fibre content, carbohydrate and energy content.

BHT ANALYSIS IN FEED
- When and at what dose levels were samples of treated food analyzed: Samples of each test diet for each growing period were taken for analysis (LC-MS/MS).
- Results of homogeneity analysis (range of values): The actual BHT content was within the nominal values and within the tolerance limits given.
- Nominal concentration (mg/kg feed): 0, 150, 1000, 1500 g/ton feed.
- Concentration analysed (mg/kg feed): Not detected, 149.0, 983.3, 1462.1 (mean) (See Table no. 3 below).
- % of nominal: -, 99.3, 98.3, 97.5
- Mixing procedure adequate and variance between nominal and actual dosage acceptable (yes/no): Yes.

Test organisms (species):

other: Chicken (Ross 708)

Details on test organisms:

TEST ORGANISM
- Common name: Chicken
- Source: Via Emilia Km 17 - Longiano (FC) - Italy. Italian AUSL code number: 018 FO 098
- Age at test initiation (mean and range, SD): The chicken used in the present study were chickens for fattening from birth to 35 days old.
- Weight at test initiation (mean and range, SD): 47.1 ± 0.9 g
- Sexes used / mixed or single sex: Males
- Disease free: yes . The animals were selected out of healthy groups in the hatchery. Animals were selected from the same parent stocks and were vaccinated in the hatchery at the day of hatching against Marek’s disease, Paracox for coccidiosis, infectious bronchitis and Newcastle Disease. The animals were also vaccinated during the study against Gumboro disease at D14 and against Newcastle disease at D16.
- Kept according to standard practices: yes

Limit test:

no

Total exposure duration (if not single dose):

35 d

Remarks:

From birth to 35 days old.

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

540 males

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Control T1: Basal diet
T2: T1 + BHT at 150 g/tonne feed (recommended dose, ca. 13.2-22.3 mgBHT/kg bw).
T3: T1 + BHT at 1000 g/tonne feed (6.7 x recommended dose, ca. 94.4-175.5 mgBHT/kg bw).
T4: T1 + BHT at 1500 g/tonne feed (10 x recommended dose, ca. 150.3-268.1 mgBHT/kg bw).

Details on test conditions:

ACCLIMATION
- Acclimation period: No.
- Feeding: During the whole study period the animals were fed ad libitum using one feeder per pen, and fresh drinking water was supplied with a bell drinker for the first 10 days and then with droplet drinkers. The water quality is analytically controlled once a year.
- Health (any disease or mortality observed): The animals were selected out of healthy groups in the hatchery.
- Fasting period before study: No.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The net floor space available in each pen was 2.24 square meter (1.4 m x 1.6 m). The pens were aligned in 4 rows with central corridors 1.4 m wide. The pens were accurately cleaned and disinfected before installing the animals in and an ‘empty period’ of at least 7 days was respected to ensure good sanitary conditions in the pens.
- Compliant to good husbandry practices: yes
- Suitable to avoid crowding stress: yes
- Caging: Grouped, 15 chickens per pen. Total of 36 pens in a room.

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 15 (T1)
- For treated: 15 (T2, T3, T4)

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: 9 (T1)
- For treated: 9 (T2, T3, T4)

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: The temperature and relative humidity were recorded every 30 minutes
- Room temperature: Within the especifications.
- Relative humidity (%): Within the especifications.
- Photoperiod: 23:1 hours light and dark.
- Ventilation: The ventilation rate varied from 0 m3/hour to the maximum ventilation rate required, according to the desired temperature and the age of the chickens.

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Twice daily, in the morning and in the afternoon.
- Remarks: General health status was checked, ensuring constant feed and water supply as well as checking the correct temperature and ventilation and unexpected events.

BODY WEIGHT
- Time schedule for examinations: Pen body weights on days 0, 10, 28 and 35. Died animals were also weighed.

FOOD CONSUMPTION
- Time schedule for examinations: Feed intake was accurately measured per pen, taking into account the amounts provided and weighed back between days 0 and
day 10, between day 10 and day 28 and between days 28 and day 35. In order to determine the net feed intake the net weight of feed administered during the trial was recorded. The total quantity administered per period was used to evaluate the intake and the feed:gain ratio (F:G) in each period.
- Remarks: Average feed intake and average body weight per pen were used to calculate the average feed:gain ratio. The adjusted feed:gain ratio was calculated considering the total feed intake per pen divided by the sum of animals live weight and the body weight of the animals died during each period for each replicate.

HAEMATOLOGY:
- Time schedule for examinations: On day 35.
- Dose groups that were examined: Blood samples were taken from one bird per pen (36 chickens for fattening in total, 9 animals per treatment).
- Parameters: RBC, Haemoglobin, Haematocrit, MCV, MCH, MCHC, WBC, Lymphocytes, Monocytes, Neutrophils, Eosinophils, Basophils, Platelets, Reticulocyte count, RPI, MPV, PCT, PDW.

BIOCHEMISTRY:
- Time schedule for examinations: On day 35.
- Dose groups that were examined: Blood samples were taken from one bird per pen (36 chickens for fattening in total, 9 animals per treatment).
- Parameters: RBC, Haemoglobin, Glucose, Calcium, Inorganic P, Cholesterol, Triglycerides, Phospholipids, Uric acid, Urea, Creatinine, Lactate dehydrogenase, Alkaline
Phosphatase, GOT (Aspartate Transaminase), GPT (Alanine Transaminase), Total Bilirubin, GGT, haptoglobin, serum albumin, serum total protein, blood clotting: quick/INR value, prothrombin time (PT), thyroid hormones (TSH, T3, T4).

PATHOLOGY
- Dose groups that were examined: The 36 animals sampled at trial end (D35) for blood collection were sacrificed and necropsied by a veterinary surgeon.
- Terminal necropsy: To assess general health and fitness for human consumption.
- Veterinary necropsy: Checks on: external skin, eyes and any injuries, feet, ears, head and tail, mouth and anus, gut – oral cavity, oesophagus, stomach, upper, mid and lower small intestine, caecum and colon, pancreas, spleen liver/gall bladder, kidneys, genitals, abdominal fat, omentum, heart and lungs, skeletal muscle and fat.

Key result

Duration (if not single dose):

35 d

Dose descriptor:

NOEL

Effect level:

>= 268.1 mg/kg bw/day (actual dose received)

Conc. / dose based on:

test mat.

Basis for effect:

other: No adverse effects observed at the higest dose tested.

Remarks on result:

other: 1500 g/tonne feed (ca. 150,3-268,1 mgBHT/kg bw)

Mortality and sub-lethal effects:

MORTALITY
- Results: The two dead animals were of the treatments T1 and T4, both occurred in the middle of the study (experimental period of 10-28d).
- Remarks: No alterations were found in organs and tissues in both animals nor symptoms of pathological diseas. This deaths were assumed to be not test item related. See Table no. 1 below.

CLINICAL SIGNS
- Results: The general performance parameters,were good and similar to those expected under commercial rearing of chickens for fattening in Italy. The veterinarian considered the animals’ health and husbandry to be generally good with a very low mortality and normal consistency of the faeces.

BODY WEIGHT
- Results: No statistical differences were found among feeding treatments for both parameters for all periods.
- Remarks: See Table no. 3 below.

FOOD CONSUMPTION (if feeding study)
- Results: No statistical differences were found among feeding treatments for both parameters for all periods. In the global study period (D0-D35) the feed:gain ratio was tendentially higher in T4 and T3 groups in comparison with T1 group (1.74 and 1.74 vs. 1.66 for T4, T3 and T1 groups respectively; P=0.0723).
- Remarks: See Table no. 4 below.

HAEMATOLOGY:
- Results: All measured haematological parameters were within the respective reference ranges. No statistical differences were found among feeding treatments with the exception of monocytes that was higher in T2 vs. T1 and T4 groups (2.80 vs. 2.21 and 2.29 % respectively; P=0.0278) but no difference occurred between T1 and T4 group. The absence of reticulocyte and RPI could be an index of a good sanitary status of the animals.
- Remarks: See Table no. 5 below.

BIOCHEMISTRY:
- Results: All measured biochemical parameters were within the respective reference ranges. Urea was lower in T3 vs. T1 and T2 groups (respectively 5.10 vs. 6.22 and 6.38 mg/dl; P = 0.0402) but no difference occurred between T1 and T4 group. Creatinine was lower in T3 vs. T1 and T2 groups (respectively 0.53 vs. 0.61 and 0.60 mg/dl; P = 0.0414) but no difference occurred between T1 and T4 group. Bilirubin was tendentially higher in T2 vs. T3 and T4 groups (respectively 0.14 vs. 0.11 and 0.11 %; P = 0.0795) but no difference occurred between T1 and T4 group. No differences were observed between the control and the treatment groups regarding neither the thyroid hormones (TSH, T3, T4) nor hepatic function related enzymes (GOT, GPT, GGT).
- Remarks: See Tables no. 6 and 7 below.

PATHOLOGY
- Results: No statistical differences were found among feeding treatments for all parameters of necropsy.
- Remarks: See Table no. 8 below.

Further details on results:

No differences were observed between the control and the treatment groups regarding neither the thyroid hormones (TSH, T3, T4) nor hepatic function related enzymes (GOT, GPT, GGT).

Reported statistics and error estimates:

Student “t” Test was used to compare the means of each group. The level of significance to indicate differences stated in the ANOVA model were P≤0.05 when the difference was statistically significant, while 0.05

Table 1. Mortality:

Experimental periods		T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000 g/ton. feed	T4 BHT 1500 g/ton. feed
0-10 d	n.	0	0	0	0
10 - 28 d	n.	1	0	0	1
28 - 35 d	n.	0	0	0	0
0 - 35 d	n.	1	0	0	1
Total mortality / culling	%	0.74	0	0	0.74
		0.37

Table 2. BHT content in feed:

Study periods	BHT, mg/kg (mean ± standard deviation, n=4)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000 g/ton. feed	T4 BHT 1500 g/ton. feed
D0-D10	Not detected	147.4±6.1	968.7±31.6	1419.4± 16.6
D10-D28	Not detected	156.7±6.4	1025.9±38.0	1448.4±58.6
D28-D35	Not detected	143.0±2.4	955.4±20.0	1518.9±35.7

Table 3. Body weights:

Live weight and average daily gain (mean; n = 9)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect (P)	Standard error of the mean
Live weight (g)
D0	47.0	47.3	47.0	47.1	0.8749	0.3091
D10	215.6	212.4	212.4	213.4	0.8467	2.8989
D28	1168.4	1169.0	1149.5	1120.9	0.1013	15.0653
D35	1655.6	1634.8	1633.0	1598.1	0.2925	20.9656
Average daily gain (g)
D0-D10	16.9	16.5	16.5	16.6	0.8087	0.2779
D10-D28	52.9	53.1	52.1	50.3	0.0676	0.7770
D28-D35	69.6	66.5	69.1	68.2	0.6973	1.9354
D0-D35	45.9	45.4	45.3	44.2	0.2818	0.6011

Table 4. Food intake and food gain:

Feed intake and feed:gain ratio (mean; n = 9)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect (P)	Standard error of the mean
Feed intake (g)
D0-D10	24.0	23.2	23.5	24.6	0.2884	0.5452
D10-D28	82.9	87.0	87.1	84.0	0.2669	1.8105
D28-D35	132.7	129.4	137.5	134.5	0.2839	2.9387
D0-D35	75.9	77.2	79.0	77.0	0.4601	1.3620
Feed:Gain ratio
D0-D10	1.42	1.40	1.42	1.48	0.3331	0.0293
D10-D28	1.57	1.64	1.67	1.67	0.1358	0.0338
D28-D35	1.91	1.95	1.99	1.99	0.4622	0.0397
D0-D35	1.66	1.70	1.74	1.74	0.0723	0.0256

Tabla 5. Haematology:

Blood haematological parameters after 35 days from the start of the study (mean; n = 9)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment effect (P)	Standard error of the mean
WBC (1,000/ml)	19.64	19.57	17.84	18.19	0.5147	1.05133
RBC (1,000,000/ml)	2.53	2.51	2.49	2.43	0.6444	0.05651
Haemoglobin (%)	12.89	12.83	12.54	12.41	0.6245	0.29756
Haematocrit (%)	31.20	29.94	29.72	30.39	0.6610	0.89137
MCV (fl)	123.56	119.33	119.44	124.44	0.2671	2.28876
MCH (pg)	50.84	51.11	50.47	51.11	0.6862	0.43197
MCHC (%)	41.37	42.86	42.21	41.28	0.4243	0.76405
Neutrophils (%)	26.83	30.50	26.67	28.84	0.7711	2.96711
Lymphocytes (%)	67.71	63.82	68.17	65.91	0.7177	2.94052
Monocytes (%)	2.21 a	2.80 b	2.50 ab	2.29 a	0.0278	0.14164
Eosinophils (%)	0.87	0.90	0.76	0.74	0.6599	0.10679
Basophils (%)	2.38	1.98	1.91	2.21	0.3768	0.20807
Platelets (1,000/mmc)	64.77	62.52	62.52	62.47	0.7332	0.72566
Reticulocyte (%)	0.00	0.00	0.00	0.00	-	-
RPI (%)	0.00	0.00	0.00	0.00	-	-
MPV (fl)	8.52	8.98	8.85	8.88	0.3404	0.18535
PCT (%)	0.39	0.39	0.44	0.40	0.7358	0.03679
PDW	19.78	18.85	19.28	19.29	0.3087	0.34608

WBC = white blood cells; RBC = red blood cells; MCV = mean volume of erythrocytes; MCH = mean content of haemoglobin; MCHC = mean concentration of haemoglobin in erythrocytes; RPI = Reticulocyte production index; MPV = mean platelet volume; PCT = platelet crit (percent volume of the blood occupied by platelets); PDW = platelet distribution width.

a,b.: Different letters in the same row = differences significant (P<0.05)

Table 6. Biochemistry:

Blood biochemical parameters after 35 days from the start of the study (mean; n = 9)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton.feed	Treatment  Effect (P)	Standard error of the mean
Glucose (mg/dl)	226.89	222.56	221.33	224.11	0.8476	4.62706
Urea (mg/dl)	6.22 b	6.38 b	5.10 a	5.79 ab	0.0402	0.32302
Uric acid (mg/dl)	5.57	5.55	5.75	5.82	0.5774	0.16109
Creatinine (mg/dl)	0.61 b	0.60 b	0.53 a	0.58 ab	0.0414	0.02035
Cholesterol (mg/dl)	142.89	140.00	131.67	132.33	0.3788	5.41952
Triglycerides (mg/dl)	87.56	89.78	86.44	90.89	0.9213	5.04318
Total Bilirubin (mg/dl)	0.12	0.14	0.11	0.11	0.0795	0.00850
GOT (U/l)	198.99	213.89	215.44	199.11	0.2819	7.86361
GPT (U/l)	12.56	13.56	13.22	13.33	0.6952	0.61802
GGT (U/l)	17.56	15.22	16.00	15.89	0.2808	0.85527
Alkaline phosphatase (U/l)	491.44	484.22	508.00	496.78	0.6483	13.43985
Lactate dehydrogenase (U/l)	926.67	976.44	913.78	925.44	0.1334	19.68379
Calcium (mg/dl)	12.28	11.46	11.20	12.22	0.1253	0.37847
Inorganic P (mg/dl)	9.19	8.60	8.40	8.97	0.7749	0.58202
Phospholipides (mg/dl)	284.56	267.89	256.67	272.56	0.4827	12.60750
Haptoglobin (mg/dl)	5.30	4.40	5.01	5.16	0.2148	0.31577
Serum albumin (g/dl)	1.15	1.14	1.09	1.17	0.1275	0.02474
Serum total protein (g/dl)	5.56	5.56	5.47	5.50	0.9132	0.10515
INR value	1.52	1.42	1.50	1.51	0.8452	0.08700
Prothrombin value (sec)	14.77	15.10	15.24	15.24	0.8672	0.45918
TSH (mU/ml)	0.33	0.39	0.33	0.29	0.5962	0.05305
T3 (mU/ml)	6.76	6.68	6.34	6.91	0.5258	0.27455
T4 (mU/ml)	0.85	0.86	0.86	0.81	0.8721	0.04954

GOT = Aspartate Transminase; GPT = Alanine Transminase, GGT = gamma-glutamyl transpeptidase, INR value = International Normalised Ratio; TSH = Thyroid stimulating hormone; T3 = Triiodothyronine; T4 = Thyroxine.

Table 7: Individual results on TSH, T3 and T4 (thyroid hormones):

Treatment	Pen	TSH	FT3	FT4
T1	1	0.22	6.5	0.77
T1	5	0.33	6.6	0.61
T1	10	0.19	5.8	0.84
T1	12	0.57	7.7	1.2
T1	20	0.31	7.5	0.74
T1	21	0.26	7.6	0.88
T1	24	0.59	6.1	0.87
T1	25	0.33	5.4	0.85
T1	31	0.16	7.6	0.85
T2	2	0.26	6.4	0.71
T2	9	0.44	5.1	0.8
T2	13	0.33	6.9	0.98
T2	17	0.22	6.7	0.65
T2	18	0.25	7.1	0.91
T2	19	0.34	7.3	0.99
T2	30	0.94	7.7	0.79
T2	32	0.17	6.8	0.87
T2	34	0.55	6.1	1.02
T3	3	0.27	5.3	0.79
T3	8	0.46	7.9	0.75
T3	11	0.41	5.6	0.77
T3	14	0.54	6.8	0.94
T3	16	0.37	6.5	0.81
T3	23	0.24	6.7	1.2
T3	27	0.19	6.8	0.78
T3	28	0.36	6.1	0.74
T3	35	0.16	5..4	0.99
T4	4	0.34	5	0.64
T4	6	0.27	7.7	0.66
T4	7	0.21	7.1	0.81
T4	15	0.34	56.7	0.78
T4	22	0.22	7.8	0.71
T4	26	0.21	7	1.01
T4	29	0.35	7	0.69
T4	33	0.24	6.6	0.99
T4	36	0.39	7.3	1

Tabla 8. Necropsy:

Necropsy results (mean; n = 9)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect(P)	Standard error of the mean
External skin	0.11	0.00	0.00	0.11	0.5787	0.07857
Leg	0.22	0.11	0.22	0.22	0.9224	0.13889
Eyes	0.00	0.00	0.00	0.00	_	_
Anus	0.11	0.11	0.00	0.00	0.5787	0.07857
Oral cavity	0.00	0.00	0.00	0.00	_	_
Oesophagus	0.00	0.00	0.00	0.00	_	_
Stomach	0.11	0.11	0.00	0.00	0.5787	0.07857
Gizzard	0.00	0.00	0.00	0.00	_	_
Small intestine	0.22	0.22	0.22	0.11	0.9224	0.13889
Caecum	0.11	0.00	0.11	0.00	0.5787	0.07857
Colon	0.11	0.11	0.11	0.11	1.0000	0.11111
Pancreas	0.00	0.00	0.00	0.00	_	_
Liver	0.22	0.33	0.22	0.22	0.9395	0.15215
Lungs	0.11	0.11	0.00	0.11	0.8013	0.09623
Kidneys	0.00	0.00	0.00	0.00	_	_
Heart	0.00	0.00	0.00	0.00	_	_
Muscle	0.22	0.00	0.11	0.00	0.2808	0.09213
Fat	0.11	0.00	0.11	0.00	0.5787	0.07857
Score: 0 = no alteration found;1=slight alterations; 2 = alteration of medium intensity; 3 = serious alteration

Validity criteria fulfilled:

not applicable

Conclusions:

The administration of BHT to chickens for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 150,3-268,1 mgBHT/kg bw), did not cause any adverse effects under the test conditions. No adverse effects were observed related neither to the thyroid hormones (TSH, T3, T4) nor hepatic function (GOT, GPT, GGT).

Executive summary:

In the present study, the tolerance of of chickens for fattening to Butylated hydroxytoluene (BHT) as technological feed additive was evaluated (GLP study). The study was carried out with 540 chickens at the start of the study. The treatments were: 1) Control group T1: animals fed with basal diet; 2) T2: animals fed basal (T1) diet supplemented with BHT at 150 g/tonne feed (recommended dose, ca. 13.2 -22.3 mgBHT/kg bw)); 3) T3: animals fed basal (T1) diet supplemented with BHT at 1000 g/tonne feed (6.7 x recommended dose, ca. 94.4 -175.5 mgBHT/kg bw) and 4) T4: animals fed basal (T1) diet supplemented with BHT at 1500 g/tonne feed (10x recommended dose, ca. 150.3 -268.1 mgBHT/kg bw). The feeds were issued to the appropriate pens for 35 consecutive days. Each feeding treatment was replicated in 9 pens. The data recorded during the feeding phase were live weight (LW) at 0, 10, 28 and 35 days from the start of the study, average daily gains (ADG), daily feed intake and feed:gain ratio (F:G) during the periods 0-10, 10-28, 28-35 and 0-35 days from the start of the study. At 35 days, blood samples were taken from one animal per pen (36 birds in total, 9 per treatment) and analysed for routine haematology and biochemistry parameters. The same 36 animals were then sacrificed and necropsied by the veterinary surgeon responsible for animal welfare. The results of the study show a low mortality. The animals were in good general health as recorded during the veterinary inspections. No statistical differences were found among feeding treatments for the all performance parameters; only in the global period of the trial (D0-D35) the feed:gain ratio was tendentially higher in T4 and T3 groups in comparison with T1 group. Concerning the blood parameters statistical differences were found for: Monocytes that were higher in T2 vs. T1 and T4 groups but no difference occurred between T1 and T4 group; Urea was lower in T3 vs. T1 and T2 groups but no difference occurred between T1 and T4 group; Creatinine was lower in T3 vs. T1 and T2 groups but no difference occurred between T1 and T4 group; Bilirubin was tendentially higher in T2 vs. T3 and T4 groups but no difference occurred between T1 and T4 group. All measured blood haematological and biochemical parameters were within the respective reference ranges. No statistical differences were found among feeding treatments for the all necropsy evaluations. In conclusion, the administration of BHT to chickens for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 150,3-268,1 mgBHT/kg bw), did not cause any adverse effects under the test conditions. It should be highlighted that no test item related adverse effects were observed regarding neither the thyroid hormones (TSH, T3, T4) nor the hepatic function (GOT, GPT, GGT).

Description of key information

In accordance with column 2 of REACH Annex X, long-term or reproductive toxicity to birds studies do not need to be conducted taking into account the large mammalian dataset that is available.

The administration of BHT to chickens for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 150,3-268,1 mgBHT/kg bw), did not cause any adverse effects under the test conditions. No adverse effects were observed related neither to the thyroid hormones (TSH, T3, T4) nor hepatic function (GOT, GPT, GGT).