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

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Residue in dairy cows milk

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2014 - 14 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Principles of method if other than guideline:

The aim of the study was the evaluation of residues of BHT as technological feed additive (E 321) in lactating dairy cows milk, when provided with Total Mixed Ration (TMR) containing the maximum authorized dose of the test product. After 14 days of pre-experimental period of adaptation to the galan gate system, 8 Italian Friesian dairy cows were exposed to 150 mg BHT/kg of complete feed ( based on a dry matter content of 88 % equivalent to 90.8 mg BHT/kg TMR assuming a dry matter content of 53.28 %) during 10 days. The animals were reared into the same pen for the entire study period. Live weight, milk production and feed intake were daily recorded. Milk composition (protein, fat, lactose, urea, somatic cells count and BHT content) was however determined at D0, D8, D9 and D10 from the beginning of the study.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

other: Residue in cow milk

Test material

Test material information

Constituent 1

Test animals

Species:

cattle

Strain:

other: Italian Friesian

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CERZOO Research Centre farm.
- Age at study initiation: 3 primiparous cows, 3 second parity cows and 2 third parity cows
- Weight at study initiation: 630.1± 42.7 kg
- Housing: Grouped in one stall characterised by a feeding aisle on concrete floor and a resting area with individual berths on straw and chip litter.
- Diet (e.g. ad libitum): Once daily (in the morning).
- Water (e.g. ad libitum): Ad libitum (internal water system network).
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS: During the whole study period the climatic conditions and lighting scheme were natural.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test product (BHT) was pre-diluted in one component of Total Mixing Ration (TMR) (corn + sorghum mix.) in CERZOO Feed mill using an horizontal mixer of 6 ton. of capacity. The components of TMR were mixed in the Labrador cart (capacity of 8 m3). The pre-diluted test product was added as last component.

With the exception of the test product, the animals did not receive any other products for the entire trial period. The TMR was given individually with calan gate system according to the procedures which are normally used in the farm. The same amount of TMR was provided to each animal.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): The components were mixed in the cart in the following order: corn silage, hays, corn mix. (corn meal + barley flakes: ratio 60:40 and corn meal + sorghum meal: ratio 80:20), concentrate and water. See Table no. 1 below.

See Table no. 1.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

BHT in milk, corn +sorghum mixture or TMR specimens was extracted in n-hexane (using ultra-turrax when necessary, to comminute during extraction). The extract was centrifuged, and milk extract defatted by freezing overnight. The supernatants was eventually diluted or concentrated in nitrogen stream, and then analysed by gas chromatography coupled to mass spectrometry (GC/MS).

The method for BHT measurement was validated in milk (recovery tests at 0.012, 0.6, 1.6 and 12 mg/kg spiking levels – two recoveries per level). A certified reference standard was used for both calibration and spiking of the samples. The limit of detection was calculated as the lowest concentration having a signal-to-noise signal of 3 as minimum. The limit of quantification, however, was set at the lowest spiking level at which both accuracy and precision are adequate.

Duration of treatment / exposure:

10 days

Frequency of treatment:

Daily

Doses / concentrations

Dose / conc.:

150 other: mg BHT/kg feed

No. of animals per sex per dose:

8

Control animals:

no

Details on study design:

Milk samples were collected in the milking parlour, where milking stalls were arranged in a herringbone pattern (6+6).

Examinations

Examinations:

CAGE SIDE OBSERVATIONS/CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and in the afternoon.
- Parameters: Checking the general health status and recording unexpected events.
*Adverse events: e.g. power failure, feed/water failures, disease outbreak, etc.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Health/illness records (daily). Diarrhoea scoring (semi-objective), culling and mortality.

BODY WEIGHT: Yes
- Time schedule: Daily, as mean of two measurements at the two milking operations (morning and afternoon).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: Daily TMR intake recorded for each animal from D1 to D10. The refused TMR was quantified after 24 hours before the new TMR administration.

FEED (TMR) ANALYSIS: Yes
- Time schedule and parameters:
*At the study start for dry matter (DM), crude protein (CP), ADF, NDF, starch, non-fibre carbohydrates (NFC) crude fat, metabolized energy (ME) and net energy lactation (NEl, calculated).
*BHT content every day from D1 to D10 as pool of TMRs provided to each of the 8 animals.
*Nutrient composition of each ingredient of the TMR: DM, CP, ADF, NDF, starch, NFC, crude fat, ME and NEl (predicted).

MILK ANALYSIS: Yes
- Time schedule for examinations: The milk samples were taken at D0 and during the last three days of the study period (D8, D9, D10) at each milking (morning and afternoon) separately.
- Parameters: BHT content and for the standard parameters as fat, protein, lactose, somatic cells and urea.
- Method: The individual milk samples were taken upon the morning and afternoon milking operation. Each milk sample was divided into 3 aliquots (A, B, C) and analysed for BHT content (aliquot A), for the standard milk quality parameters: fat, protein, lactose, somatic cells and urea (aliquot B). The analytical methods used were infrared spectrometry for fat, protein, lactose and urea and optofluorometry for somatic cells.

Results and discussion

Details on results:

Health conditions of the animals:
During the study no animals were excluded. No health problems or situation had marked effect on milk quality and production or had effect on anticipated reduction of milk yield during lactation were recorded. See Table no. 2.

TMR composition:
The BHT content in the TMR sampled every day from each animal and pooled was in accordance with the expected value of 89.5 mg BHT/kg TMR with a D0 measured dry matter content of 52.49 %. The actual mean value resulted in 99 ± 5 % of the intended dosage. See Tables no. 3 and 4.

Dairy cows body weight, milk production, TMR and BHT intake:
The mean BHT daily intake resulted in 3825 mg/d. See Tables no. 5 and 6.

Milk composition:
See Tables no. 7 and 8.

Milk components outputs:
See Tables 9 and 10.

BHT residues in milk:
The animals were kept in sustainable conditions and their performance (live weight, milk production and feed intake) and of the milk quality (milk composition and milk component output) were in line with the physiological processes associated with lactation and/or growth. The animals variability didn’t affect the milk residual content of BHT: the analysis of each milk sample collected at each milking from all animals during the last three days (D8, D9, D10) of the study period (D1-D10) didn’t show residual content of BHT.
Recovery tests demonstrated that the method applied was fitting for purposes in the range 0.012 mg/kg to 12 mg/kg, being mean recovery (used to express accuracy) 103 %, with a relative standard deviation (used to express precision) of 3 %. Limit of detection, calculated as the lowest concentration having a signal-to-noise of 3 as minimum, was 0.005 mg/kg. Limit of quantification was set as the lowest spiking level having adequate accuracy and precision; actual value was 0.012 mg/kg.
See Tables no. 11 and 12.

Any other information on results incl. tables

Table 1. Chracteristics of the animals at D-14:

Cow n.	Number of parity	Days of lactation	Live Weight (kg)	Milk production (kg)
66	1	177	618	28.5
67	1	166	596	31.5
413	3	216	672	32.4
465	2	229	626	29.4
472	2	158	591	40.7
481	3	287	665	31.9
485	2	188	695	37.1
520	1	224	578	31.2
Mean士SD	1.88±0.83	205.6±42.4	630.1±42.7	32.8±4.1

Table 2. Adverse effects:

Period	Date	Cow n.	Event
D-14-DO	September 18,2014	66	Slight pneumonia
D1-DIO	September 29,2014	472	Sole ulcer
	September 30,2014	413	Nipple lesion
	October 7,2014	465	Sole ulcer

For sole ulcers the farrier applied podal soles, nipple lesion was locally disinfected with chlorhexidine, while for the slight pneumonia no antibiotics were used.

Table 3. Analytical characteristics of the raw materials used for TMR production:

	Corn silage (1)	Concentrate (2)	Corn meal+ barley flake mix. (60:40) (3)	Corn meal + Sorghum meal mix.(80:20) (3)	Rye grass Hay (4)	Dehydrated alfalfa hay (4)
Number of lot analysed	1	1	1	1	1	1
Dry matter (% FM)	36.34	89.66	84.92	89.69	90.02	97.14
Crude protein (% DM)	8.47	35.12	9.64	12.36	10.65	16.99
Crude fat (% DM)	2.46	4.35	2.91	3.95	1.12	1.14
Starch (% DM)	23.24	12.92	61.99	65.58	3.24	4.34
ADF (% DM)	25.64	8.26	3.07	4.12	36.02	31.12
NDF (% DM)	47.50	18.69	9.63	13.24	56.57	42.14
NFC (% DM)	37.72	27.40	76.31	69.03	25.61	33.87
Ash (% DM)	4.75	14.89	1.79	1.72	9.25	9.74
Metabolisable energy (5) (Mcal/kg DM)	2.27	2.80	3.18	3.09	1.95	2.04
Net Energy Lactation (5) (Mcal/kg DM)	1.41	1.78	2.04	1.99	1.18	1.24

Note: 1) Produced in the CERZOO facility, 2) Provided by LUIGI FERRARI, Sarmato (PC) Italy, 3) Provided by Consorzio Agrario of Piacenza (PC) Italy; 4) Provided by ALIVERDE (RA) Italy; 5) Predicted.

Table 4. TMR analysis:

Dry matter	%FM	52.49
Crude protein	%DM	14.63
Crude fat	%DM	3.66
Starch	%DM	28.26
ADF	%DM	22.54
NDF	%DM	37.12
Ash	%DM	5.12
NFC	%DM	40.88
Metabolizaole energy	Mcal/kg DM	2.63
Net Energy Lactation	Mcal/kg DM	1.66
BHT (1,2)	mg/kg FM	88.3(±4.9)

Note 1: The actual amountof BHT in the com + sorghum mix. was 824士69 mg/kg FM.

Note 2: Mean value content in the 10 daily TMR samples.

Table 5. Body weight, milk production, feed intake at D0:

Cow n.	Body weight (kg)	Milk production (kg/d)	Feed intake (kg as fed)
66	629	32.4	46.3
67	605	31.2	40.9
413	680	32.6	46.3
465	637	31.3	42.1
472	582	38.8	43.9
481	661	31.6	41.5
485	712	36.9	46.3
520	585	29.9	45.5
Mean ± SD	636.4±46.1	33.1±3.1	44.1±2.3

Table 6. Body weight, milk production, feed intake and BHT intake during the experimental period (D1 -D10):

Cow n.	Body weight (kg)	Milk production (kg/d)	Feed intake (kg as fed)	BHT intake (1) (mg/d)
66	631	30.6	45.35	3998
67	591	28.5	40.63	3577
413	688	27.1	46.30	4079
465	648	31.2	41.61	3677
472	584	36.6	44.86	3953
481	670	31.6	39.92	3517
485	698	35.1	46.21	4072
520	589	30.8	42.38	3730
Mean^2	638	31.4	43.41	3825
SEM^J	2.7121	0.5150	0.6245	71.4981

Note 1: Mean value of the 10 daily data; Note 2: Arithmetic mean; Note 3: Standard error of the mean.

Table 7. Milk composition at D0:

Cow n.	Milk fat (%)	Milk protein (%)	Milk lactose (%)	Milk urea (mg/dL)	Somatic cells count (n. x 1000/mL)
66	3.80	3.36	4.75	20.5	56
67	3.99	3.19	5.06	23.5	35
413	4.47	3.44	4.70	21.6	32
465	3.73	3.35	4.71	22.6	26
472	4.07	3.18	4.75	20.9	22
481	4.37	3.42	4.69	23.9	27
485	3.86	3.46	4.91	22.9	53
520	3.61	3.21	5.04	23.8	338
Mean±SD	3.99±0.30	3.33±0.12	4.83±0.15	22.46±1.32	73.63±107.55

Table 8. Milk composition in the last 3 days of the experimental period:

Cow n.	Milk fat (%)	Milk protein (%)	Milk lactose (%)	Milk urea (mg/dL)	Somatic cells count (n. x 1000/mL)
66	3.69	3.24	4.76	29.6	88
67	4.04	3.26	5.02	30.6	17
413	4.61	3.49	4.30	25.0	260
465	3.69	3.34	4.67	27.1	66
472	3.68	3.14	4.78	31.0	85
481	4.44	3.39	4.58	26.7	73
485	3.94	3.42	4.90	30.1	76
520	3.21	3.25	5.11	34.3	181
Mean (1)	3.92	3.32	4.77	29.3	106
SEM	0.0704 (2)	0.0813 (2)	0.0584	1.9490	48.6227

Note 1:Arithmetic mean; Note 2: Harmonic mean.

Table 9. 4% FCM and milk components outputs at D0:

Cow n.	4%FCM	Milk fat	Milk protein	Milk lactose
	(kg/d)	(kg/d)	(kg/d)	(kg/d)
66	31.43	1.23	1.09	1.54
67	31.13	1.24	1.00	1.58
413	34.90	1.46	1.12	1.53
465	30.01	1.17	1.05	1.47
472	39.21	1.58	1.23	1.84
481	33.35	1.38	1.08	1.48
485	36.10	1.42	1.27	1.81
520	28.15	1.08	0.96	1.51
Mean±SD	33.04±3.59	1.32±0.17	1.10±0.11	1.60±0.15

Table 10. 4% FCM and milk components outputs in the last 3 day of the experimental period:

Cow n.	4%FCM (kg/d)	Milk fat (kg/d)	Milk protein (kg/d)	Milk lactose (kg/d)
66	27.80	1.08	0.95	1.39
67	30.03	1.21	0.98	1.50
413	28.19	1.19	0.90	1.11
465	28.88	1.12	1.01	1.41
472	36.45	1.41	1.20	1.83
481	33.48	1.39	1.06	1.44
485	35.43	1.41	1.22	1.75
520	27.80	1.02	1.01	1.59
Mean (1)	31.01	1.23	1.04	1.50
SEM	0.7470	0.0449(2)	0.0160	0.0439(2)

Note 1:Arithmetic mean; Note 2: Harmonic mean.

Table 11. BHT residues in milk at D0 and in the last 3 days of experimental period:

	DO		D8		D9		D10
Cow n.	A.M. milking	P.M. milking	A.M. milking	P.M. milking	A.M. milking	P.M. milking	A.M. milking	P.M. milking
66	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
67	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
413	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
465	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
472	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
481	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
485	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D
520	N.D.	N.D	N.D.	N.D	N.D.	N.D	N.D.	N.D

Table 12. BHT recovery tests in milk:

Spiking level (mg/kg)	Measured concentration (mg/kg)	Recovery (%)	Meanrecovery (%)	Relative Standard Deviation (%)
0.012	0.013	109%	106	3
	0.013	104%
0.6	0.671	112%	111	1
	0.665	111%
1.6	1.675	105%	103	3
	1.612	101%
12	9.822	82%	93	17
	12.434	104%
Overall			103	9

Applicant's summary and conclusion

Conclusions:

The Butylated hydroxytoluene (BHT) when provided to lactating dairy cows with Total Mixed Ration at the maximum authorized level (150 mg BHT/kg of complete feed based on a dry matter content of 88 % equivalent to 89.5 mg BHT/kg TMR with a dry matter content of 52.49 %) for 10 consecutive days does not determine the occurrence of detectable residues in milk considering a LOD of 0.005 mg/kg.

Executive summary:

The study evaluated the residues of Butylated hydroxytoluene (BHT) in lactating dairy cows’ milk when administered with Total Mixed Ration (TMR) at the maximum authorized dose of test product (150 mg BHT/kg feed) (GLP study). After 14 days of pre-experimental period of adaptation to the galan gate system, 8 Italian Friesian dairy cows were exposed to 150 mg BHT/kg of complete feed ( based on a dry matter content of 88 % equivalent to 90.8 mg BHT/kg TMR assuming a dry matter content of 53.28 %) during 10 days. The animals were reared into the same pen for the entire study period. Live weight, milk production and feed intake were daily recorded. Milk composition (protein, fat, lactose, urea, somatic cells count and BHT content) was however determined at D0, D8, D9 and D10 from the beginning of the study. The daily analysis of the TMR (pool of the TMR sampled to each of the 8 animals) during the 10 days of the study showed a mean intake of 88.3 ± 4.9 mg BHT / kg of TMR, corresponding to 99 % of the intended level. The physiological individual variability of the performance of the animals (live weight, milk production and feed intake) and of the milk quality (milk composition and milk component output) didn’t affect the milk residual content of BHT: the analysis of each milk samples collected at each milking from all animals during the last three days of the administration period (D1-D10) didn’t show residual content of BHT. Indeed, BHT residues were not detectable (< 0.005 mg/kg, LOD) in all cases.