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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
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

Objective of study:
other: The aim of the study was to evaluate 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.
Principles of method if other than guideline:
The study was carried out with 8 Italian Friesian dairy cows. The total study duration was 24 days as sum of 14 days of pre-experimental period for animals’ adaptation to the calan gate system and 10 consecutive days of Butylated hydroxytoluene (BHT) administration. The data recorded during each of the 10 days of BHT administration for each animal were: live weight, milk production and feed intake. 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no data
Specific details on test material used for the study:
EU Nº: Feed additive E 321
Dosage: 150 mg/kg complete feed with a dry matter content of 88 % equivalent to 90.8 mg BHT/kg TMR with a dry matter content of 53.28 %
Trade Name: Rovelox® 100
Batch No.: A 19112013
Formulation: Butylated hydroxytoluene (BHT)
Physical
presentation: powder
Radiolabelling:
no

Test animals

Species:
other: diary cow
Strain:
other: The study was carried out with 8 (+ 4 spare animals) Italian Friesian dairy cows.
Sex:
female
Details on test animals and environmental conditions:
The study was carried out with 8 (+ 4 spare animals) Italian Friesian dairy cows. The total study duration was 24 days as sum of 14 days of pre-experimental period for animals’ adaptation to the calan gate system and the new disposition in the stall and 10 consecutive days of BHT administration. The animals were selected out of groups in the Assay Centre breeding without disease problem, and assigned to the treatment in order to achieve maximum possible homogeneity between animals.

Study site location and setup
The study took place at the CERZOO facility. The housing management, feeding and husbandry conditions are considered as representative for a modern commercial operation in Europe.

Housing system and cows handling at study start
The animals used for the study were reared in one stall characterised by a feeding aisle on concrete floor and a resting area with individual berths on straw and chip litter. Rearing stalls were thoroughly cleaned and the litter was replaced before animals were introduced. Before groups formation, the animals were observed daily for 14 consecutive days during the adaptation period (from D-14 to D0). At the end of this period a general check-up was performed by the veterinary designed for animal welfare in order to evaluate whether animals were eligible to be used in the study. All the animals were reared into the same pen. The TMR was given individually with calan gate system according to the procedures which are normally used in the farm. The animals were identified by the farm number written on coloured ear tags.

Feeding and watering equipment
The animals were fed with a TMR containing mainly corn silage, alfalfa hay, grass hay, concentrates and water. The stated feeding form was the same throughout the study. With the exception of the test product, the animals did not receive any other products for the entire trial period. The TMR was given once daily (in the morning). 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. The discharge was performed by a lateral outlet in the cart and controlled manually by the operator. The same amount of TMR was provided to each animal. Drinking water was supplied ad libitum by an internal water system network. To ensure potability, the water is analysed annually for sodium, chloride, potassium, calcium content, Salmonella spp. and E. coli.
8.4.- Environmental control
During the whole study period the climatic conditions and lighting scheme were natural.
8.5.- Milking of animals
Milk samples were collected in the milking parlour, where milking stalls were arranged in a herringbone pattern (6+6). Every stall was equipped with AFIMILK control panel which shows the company number of the cow identified through the pedometer. The cow’s production data were recorded every day.
8.6.- Animals
The 8 animals selected for the study from the 12 animals available at the study start (8 + 4 spare animals), were singled out among those present at the CERZOO Research Centre farm in such a way that they were typical of the age of the animal breaded in Italian farm (n. of lactations: 3 primiparous cows, 3 second parity cows and 2 third parity cows). The animals that were singled out were identified basing on the farm numbers written on coloured ear tags.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Duration and frequency of treatment / exposure:
Ten days daily.
Doses / concentrations
Dose / conc.:
4 157 other: mg BHT/animal and day
No. of animals per sex per dose:
Eight diary cows per dose.
Control animals:
no
Positive control:
None.
Details on study design:
Background
This study was carried out to provide data concerning the safety for the consumer regarding the milk produced by target animals species (dairy cows) fed Total Mixed Ration (TMR) containing Butylated hydroxytoluene (BHT).

Objective of the study
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 TMR containing the maximum authorized dose of the test product. Equipment, animals and husbandry conditions available today and regarded as representative of the present state of art have been used.

Animals
The 8 animals selected for the study from the 12 animals available at the study start (8 + 4 spare animals), were singled out among those present at the CERZOO Research Centre farm in such a way that they were typical of the age of the animal breaded in Italian farm (n. of lactations: 3 primiparous cows, 3 second parity cows and 2 third parity cows). The animals that were singled out were identified basing on the farm numbers written on coloured ear tags.

Study design and animal allotment
The animals were selected among the animals available according to their milk production, parity and DIM (Days in Milk) so to have an homogeneous group. Lactating dairy cows were fed according to standard feeding plan, following the removal and weighed of the previous day’s refusal TMR (Total Mixed Ration).
Details on dosing and sampling:
Treatments
The test product was provided to all the 8 selected animals at the nominal level of 150 mg/kg of complete feed based on a dry matter content of 88 % equivalent to 90.8 mg BHT/kg TMR with a theoric dry matter content of 53.28 %. Following the determinations of the TMR dry matter content at the study start (52.49 %), the BHT provided per kg of TMR was 89.5 mg. The daily amount of the test product was calculated as follows:
- Dosage: 150 mg BHT/kg complete feed with a dry matter content of 88 % equivalent to 89.5 mg BHT/kg TMR with a measured dry matter content of 52.49 %;
- Daily quantity of TMR provided to each animal: 46.45 kg as fed;
- Daily quantity of BHT provided to each animal (46.45 kg TMR x 89.5 mg BHT): 4157 mg BHT/animal and day.

Analyses
- Milk composition
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 residual amount of milk will be stored in CERZOO at -20°C for three months from the end of the study (aliquot C).
Statistics:
Data of milk yield, animals’ weight, TMR intake, BHT content in milk, milk quality parameters recorded during the 10 days of treatment were analyzed as repeated measurements in a completely randomized design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, release 9.3, 2002-2010).

Results and discussion

Preliminary studies:
No data.
Main ADME results
Type:
excretion
Results:
BHT, when provided to lactating dairy cows at the maximum authorized dosage level (150 mg BHT/kg of complete feed) for 10 consecutive days, did not determine the occurrence of detectable residues in milk considering a LOD of 0.005 mg/kg.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
No data.
Details on distribution in tissues:
No data.
Details on excretion:
The Butylated hydroxytoluene (BHT), when provided to lactating dairy cows with Total Mixed Ration at the maximum authorized dosage level (150 mg BHT/kg of complete feed based on a dry matter content of 88 %, therefore equivalent to 89.5 mg BHT/kg of TMR considering a dry matter content of 52.49 %) for 10 consecutive days, did not determine the occurrence of detectable residues in milk considering a LOD of 0.005 mg/kg.
Toxicokinetic parameters
Key result
Test no.:
#1
Toxicokinetic parameters:
other: BHT, when provided to lactating dairy cows at the maximum authorized dosage level (150 mg BHT/kg of complete feed) for 10 consecutive days, did not determine the occurrence of detectable residues in milk considering a LOD of 0.005 mg/kg.

Metabolite characterisation studies

Metabolites identified:
not measured

Bioaccessibility

Bioaccessibility testing results:
No data.

Applicant's summary and conclusion

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

The aim of the study was to evaluate 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.

The study was carried out with 8 Italian Friesian dairy cows. The total study duration was 24 days as sum of 14 days of pre-experimental period for animals’ adaptation to the calan gate system and 10 consecutive days of Butylated hydroxytoluene (BHT) administration. The animals were reared into the same pen for the entire study period. The TMR was given individually with calan gate system. The test product was provided to all the 8 animals at the nominal level of 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 %. The data recorded during each of the 10 days of BHT administration for each animal were: live weight, milk production and feed intake. 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 TMR provided to the animals at D0 had an analytical dry matter content of 52.49 %. Consequently, the actual level of inclusion of BHT (pre-diluted in one component of the TMR) during each of the 10 days of the study and to each animals was 89.5 mg BHT/kg of TMR. 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) in all cases.

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