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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
The tests was conducted based on OECD-GLP standard and OECD test guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Oryzias latipes
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
30 d
Remarks on exposure duration:
The Ministry of the Environment in Japan states that the test was conductec according to OECD guidelines (210), therefore it can be assumed that the duration was the one recommended by the OECD guideline i.e. 30 days post hatch for Oryzias latipes.
Reference substance (positive control):
not specified
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.053 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Key result
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Validity criteria fulfilled:
not specified
Conclusions:
The NOEC for the test subtance BHT in Oryzias latipes in the early-life stage toxicity test was 0.053 mg/L and the NOEC was 0.14 mg/L.
Executive summary:

An early-life stage toxicity test was conducted by the Ministry of the Environment in Japan based on OECD GLP-standards and OECD guideline 210. The NOEC of BHT in Oryzias latipes was 0.053 mg/L and the LOEC was 0.14 mg/L

Endpoint:
long-term toxicity to fish, other
Type of information:
experimental study
Adequacy of study:
key 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
Principles of method if other than guideline:
The objectives of the study was to evaluate the efficacy and tolerance of dietary BHT by its effects on growth performance, lipid metabolism, inflammation, anti-oxidant response, apoptosis and hepatosis induced by oxidative stress in largemouth bass. 30 fish per replicate (6) and dose feed with experimental diets with BHT supplement levels of 0, 150, 300 and 1500 mg/kg in diet (corresponding to 2.32, 4.72 and 23.8 (B1500) mg/kg bw/day respectively). The fish from each tank were batch weighed at the end of the growth trial. Twelve fish for each treatment (2 fish from each tank) were randomly selected and anaesthetized with chlorobutanol (300 mg/mL). The body weight, body length, liver and viscera weight were recorded individually to calculate condition factor (CF), hepatosomatic index (HSI), and viscerosomatic index (VSI), respectively. Blood samples were drawn from the caudal part for analysis and two liver samples near to the bile duct were collected for histology, and for antioxidant analysis and RNA isolation.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: henglong Chemical Co., Ltd, Shandong, China
- Substance name: BHT
- Purity: 99%
Analytical monitoring:
not specified
Vehicle:
yes
Remarks:
fish oil
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Each diet was extruded into 2 mm diameter pellets under the following extrusion condition as: feeding section (90 °C/5 s), compression section (150 °C/5 s) and metering section (120 °C/4 s) using a Twin-screwed extruder. All diets were stored at room temperature (25–30 °C) in the summer and kept out of the sun for 10 weeks, the same storage conditions used at fish farms.
- Vehicle: BHT was added into fish oil (0 (control), 0.15, 0.30 and 1.50 g/kg in diet).
Test organisms (species):
Micropterus salmoides
Details on test organisms:
TEST ORGANISM
- Common name: Largemouth bass
- Source: Sanshui platinum Aquafarm (Foshan, Guangdong, China)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Weight at study initiation (mean and range, SD): 6.20 ± 0.01 g
- Food type: See Table no. 1 below.
- Amount: Fish were fed to apparent satiation.
- Frequency: Twice daily (8:00 and 16:00)

ACCLIMATION
- Acclimation period: 4 weeks
- Acclimation conditions (same as test or not): Same as the test
- Type and amount of food: Control experimental diet without BHT (B0)
- Feeding frequency during acclimation: 24 h of starvation
- Health during acclimation (any mortality observed): No mortality observed
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
70 d
Test temperature:
23 ± 1 °C
pH:
pH = 7.5–8.5
Dissolved oxygen:
> 7.0 mg/L
Nominal and measured concentrations:
BHT supplement levels:
0 (B0), 150 (B150), 300 (B300) and 1500 (B1500) mg/kg in diet.
Resulting in: 2.32 (B150), 4.72 (B300) and 23.8 (B1500) mg/kg bw/day.
Details on test conditions:
TEST SYSTEM
- Test vessel: Tanks
- Size of vessel: 256 L
- Aeration: 24 h per day
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Photoperiod: 12 hr dark, 12 hr light.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The fish from each tank were batch weighed at the end of the growth trial. Twelve fish for each treatment (2 fish from each tank) were randomly selected and anaesthetized with chlorobutanol (300 mg/mL). The body weight, body length, liver and viscera weight were recorded individually to calculate condition factor (CF), hepatosomatic index (HSI), and viscerosomatic index (VSI), respectively. Blood samples were drawn from the caudal part of the sedated fish using anticoagulant syringes with 2% NaF and 4% potassium oxalate. Blood samples were centrifuged at 4000 rpm for 10 min at 4 °C to obtain plasma. Two liver samples near to the bile duct were collected for histology (fixed in 10% phosphate-buffered formalin solution with a pH of 7.2) or fast frozen in liquid nitrogen for antioxidant analysis and RNA isolation. All samples were stored at −80 °C until analysis. See details below.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2-5 fold
- Justification for chosing the doses: The highest level (1500 mg/kg) was designed to be 10-fold of the maximum approved level (150 mg/kg) for the tolerance test to
obtain the safety margin of BHT utilized in fish feed.
Reference substance (positive control):
no
Key result
Duration:
70 d
Dose descriptor:
NOEC
Effect conc.:
>= 23.8 other: mg/kg bw/day
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: No adverse effects observed at the highest dose tested
Remarks on result:
other: (1500 mg/kg in diet)
Details on results:
Growth performance and morphometric parameters:
All of the experimental groups showed high survival (> 97%), and there was no difference among groups. With an increased BHT intake, there were no significant differences in specific growth rates (GR) and feeding rates (FR) among the groups. The feed conversion ratio (FCR) in the B150 group was significantly lower than that in the B0 and B1500 groups (P < 0.05), but it was not different from the B300 group (P > 0.05). The condition factor in the B150 and B300 groups were significantly higher than in the B1500 group (P < 0.05). The hepatosomatic index and viscerasomic index of the B150 group were significantly higher than in the B300 group (P < 0.05).
See Table no.1 below.
Authors stated that carnivorous fish always have higher lipid requirements than poultry and most mammals, especially the largemouth bass is a fish species
very sensitive to peroxidation. Hence, they suppose that, for this species, it could need higher level of antioxidant in diet than 150 mg/kg.

Hematological parameters:
Lipid metabolism parameters were improved by BHT inclusion. TC, TG and LDL-C were significantly decreased and the HDL-C/TC ratio was increased in the BHT inclusion groups (P < 0.05). Significantly reduced plasma ALT after BHT feeding indicated that BHT ameliorated liver dysfunction in the largemouth bass (P < 0.05). Increased plasma AKP and TBA are biomarkers of clinical cholestasis. BHT inclusion reduced plasma AKP significantly (P < 0.05) but did not affect TBA among groups (P > 0.05).
See Table no. 2 below.
In the present study, plasma clinical parameters data clearly demonstrated the efficacy of BHT in all groups for improving lipid metabolism and antioxidant responses by decreasing plasma TC, TG, LDLC, and MDA concentrations and increasing HDL-C/TC, GSH-Px and TAOC. On the other hand, the increased plasma ALT and AKP levels of the B0 group indicated that oxidative stress could induce hepatic dysfunction and hepatotoxicity in largemouth bass. In contrary, administration of BHT markedly improved hepatic function via decreasing plasma AKP and ALT activities in largemouth bass.

Antioxidant response:
Plasma MDA in the B150 group was significantly lower than that of the B0 group (P < 0.05). There were no significant differences in plasma SOD and CAT activities (P > 0.05). Extremely increased GSH-Px in plasma was observed in all groups fed BHT, and fish in the B300 group had higher T-AOC than those in the B150 group (P < 0.05). In liver tissue, T-AOC was significantly enhanced and O2−% was significantly decreased in each BHT inclusion group compared to the B0 group (P < 0.05), as well as hepatic MDA content was significantly decreased in B1500 group (P < 0.05). There were no significant differences in hepatic CAT and SOD activities (P > 0.05). The results indicated that BHT could prevent the accumulation of free radicals and improve antioxidant activities in the liver of largemouth bass.
See Table no. 3 below.

Lipid metabolism genes expression:
Compared with the B0 group, the mRNA level of PPAR-γ was significantly reduced in the B1500 group (P < 0.05). Dietary BHT positively improved the hepatic cholesterol transport and metabolism via significantly up-regulating the APOA1 mRNA levels and down-regulating the APOB mRNA levels in each BHT inclusion groups (P < 0.05), which are protein coding genes related to the synthesis of HDL and LDL (including very low-density lipoprotein, VLDL and chylomicrons), respectively. The key enzyme in classical pathway of bile acid biosynthesis CYP7A1 mRNA level was significantly increased in B300 group and CYP8B1 was significantly increased in all BHT inclusion groups (P < 0.05).
Given all that, it suggested that BHT supplementation could improve hepatic lipid metabolism by enhancing cholesterol transport and improving hepatic bile acid biosynthesis in largemouth bass.

Inflammatory cytokines genes expression:
BHT inclusion reduced the mRNA levels of pro-inflammatory cytokines genes, such as TNFα and IL-11β expression in the B300 group and IL8 expression in the B150 and B1500 groups (P < 0.05). The decreased mRNA levels of proinflammatory cytokines in liver tissues indicated that BHT serves as a therapeutic agent for oxidative-induced liver fibrosis by regulating proinflammatory cytokines.

Hepatic histopathological and immunofluorescence (IF) examination:
Three typical phenotypes were observed from hepatic samples of the largemouth bass. Phenotype I showed serious liver fibrosis symptom and highlighted activated caspase-3 signals. Hepatic steatosis was defined as phenotype II, which had intense vacuoles in the hepatocytes resembling lipids and a slight cleaved caspase-3 signal. Phenotype III had normally shaped hepatocytes with regular gross morphology and clearly located cell nuclei, normal sinusoidal smooth muscle collagen markers and a negative response to cleaved caspase-3. In the B0 group, six samples showed serious hepatic fibrosis, 5 samples had apparent steatosis symptoms, and only one sample had normally shaped hepatocytes. In the B150 group, four samples had hepatic steatosis and eight samples had normally shaped hepatocytes. In the B300 group, eleven samples had normally shaped hepatocytes and one exhibited mild hepatic steatosis. In the B1500 group, three samples had enlarged hepatocytes and mild hepatic steatosis, nine of the samples had normally shaped hepatocytes. No fibrosis phenotypes were observed in the BHT inclusion groups. The mRNA levels of TGF-β1 and α-SMA, the biomarkers of hepatic fibrosis development, were downregulated in BHT inclusion groups, and the B300 group had the lowest levels (P < 0.05). The mRNA levels of caspase-9 were significantly (P < 0.05) decreased in the B300 and B1500 groups compared to those in the B0 group. There were no differences on caspase-3, caspase-8 and caspase-10 mRNA levels (P > 0.05).
See Table 4. below.
Taking into account on the one hand, the serious hepatic fibrosis and steatosis observed in the B0 group, combined with high pro-inflammatory cytokines (including TNFα), caspase-9 gene expression and strong activated caspase-3 signaling in liver tissue, and on the other hand, caspase-9 mRNA levels in B300 and B1 500 BHT being significantly down-regulated, and the normally shaped liver tissued of fish in B300, it suggests that BHT may act as a therapeutic agent for inflammatory disease and hepatocellular damage by inhibiting the ROS products, regulating pro-inflammatory cytokines, and intrinsic apoptosis reactions. In the present study, although the TNFα mRNA level was significantly higher in the B0 group, it is not confirmed whether extrinsic apoptosis occurred because of the similar expression of caspase-8 and 10 among groups.
Moreover, based on the mRNA levels of TGF-β1 and the reduction of a-SMA in the B300 group, the authors stated that BHT may play an important role in preventing hepatic stellate cell (HSC) activation and hepatic fibrosis development in largemouth bass.
Histological examination of liver morphology with H & E and Sirius red staining indicated that BHT treatment resulted in a reduction in indices of hepatocellular damage such as ballooning of hepatocytes, reduced infiltration of inflammatory macrophages and marked decrease in collagen deposition, which suggested that BHT is important for the prevention of hepatocyte injury and liver fibrosis after oxidative stress.
Reported statistics and error estimates:
All data were subjected to one-way analysis of variance (one-way ANOVA). Homogeneity of variance was confirmed by Levene's test before ANOVA and differences among the means were analyzed by Duncan's multiple-range test. The differences were regarded as significant when P < 0.05, and data were reported as mean value ± standard error of mean (S.E.M).

Table 1. Growth performance and morphometric parameters (means ± SEM, n = 6):

 

B0

B150

B300

B1500

Survival %(b)

97.8±1.65

98.9±1.11

100±0.00

98.9±0.70

SGR %/d(c)

2.73±0.05

2.74±0.05

2.76±0.06

2.73±0.05

FR%bw/d (d)

1.58±0.03

1.55±0.03

1.57±0.03

1.59±0.03

BHT intake (mg/kg bw/d) (e)

/

2.32±0.04a

4.72±0.09b

23.8±0.43c

FCR (f)

0.72±0.01a

0.70±0.01a

0.71±0.01ab

0.72±0.01b

CF (g)

2.03±0.03b

2.07±0.03b

1.99±0.03b

1.98±0.02a

HSI (h)

2.76±0.27ab

3.23±0.37b

2.25±0.24a

2.58±0.28ab

VSI (1)

7.78±0.26ab

8.09±0.34b

7.20±0.23a

7.78±0.29ab

“/” means: not intake.                            

a Within the same row, values with different superscripts are significantly different (P < 0.05).

b Survival, % = 100 x final fish number/initial fish number.       

c SGR (specific growth rate, %/d) = 100 x [Ln (FBW)/Ln (IBW)]/days.

d FR (feeding rate, %) = 100 x feed intake/[(Wf + Wi + Wd)/2]/days. Wf is the final total weight, Wd is the total weight of dead fish, Wi is the initial total weight. The same below.                     

e BHT intake (mg/kg bw) = BHT contents x feed intake/[(Wf + Wi + Wd)/2]/days.

f FCR (feed conversion ratio) = feed intake/(Wf + Wd - Wi).       

g CF (condition factor) = 100 x (body weight, g)/(body length, cm)3.

h HSI (hepatosomatic index, %) =10C x liver weight/whole body weight.

i VSI (viscerasomatic index, %) =100 x viscera weight/whole body weight.

Table 2. Hematological parameters oflargemouth bass (means ± SEM. n = 12):

 

B0

B150

B300

B1500

Lipid metabolism

TC (mmol/L)

5.78±0.47b

3.80±0.27a

3.59±0.19a

3.37±0.17a

HDL-C(mmol/L)

1.45±0.12

1.27±0.19

1.14±0.16

1.02±0.09

HDL-C/TC

0.26±0.02a

0.40±0.05b

0.35±0.03ab

0.31±0.03ab

TG (mmol/L)

3.31±0.46b

1.95±0.26a

1.67±0.45a

2.60±0.53ab

LDL-C(mmol/L)

2.83±0.37b

1.64±0.42a

1.69±0.19a

1.17±0.29a

Liver function

ALT(U/L)

7.82±1.61b

3.05±0.59a

4.94±0.88ab

4.59±0.60a

AST(U/L)

13.3±3.73

9.07±2.43

8.85±2.78

7.46±3.74

Cholestasis biomarkers

AKP (U/L)

213±16.0b

148±13.8a

156±20.3a

125±10.6a

TBA(umol/L)

75.3±10.4

75.0±9.79

52.4

73.7±10.2

a Values (mean ± SEM) in the same row with different superscript letters are significantly different (P < 0.05).

Table 3. Plasma and hepatic antioxidant responses (means ± SEM. n =12):

 

B0

B150

B300

B1500

Plasma

SOD (U/mL)

10.4 ± 1.69

7.38 ± 0.99

9.92 ± 1.12

9.03 ± 1.52

MDA (nmol/mL)

10.1 ± 1.12b

6.59 ± 0.51a

7.76 ± 1.11ab

8.93 ± 0.58ab

T-AOC (U/mL)

3.84 ± 0.60ab

3.01 ± 0.41a

4.60 ± 0.18b

3.12 ± 0.63ab

CAT (U/mL)

0.03 ± 0.01

0.04 ± 0.01

0.05 ± 0.01

0.04 ± 0.01

GSH-Px (U/mL)

81.8 ± 13.6a

538 ± 54.0b

520 ± 41.4b

506 ± 50.6b

Liver

SOD(U/mg protein)

6.65 ± 0.60

5.67 ± 1.12

6.71 ± 0.49

5.26 ± 0.48

MDA(nmol/mg protein)

1.74 ± 0.49b

1.50 ± 0.52b

1.45 ± 0.22ab

0.80 ± 0.09a

O2- • (U/mg protein)

0.47 ± 0.02c

0.31 ± 0.02a

0.29 ± 0.01a

0.35 ± 0.02b

T-AOC(U/mg protein)

1.12 ± 0.07a

1.70 ± 0.18b

1.95 ± 0.12b

1.95 ± 0.11b

CAT(U/mg protein)

0.77 ± 0.15

0.82 ± 0.77

1.08 ± 0.11

1.02 ± 0.95

a Values (mean ± SEM) in the same row with different superscript letters are significantly different (P < 0.05).

Table 4. Phenotype of hepatic histopathological examination of (n = 12):

 

B0

B150

B300

B1500

Hepatic fibrosis (I)

6

0

0

0

Fatty liver (II)

5

4

1

3

Normal liver (III)

1

8

11

9

Validity criteria fulfilled:
not applicable
Conclusions:
BHT did not cause adverse effects on growth performance, lipid metabolism, inflammation, anti-oxidant response, apoptosis and hepatosis induced by oxidative stress in largemouth bass (Micropterus salmoides) after 70 dietary exposure up to 1500 mg/kg (23.80 mg/kg bw/day) test item.
Executive summary:

A 70 days growth study was performed to investigate the efficacy and tolerance of dietary butylated hydroxytoluene (BHT) by evaluating growth performance, lipid metabolism, inflammation, anti-oxidant response, apoptosis and hepatosis induced by oxidative stress in largemouth bass (Micropterus salmoides). 30 fish per replicate (6) and dose feed with experimental diets with BHT supplement levels of 0, 150, 300 and 1500 mg/kg in diet (corresponding to 2.32, 4.72 and 23.8 (B1500) mg/kg bw/day respectively). The fish from each tank were batch weighed at the end of the growth trial. Twelve fish for each treatment (2 fish from each tank) were randomly selected and anaesthetized with chlorobutanol (300 mg/mL). The body weight, body length, liver and viscera weight were recorded individually to calculate condition factor (CF), hepatosomatic index (HSI), and viscerosomatic index (VSI), respectively. Blood samples were drawn from the caudal part for analysis and two liver samples near to the bile duct were collected for histology, and for antioxidant analysis and RNA isolation. The BHT inclusion level did not affect the specific growth rate, but fish in the B150 group showed the lowest feed conversion rate (P < 0.05). Authors pointed out that carnivorous fish always have higher lipid requirements than poultry and most mammals, especially the largemouth bass is a fish species very sensitive to peroxidation. Hence, they suppose that, for this species, it could need higher level of antioxidant in diet than 150 mg/kg. B. Plasma clinical parameters data clearly demonstrated the efficacy of BHT in all groups for improving lipid metabolism and antioxidant responses by decreasing plasma TC, TG, LDLC, and MDA concentrations and increasing HDL-C/TC, GSH-Px and TAOC. The increased plasma ALT and AKP levels of the B0 group indicated that oxidative stress could induce hepatic dysfunction and hepatotoxicity in largemouth bass. In contrary, administration of BHT markedly improved hepatic function via decreasing plasma AKP and ALT activities in largemouth bass. In reference to the antioxidant defense system, boosted plasma GSH-Px in BHT inclusion groups were observed. A significantly lower plasma MDA concentration in the B150 group compared with B0 group was detected, while hepatic T-AOC was significantly enhanced and O2−% was significantly decreased in each BHT inclusion group, as well as hepatic MDA was significantly decreased in B1500 group, indicating that BHT could prevent the accumulation of free radicals and improve antioxidant activities in the liver of largemouth bass. Dietary BHT inclusion down-regulated the hepatic mRNA levels of inflammation, apoptosis and fibrosis related genes, including TNFα, TGF-β1, α-SMA, IL8, IL11β and caspase-9. The immunofluorescence examination revealed significantly decreased cleaved caspase-3 signals in the BHT groups. This suggests that BHT may act as a therapeutic agent for inflammatory disease and hepatocellular damage by inhibiting the ROS products, regulating pro-inflammatory cytokines, and intrinsic apoptosis reactions and that it may play an important role in preventing hepatic stellate cell (HSC) activation and hepatic fibrosis development in largemouth bass. Moreover, BHT could improve hepatic lipid metabolism via up-regulating the mRNA levels of APOA1, CYP7A1, CYP8B1, and down-regulating the mRNA levels of PPAR-γ and APOB. Histological examination of liver morphology indicated that BHT treatment resulted in a reduction in indices of hepatocellular damage such as ballooning of hepatocytes, reduced infiltration of inflammatory macrophages and marked decrease in collagen deposition, which suggested that BHT is important for the prevention of hepatocyte injury and liver fibrosis after oxidative stress. In conclusion, consuming up to 23.80 mg/kg bw/d (1500 mg/kg in diet) of BHT effectively improved the plasma and hepatic lipid metabolism, antioxidant response as well as reduced ROS production, protecting hepatic cells from injury. No adverse effects were observed relating the growth performance of largemouth bass up to 23.80 mg/kg bw/day (1500 mg/kg in diet) under test conditions.

Description of key information

The NOEC (30d) of BHT in Oryzias latipes was 0.053 mg/L and the LOEC (30d) was 0.14 mg/L.

BHT did not cause adverse effects on growth performance, lipid metabolism, inflammation, anti-oxidant response, apoptosis and hepatosis induced by oxidative stress in largemouth bass (Micropterus salmoides) after 70 dietary exposure up to 1500 mg/kg (23.80 mg/kg bw/day) test item.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.053 mg/L

Additional information

Key study: Test method according to OECD guideline 210: The NOEC (30d) of BHT in Oryzias latipes was 0.053 mg/L and the LOEC (30d) was 0.14 mg/L

BHT did not cause adverse effects on growth performance, lipid metabolism, inflammation, anti-oxidant response, apoptosis and hepatosis induced by oxidative stress in largemouth bass (Micropterus salmoides) after 70 dietary exposure up to 1500 mg/kg (23.80 mg/kg bw/day) test item.