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Endpoint:
additional ecotoxicological information
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Effects of Bisphenol A and Tetrabromobisphenol A on sex organ development in quail and chicken embryos
Author:
Berg C, Halldin K & Brunström B
Year:
2001
Bibliographic source:
Environ. Toxicol. Chem. 20: 2836 - 2840

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The primary objective of the present study was to determine whether or not the test material can cause oestrogen-like effects on sex organ development in avian embryos. A second objective of this study was to compare the oestrogenic potency of the test material with that of the synthetic oestrogen diethylstilbestrol. The third objective was to compare embryos of Japanese quail (Coturnix japonica) and domestic fowl (Gallus domesticus) in terms of sensitivity to oestrogen-like compounds.
GLP compliance:
not specified
Type of study / information:
The test material was examined for oestrogen-like developmental effects on the reproductive organs in avian embryos.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): 3,3’,5,5’-Tetrabromobisphenol A
- Abbreviation: TBBPA
- Purity: >99 % (recrystallised)
- Supplier: Sigma Chemical (St. Louis, MO, USA)

Results and discussion

Any other information on results incl. tables

Mortality

The highest dose of the test material (45 µg/g egg) inflicted high mortality in both quail and chicken embryos (Table 1). Diethylstilbestrol treatment did not cause any increase in mortality rate in either species.

 

Gross morphology of the Müllerian ducts

The test material had no effect on the MDs in either female or male quail embryos (Table 2) and no significant effects on the MDs were observed in the chicken embryos exposed (Table 3).

Both 2 and 20 ng DES/g egg caused a significant increase in MD malformation frequency in female quail embryos (Table 2) (p = 0.02 and p < 0.0001, respectively). DES exposure resulted in retained MDs in 8 of 10 male embryos at 20 ng/g egg (p < 0.0001), while no effects were observed at 2 ng DES/g egg. No significant effects were observed in chicken embryos exposed to DES.

 

Histology of the left testis

At hatching, the testis normally consists of testicular cords containing germ cells. The cords are surrounded by a few cell layers–thick epithelium, the tunica albuginea. As opposed to the medullary location of the germ cells in the male gonad, the ovarian germ cells are located in the cortex. The female germ cells enter meiotic prophase during embryogenesis and are identified by their large nucleus containing condensed chromatin. In most of the control quails, no germ cells were found in the cortical area of the left testis. However, quite a large number of control gonads were classified as ovotestes, i.e., presence of five or more germ cells in prophase in the cortex. None of the control chicken embryos had an ovotestis according to this definition.

In quail embryos the test material did not cause an increased ovotestis frequency compared with the control frequency; the test material did not induce ovotestis formation in chicken embryos.

Ovotestis frequency was significantly increased in quail embryos at 20 ng DES/g egg (p = 0.024) and in chicken embryos at 200 ng DES/g egg (p = 0.0003).

 

Discussion

The test material was lethal to both quail and chicken embryos at 45 µg/g egg. No oestrogen-like effects of this compound were noted. The risk for reproductive toxicity in avian wildlife is probably low, as the doses required for effects in this study were fairly high and the test material seems to be rapidly biotransformed and excreted.

 

Table 1: Mortality by day 15 in quail embryos and day 19 in chicken embryos

Treatment (µg/g egg)

Mortality (%)

Quail

Chicken

Control A

26 (11/43)

24 (20/83)

Control B

13 (6/47)

8 (2/24)

15

23 (10/44)

25 (6/24)

45

80 (20/25)***

96 (24/25)***

Positive Control (0.002)

27 (8/30)

32 (11/34)

Positive Control (0.02)

22 (7/32)

21 (15/71)

Positive Control (0.2)

-

35 (12/34)

The ratio (in parentheses) represents the number of dead embryos divided by the number of eggs treated. The mortality in the treatment groups was compared with the control frequency using Fisher’s exact test.

Control A: Eggs treated with the propylene glycol-based emulsion served as controls for the DES- treated eggs.

Control B: Eggs treated with the oil/lecithin/water emulsion served as controls for the test material-treated eggs.

*** p < 0.001

 

Table 2: Frequencies of 15-d-old quail embryos exhibiting malformations of the reproductive organs

Treatment (µg/g egg)

Females with abnormal Müllerian ducts (%)

Males with an ovotestis¹ (%)

Control A

7 (1/15)

33 (4/12)

Control B

0 (0/11)

45 (5/11)

15

0 (0/5)

50 (9/18)

Positive Control (0.002)

55 (6/11)*

63 (5/8)

Positive Control (0.02)

87 (13/15)***

89 (8/9)*

The ratio (in parentheses) represents the number of affected embryos divided by the number of embryos examined. The malformation frequencies in the treatment groups were compared with the control frequency using Fisher’s exact test.

¹Defined as presence of a cortex containing five or more germ cells in meiotic prophase in at least one of the sections of the left testis.

Control A: Eggs treated with the propylene glycol-based emulsion served as controls for the DES- treated eggs.

Control B: Eggs treated with the oil/lecithin/water emulsion served as controls for the test material-treated eggs.

* p< 0.05,*** p < 0.001

 

Table 3: Frequencies of 19-d-old chicken embryos exhibiting malformations of the reproductive organs

Treatment (µg/g egg)

Females with abnormal Müllerian ducts (%)

Males with an ovotestis¹ (%)

Control A

10 (3/30)

0 (0/20)

Control B

0 (0/9)

0 (0/13)

15

0 (0/12)

0 (0/5)

Positive Control (0.002)

0 (0/10)

0 (0/9)

Positive Control (0.02)

7 (1/15)

4 (1/23)

Positive Control (0.2)

36 (5/14)

63 (5/8)***

The ratio (in parentheses) represents the number of affected embryos divided by the number of embryos examined. The malformation frequencies in the treatment groups were compared with the control frequency using Fisher’s exact test.

¹Defined as presence of a cortex containing five or more germ cells in meiotic prophase in at least one of the sections of the left testis.

Control A: Eggs treated with the propylene glycol-based emulsion served as controls for the DES- treated eggs.

Control B: Eggs treated with the oil/lecithin/water emulsion served as controls for the test material-treated eggs.

*** p < 0.001

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was lethal to both quail and chicken embryos at 45 µg/g egg. No oestrogen-like effects of this compound were noted.
Executive summary:

A study was conducted in which the primary objective was to determine whether or not the test material can cause oestrogen-like effects on sex organ development in avian embryos. A second objective of this study was to compare the oestrogenic potency of the test material with that of the synthetic oestrogen diethylstilbestrol (DES). The third objective was to compare embryos of Japanese quail (Coturnix japonica) and domestic fowl (Gallus domesticus) in terms of sensitivity to oestrogen-like compounds. The study was carried out in accordance with sound scientific principles.

The test material was dissolved in a mixture of peanut oil and lecithin, and an emulsion in water was prepared for use as the vehicle. DES was administered in an oil/lecithin/propylene glycol emulsion. Two control groups were formed, one for each emulsion.

The test material and DES were injected into the yolk via a small hole at the blunt end of the egg. The quail eggs were treated on day 3 of incubation (20 µL/egg) and the chicken eggs on day 4 (100 µL/egg). Doses were 15 and 45 µg test material/ g egg and 2 and 20 ng DES/g egg. A further group of chicken eggs was dosed with 200 ng DES/g egg. A minimum of 24 embryonated eggs per dose were treated.

The embryos were dissected 2 d before anticipated hatching. The embryos were examined for gross abnormalities of the MDs (Müllerian ducts) in situ under a dissecting microscope. Left testes were subjected to histological examination and if the testis had a cortex containing five or more germ cells in meiotic prophase (oocyte like), it was classified as an ovotestis.

The highest dose of the test material (45 µg/g egg) inflicted high mortality in both quail and chicken embryos. The test material had no effect on the MDs in either female or male quail embryos and no significant effects on the MDs were observed in the chicken embryos exposed.

In most of the control quails, no germ cells were found in the cortical area of the left testis. However, quite a large number of control gonads were classified as ovotestes. None of the control chicken embryos had an ovotestis. In quail embryos the test material did not cause an increased ovotestis frequency compared with the control frequency; the test material did not induce ovotestis formation in chicken embryos.

DES treatment did not cause any increase in mortality rate in either species. Both 2 and 20 ng DES/g egg caused a significant increase in MD malformation frequency in female quail embryos. DES exposure resulted in retained MDs in 8 of 10 male embryos at 20 ng/g egg, while no effects were observed at 2 ng DES/g egg. No significant effects on MDs were observed in chicken embryos exposed to DES. Ovotestis frequency was significantly increased in quail embryos at 20 ng DES/g egg and in chicken embryos at 200 ng DES/g egg.

Under the conditions of this study, the test material was lethal to both quail and chicken embryos at 45 µg/g egg. No oestrogen-like effects of this compound were noted. The risk for reproductive toxicity in avian wildlife is probably low, as the doses required for effects in this study were fairly high and the test material seems to be rapidly biotransformed and excreted.