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Toxicity to birds

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Endpoint:
short-term toxicity to birds: acute oral toxicity test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from July 2018 to June 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
documentation of sexes and detailed test conditions are missing
Qualifier:
no guideline followed
Principles of method if other than guideline:
acute oral exposition via feed
Specific details on test material used for the study:
Formalin (40% aqueous solution of stock FA powder)
Dose method:
mixed with feed (accounts for granular/pelleted test items)
Remarks:
2.5 mL formalin or 1000 mg/kg feed
Details on preparation and analysis of diet:
DIET PREPARATION
- Preparation of doses: Formalin (40% aqueous solution of stock FA powder) was appropriately mixed with pigeon feeds (2.5 mL formalin or 1000 mg/kg feed)
Test organisms (species):
Colomba livia
Details on test organisms:
TEST ORGANISM
- Common name: rock pigeon
- Age at test initiation (mean and range, SD): young pigeons: 18-days-old, adults: >12-month-old
- Weight at test initiation (mean and range, SD): young pigeons: 136-166 g, adults: 203-215 g
- Hausing and feed: cages, feed: standard diet (mastered, rice, sesame, and wheat) twice daily and filtered tube-well water ad libitum.
- Pretest: previous studies have shown that the 2.5 mL formalin/kg feed in broiler chicken, Japanese quails, and pigeons has no adverse effect on body weight and feed intake that suggestive of a beneficial dose
Limit test:
yes
Total exposure duration (if not single dose):
7 d
Control animals:
yes, plain diet
Nominal and measured doses / concentrations:
nominal: 2.5 mL formalin or 1000 mg/kg feed
Details on test conditions:
ACCLIMATION
- Acclimation period: 1wk
- Acclimation conditions (same as test or not): yes

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 5 young and 5 adults
- For treated: 5 young and 5 adults


RANGE FINDING STUDY
Previous study: 2.5 mL formalin/kg feed in broiler chicken, Japanese quails, and pigeons has no adverse effect on body weight and feed intake that suggestive of a beneficial dose. In the experiment, this dose was used.
Details on examinations and observations:
BODY WEIGHT
- Time schedule for examinations: after 7 d
- Remarks: euthanized under deep anesthesia using 5 mL chloroform-soaked cotton in the vacuum glass chamber for 2-3 min.


SERUM ENZYME ANALYSIS
- Dose groups that were examined: all
- Remarks: blood was collected from the heart. The blood containing the test tube was placed in a slanting
position at room temperature (32°C) for 30 min. The serum was separated from the clotted blood by centrifugation
at 3000 rpm for 20 min and again for 10 min. The supernatant was then collected by micro-pipette
in the Eppendorf tube and stored in the refrigerator at -20°C until analysis of aspartate transaminase (AST). The serum test was evaluated by spectrophotometry using commercially available kits

HISTOPATHOLOGY
- Dose groups that were examined: all
- Organs: liver
- Remarks: the internal visceral organs, including the liver, was visualized through the ventral mid-line thoracoabdominal opening, and the gross anatomy was observed very carefully. Then, the liver was removed, and its color and gross texture were observed. The weight of the liver was measured and recorded. Subsequently, the liver tissues from the different lobes were collected and immediately fixed to 10% neutral buffered formalin (NBF) by standard method. Then, NBF-fixed tissues were dehydrated with ascending graded alcohols and embedded in paraffin and lastly sectioned at 6 μm in thickness using a sliding microtome (EuromaxR, Japan). The deparaffinized sections were stained with hematoxylin and eosin (H and E) for histopathological examination. The formalin and urea induced histopathological changes were studied using a light microscope.

Duration (if not single dose):
7 d
Dose descriptor:
LOEC
Remarks:
derived by the applicant
Effect level:
1 000 mg/kg diet
Conc. / dose based on:
test mat.
Basis for effect:
other: liver lesions and increased AST (aspartate aminotransferase)
Duration (if not single dose):
7 d
Dose descriptor:
NOEC
Remarks:
derived by the applicant
Effect level:
> 1 000 mg/kg diet
Conc. / dose based on:
test mat.
Basis for effect:
body weight
Duration (if not single dose):
7 d
Dose descriptor:
NOEC
Remarks:
derived by the applicant
Effect level:
1 000 mg/kg diet
Conc. / dose based on:
test mat.
Basis for effect:
organ weights
Remarks:
liver
Reported statistics and error estimates:
Student’s t-test using SPSS, version 18.0 software (IBM Corp., NY, USA). p≤0.05
was considered statistically significant. All the results were expressed as mean ±SE.

Effects on body weight, relative organ weight, and gross morphology of liver of pigeons


This study found that there is no significant (p>0.05) difference in body weight gain among FA treated young and adult pigeons. The relative organ weight indicated that the weight of the liver of FA exposed young and adult pigeons were not significantly different from control pigeons. The color, size, and shape of the liver of both young and adult control pigeons were normal. There were no changes in the size and shape of the liver of FA treated pigeons. However, the liver revealed pale color with petechial hemorrhage on the parietal and visceral surfaces in FA-exposed young and adult pigeons.


 


Effects on serum hepatic marker enzyme


H and E stained sections of the liver of control pigeons showed normal hepatic cord, hepatocytes with acidophilic granular cytoplasm, and central vesicular nuclei as well as well-preserved cytoplasm and well-defined nucleus. The portal vein and the bile ducts were not dilated or enlarged. The arrangements of parenchymal hepatocytes as hepatic cords and non-parenchymal cells are regular in form. Therewere no signs of necrosis or cellular damage in control pigeons.


The serum enzyme AST (aspartate aminotransferase) threshold in test material exposed pigeons was significantly increased in both young and adult pigeons compared to control pigeons. However, in FA treated young and adult pigeons liver showed, severe coagulation necrosis with infiltration of inflammatory cells surrounding the central and portal veins. The sinusoids were congested and dilated with mononuclear cellular infiltration due to coagulative necrosis, hemorrhage, and apoptotic hepatocytes around the congested portal vein in FA exposed adult pigeon. The liver lesions were more predominant in FA exposed adult pigeons compared to the liver of young pigeons.

Executive summary:

In an experimental study five young (18 days old) and five adult (>12 month) pigeons (Columba livia) each was conducted. The test organisms were exposed to a 40 % aqueous solution of the test item via feed in a concentration of 1000 mg/kg feed for 7 d, twice a day.  After 7 days the body weight of control and treated organisms was determined after euthanized. In addition, the serum enzymes were analyzed in the blood collected from the heart and the liver was examined histopathological.  In comparison to control no significant effect on body and relative organ weight was observed. As well as in shape and size of the liver. Effects on the serum hepatic marker enzymes were observed. In treated young and adult pigeons liver showed, severe coagulation necrosis with infiltration of inflammatory cells surrounding the central and portal veins. The sinusoids were congested and dilated with mononuclear cellular infiltration due to coagulative necrosis, hemorrhage, and apoptotic hepatocytes around the congested portal vein in exposed adult pigeon. The liver lesions were more predominant in exposed adult pigeons compared to the liver of young pigeons.


The study is reliable and meets generally accepted scientific principles but details about sexes of the test organisms and test conditions are not reported. Furthermore, no effect values are reported and only the mean values for the results are reported (no information about the amount of affected organisms). Based on this, a NOEC of 1000 mg/kg diet for the body and organ weight was derived by the author. For the liver effects an LOEC ≤ 1000 mg/kg diet was derived. Therefore, the study is adequate as supporting information.

Endpoint:
long-term toxicity to birds
Data waiving:
exposure considerations
Justification for data waiving:
other:

Table 1: Mackay Level I calculation (BASF, 2008)






































Compartment



Percent distribution in media (%)



Air



1.16



Water



98.8



Soil



0



Sediment



0



Susp. sediment



0



Biota



0



Aerosol



0



 

Description of key information

Exposure of terrestrial organisms and secondary poisoning is unlikely.

Key value for chemical safety assessment

Additional information

In Annex X of the Regulation (EC) No 1907/2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), it is laid down that "the chemicals of concern with respect to secondary poisoning include lipophilic organic chemicals and some metal compounds". Since the substance exhibits a low log Kow (0.35, Hansch et al., 1995) and does not tend to bioaccumulate in organisms, secondary poisoning is unlikely to be a relevant exposure route. Furthermore, substance has a low potential for adsorption and exhibits a very high solubility in water or soil moisture (water solubility = 550 g/L, c.f. 4.8.). This is underlined by valid Mackay Level I distribution modelling (BASF, 2008). According to this calculation soil is not a target compartment of the test item. When released into the environment 98.8 % will remain in the water phase and 1,16 % will evaporate to the air (see table 1). In order to this, a distribution to soil and sediment is not expected.


Table 1: Mackay Level I calculation (BASF, 2008)






































Compartment



Percent distribution in media (%)



Air



1.16



Water



98.8



Soil



0



Sediment



0



Susp. sediment



0



Biota



0



Aerosol



0



 


 


However, reliable experimental data on birds after acute oral exposure are available to support the assumptions (Hasan, 2021). In an experimental study five young (18 days old) and five adult (>12 month) pigeons (Columba livia) each was conducted. The test organisms were exposed to a 40 % aqueous solution of the test item via feed in a concentration of 1000 mg/kg feed for 7 d, twice a day.  After 7 days the body weight of control and treated organisms was determined after euthanized. In addition, the serum enzymes were analyzed in the blood collected from the heart and the liver was examined histopathological.  In comparison to control no significant effect on body and relative organ weight was observed. As well as in shape and size of the liver. Effects on the serum hepatic marker enzymes were observed. The serum enzyme AST (aspartate aminotransferase) threshold in test material exposed pigeons was significantly increased in both young and adult pigeons compared to control pigeons. In treated young and adult pigeons liver showed, severe coagulation necrosis with infiltration of inflammatory cells surrounding the central and portal veins. The sinusoids were congested and dilated with mononuclear cellular infiltration due to coagulative necrosis, hemorrhage, and apoptotic hepatocytes around the congested portal vein in exposed adult pigeon. The liver lesions were more predominant in exposed adult pigeons compared to the liver of young pigeons. No effect values were determined by the authors of the study. Since no effects were observed based on body and relative organ weight a NOEC >1000 mg/kg diet was derived by the applicant for both endpoints. Only the mean values are reported as results, therefore a LOEC ≤ 1000 mg/kg diet was derived by the applicant based on the histopathological effects on the liver and the AST level in the serum.


Although histopathological effects on the liver of pigeons was observed, secondary poisoning is unlikely due to the previous mentioned low exposure probability and the low ability of the test item to accumulate in organism. The test scenario with contaminated food does therefore lead to an overestimation of the toxicity to birds.