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Endpoint:
long-term toxicity to birds
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
26 January 1998 through 25 June 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, scientifically sound study with sufficient information presented on materials and methods to adequately evaluate results.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Metal
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
reference to other study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register. 1986. Nontoxic shot approval procedures for shot. Federal Register 51: 42098-42102.
Principles of method if other than guideline:
The study design was based on a published protocol (Federal Register, 1986) and modified as requested by the USFWS.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Dose method:
gavage
Analytical monitoring:
not required
Vehicle:
no
Details on preparation and analysis of diet:
Not relevant, dosages were not presented in the diet but via gavage. Ducks were dosed with pellets introduced into the proventriculus by means of a funnel and 21.6 cm long latex tube (0.953 cm I.D.) through the esophagus. About 5 ml of water helped flush shot into the proventriculus (Kelly et al, 1998).
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: mallard/duck (game-farm)
- Source: obtained from Whistling Wings, Inc. (Hanover, Illinois, USA).
- Age at test initiation (mean and range, SD): 5 months
- Sexes used: female and male
- Cultural background:
- Disease free: no data
- Kept according to standard practices: no data
- Breeding population (e.g. reproductive history): Two generations removed from wild stock
Limit test:
yes
Total exposure duration (if not bolus):
120 d
Post exposure observation period:
The last dose was administered on day 120, but the trial continued until day 150.
No. of animals per sex per dose and/or stage:
54 males and 54 females/thirty-two bird groups (sexes equal)
Control animals:
other: Steel shot dosed mallards were considered the control by USFWS.
Nominal and measured doses / concentrations:
The rate of shot erosion was highest for tungsten-polymer shot (99%), followed by tungsten-iron (72%), and steel (55%) shot. The initial pellet weight for tungsten-polymer shots was reported to be 0.186 g (Mitchell et al, 2001a). Therefore, assuming an average erosion rate of 99% for the tungsten-polymer shots, and assuming equal corrosion of both the tungsten and nylon constituents in the shot, the ducks in this group were exposed to 0.176 g/pellet (0.186 g/pellet X 95.5% tungsten X 99% erroded). Eight pellets were administered per dose (5 dosing periods occurred over the 150 day trial), thus a total exposure of 1.41 g tungsten was received per dose. The average bodyweight of the mallards in the tungsten-polymer shot group, at the end of the study was 1.167 kg, resulting in a received dose of approximately 1.21 g solubilized tungsten/kg bodyweight.
Details on test conditions:
ACCLIMATION
- Acclimation period: The 26-day acclimation period was from 30 December 1997 to 25 January 1998.
- Acclimation conditions (same as test or not): no data
- Feeding: Provided feed and water ad libitum throughout the trial. The ducks were fed a pelleted duck grower ration (Purina Mills, St. Louis, Missouri,
USA) during the acclimation period, shelled corn during the first 60 days of the trial (26 January 1998 to 27 March 1998), and a layer mash (Mazuri, Brentwood, Missouri, USA) during the subsequent 90-day reproduction phase of the trial (28 March to 25 June 1998).
- Health (any disease or mortality observed): no data

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: cages measuring 0.914 m long 3 0.914 m wide 3 0.457 m high in a minimally heated pole barn.
- Caging: housed randomly as male-female pairs

NO. OF BIRDS PER REPLICATE
- For negative control: Steel-dosed mallards (16 pairs); referred to as "control"
- For positive control: Lead dosed mallards (6 pairs)

NO. OF REPLICATES PER GROUP
- For negative control: Received 8 #4 pellets on days 0, 30, 60, 90, and 120
- For positive control: Received 8 # 4 pellets on day 0

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: The temperature, which was continuously monitored, was maintained above 0 degree C by a propane gas heater suspended from the ceiling in the middle of the room.
- Photoperiod: Photoperiod was controlled by a timer on incandescent lights and maintained at 8 hr light:16 hr dark through the first 60 days of the trial and increased in increments over six weeks to 18 hr light:6 hr dark during the last 90 days of the study.



Details on examinations and observations:
MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Observed all ducks twice daily for general well-being.
- Remarks: Clinical signs including inappetence, apparent weight loss, ataxia, lethargy, and discolored excreta were recorded. Ducks that died before day 150 were weighed and taken to the MSU Animal Health Diagnostic Laboratory (East Lansing, Michigan, USA) for necropsy.

BODY WEIGHT
- Time schedule for examinations: On day 150 of the trial, all surviving mallards were weighed.

PATHOLOGY
- Dose groups that were examined: Liver, kidney, and ovary/testes samples from 16 (sexes equal) mallards in the control (negative control) and tungsten-polymer groups and from the 12 mallards in the lead (postive control) group were assessed without knowledge of treatment.
- Remarks: Collected and stored samples of the liver, kidneys, and testes/ovary from each duck in 10% formalin-saline solution (10% formalin in 0.9% sodium chloride) for histopathology.

ORGAN WEIGHTS
- Dose groups that were examined: Liver, kidney, and ovary/testes samples from 16 (sexes equal) mallards in the control (negative control) and tungsten-polymer groups and from the 12 mallards in the lead (postive control) group were assessed without knowledge of treatment.
- Organs: Removed and weighed the brain, gizzard, heart, liver, spleen, kidneys, and testes or ovary.

Tissues were embedded in paraffin, trimmed to 8 um, and stained with hematoxylin and eosin. Selected liver sections from control and tungsten-polymer-dosed mallards were stained with Prussian blue for determination of iron pigment.

OTHER: Approximately 10 days after each dosing, the ducks were transported to the MSU Large Animal Veterinary Clinic (East Lansing, Michigan, USA) for fluoroscopy to verify the presence of pellets in the gizzard.

The ducks were sacrificed and the gizzards opened for inspection of mucosal lining and presence of shot. the number of shots were counted and weighed in each gizzard for determination of shot retention and shot erosion, respectively. Shot retention could not be determined from the radiographs because it was difficult to count individual pellets after the second dosing on day 30.

The identification of recovered pellets as to the day of dosing could not be done because of the large number of doses.
Details on reproductive parameters:
The following parameters were examined per parental pen per week:
- Eggs laid
- Eggs cracked; eggs broken
Reference substance (positive control):
yes
Remarks:
lead shots
Duration (if not bolus):
120 d
Dose descriptor:
NOEL
Effect level:
>= 1.21 other: g solubilized tungsten/kg bodyweight
Conc. / dose based on:
other: 120 days worth of gavage dosing of tungsten polymer shot.
Basis for effect:
other: mortality, body weights, organ weights, and histology of the liver and kidneys not effected
Remarks on result:
other: Exact tungsten levels not provided
Repellency factors (if applicable):
n/a
Mortality and sub-lethal effects:
MORTALITY
- No ducks in the control (negative control) or tungsten-polymer dosed groups died during the 150-day trial.
BODY WEIGHT
- Results: There was no evidence of significant differences in body weight among the control and tungsten-polymer groups from day 0 through day 60. Tungsten-polymer-dosed mallards were significantly heavier than control mallards from day 90 through day 150.

ORGAN WEIGHTS
- Results: There were no significant differences in relative (expressed as a % of body weight) heart, kidney, brain, liver, and testis or ovary weights among the control and tungsten-polymer groups. The relative gizzard weight of tungsten-polymer-dosed mallards was significantly lower compared to controls

HISTOPATHOLOGY
- Results: Mallards dosed with steel or tungsten-polymer shot that were examined had normal kidneys and no indication of liver biliary stasis. All of the controls and one tungsten-polymer-dosed male examined had liver hemosiderosis (deposition of iron in the form of hemosiderin) ranging from mild to moderate. Liver hemosiderosis ranging from mild to moderate was apparent in five of eight controls. Mallards dosed with steel, lead, or tungsten-polymer shot had diffuse hepatocellular vacuolation. The testes and ovary were inactive in the lead-dosed mallards and appeared normal in the control, and tungsten-polymer dosed ducks that were examined.
Effects on reproduction:
- Number of eggs laid per hen and per day: During the 90 day reproduction phase, two control and three tungsten-polymer-dosed females did not lay any eggs. One control female had a small egg blocking the lumen of the magnum and the other control duck had scar tissue obstructing the oviduct.
- Number of broken eggs: There was egg yolk peritonitis in two tungsten-polymer dosed ducks, whereas the third tungsten-polymer-dosed female appeared normal.
- Description of abnormal behaviour of chicks, of severe birth defects and their general state of health, during the first 14 days after hatching, clinical signs of toxicity: All other ducks in the control and tungsten-polymer dosed groups appeared normal except for one tungsten-polymer female that had a fibrous tag on a focal area of the liver.
Results with reference substance (positive control):
MORTALITY
- All mallards dosed with lead shot (positive control) died within the first 25 days of the 150-day trial. Average time to death was 16.7 days for males and 11.0 days for females; range of 9 to 25 days for both sexes.

CLINICAL SIGNS
- Only lead-dosed mallards had obvious clinical signs of poisoning during the trial. Green-stained excreta was apparent in these ducks within 24 hr of dosing. By day 5, all lead-dosed mallards had marked tail and wing droop. Prior to death, ducks were emaciated, lethargic, and ataxic.

HISTOPATHOLOGY
- Breast muscle of all lead-dosed mallards was atrophied severely and there was minimal subcutaneous or abdominal fat. The gizzard of six lead-dosed ducks (three males, three females) had a discolored mucosal lining. The vent area of two male and one female lead-dosed mallards was
stained with bile and the gallbladder of one male and two female ducks was enlarged. One lead-dosed female had urate crystals surrounding the heart, while one lead-dosed male had a focal area of the liver with a firm, gray covering on the subcapsular surface.

- All lead-dosed mallards except one female had renal nephrosis ranging from mild to moderate and all lead-dosed mallards had mild to moderate liver biliary stasis.
Further details on results:
Approximately 90% of the lead pellets administered were recovered when the ducks were necropsied between day 9 and day 25 of the trial. Erosion of lead shot was 18%. Over half of the steel pellets and less than 3% of the tungsten-polymer pellets were recovered on day 150. Erosion of steel and tungsten-polymer shot was 55%, 72%, and 99% respectively. Fluoroscopy of the mallards during the trial substantiated the relatively rapid erosion of the two types of tungsten shot compared to steel shot.
Reported statistics and error estimates:
All statistical analyses were performed using SAS software (SAS; Statistical Analysis Systems, Release 6.12, Cary, North Carolina, USA). Since all lead-dosed mallards died by day 25 of the 150-day trial, their data were not included in the statistical analyses. Body weights were analyzed by analysis of variance (ANOVA) involving the factors treatment and sex, with repeated measurements on ducks, when applicable, over a third factor, days. SAS PROC MIXED was used to model a first-order autoregressive correlation structure for repeated measurements over days within ducks, as residuals involving measurements taken at adjacent time periods are more likely to be highly correlated than measurements taken further apart in time.

Reported statistics and error estimates (Cont'd)

Where applicable, all two-way interactions between treatment, sex, and days were modeled. Body weights were analyzed separately over two time periods due to changes in reproductive status over these periods. Body weight differences from day 0 to day 60 were analyzed with mean weight differences compared among the three treatment groups. Next, body weight differences were analyzed over the time period that ducks were reproductively active (day 90 through day 150). Organ weights and percent shot erosion were also analyzed using an ANOVA involving the factors treatment and sex. Percent shot erosion and adult relative organ weights were percentage (p) data subjected to arcsine, square root transformation prior to statistical analysis. As standard errors are not readily back-transformed, the reported means and 95% confidence intervals for treatment means of percent shot erosion and adult relative organ weights were back-transformed to the scale of observation. Residual plots were used to check for homogeneity of variance and for aberrant values. Treatment group means were reported as the least squares mean plus or minus the standard error. Treatment means were reported separately for each sex and/or day, if treatment by sex and/or treatment by day interactions, respectively, were statistically significant. Otherwise, reported treatment means and mean differences were based on pooling information over the sexes and/or days. To control for experimental Type 1 error rates, a Fisher’s protected least significant difference (LSD) was used to test comparisons between means based on the total number of pairwise comparisons. In the following sections reference to significant differences (higher or lower) across compared values indicate statistical differences at P </= 0.05.

Conclusions:
Tungsten-polymer shot repeatedly administered to adult mallards did not have deleterious health effects during the 150-day trial based on mortality, body weights, organ weights, and histology of the liver and kidneys. Essentially all of the tungsten-polymer shot eroded (99%).
Executive summary:

No toxicity to birds data of sufficient quality are available for fused tungsten carbide (target substance). However, toxicity to birds data are available for tungsten metal (source substance), which will be used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
long-term toxicity to birds
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
26 January 1998 to 25 June 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, scientifically sound study with sufficient information presented on materials and methods to adequately evaluate results.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Metal
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
reference to other study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register. 1986. Nontoxic shot approval procedures for shot. Federal Register 51: 42098-42102.
Principles of method if other than guideline:
The study design was based on a published protocol (Federal Register, 1986) and modified as requested by the USFWS.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Dose method:
gavage
Analytical monitoring:
not required
Vehicle:
no
Details on preparation and analysis of diet:
Not relevant, dosages were not presented in the diet but via gavage. Ducks were dosed with pellets introduced into the proventriculus by means of a funnel and 21.6 cm long latex tube (0.953 cm I.D.) through the esophagus. About 5 ml of water helped flush shot into the proventriculus (Kelly et al, 1998).
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: mallard/duck (game-farm)
- Source: obtained from Whistling Wings, Inc. (Hanover, Illinois, USA).
- Age at test initiation (mean and range, SD): 5 months
- Sexes used: female and male
- Cultural background:
- Disease free: no data
- Kept according to standard practices: no data
- Breeding population (e.g. reproductive history): two generation removed from wildstock.
Limit test:
yes
Total exposure duration (if not bolus):
120 d
Post exposure observation period:
The last was administered on day 120, but the trial continued until day 150.
No. of animals per sex per dose and/or stage:
16 male and female pairs.
Control animals:
yes
Nominal and measured doses / concentrations:
The rate of shot erosion was highest for tungsten-polymer shot (99%), followed by tungsten-iron (72%), and steel (55%) shot. The initial pellet weight for tungsten-polymer shots was reported to be 0.186 g (Mitchell et al, 2001a). Therefore, assuming an average erosion rate of 99% for the tungsten-polymer shots, and assuming equal corrosion of both the tungsten and nylon constituents in the shot, the ducks in this group were exposed to 0.176 g/pellet (0.186 g/pellet X 95.5% tungsten X 99% erroded). Eight pellets were administered per dose (5 dosing periods occurred over the 150 day trial), thus a total exposure of 1.41 g tungsten was received per dose. The average bodyweight of the mallards in the tungsten-polymer shot group, at the end of the study was 1.167 kg, resulting in a received dose of approximately 1.21 g solubilized tungsten/kg bodyweight.
Details on test conditions:
ACCLIMATION
- Acclimation period: The 26-day acclimation period was from 30 December 1997 to 25 January 1998.
- Acclimation conditions (same as test or not): no data
- Feeding: Provided feed and water ad libitum throughout the trial. The ducks were fed a pelleted duck grower ration (Purina Mills, St. Louis, Missouri,
USA) during the acclimation period, shelled corn during the first 60 days of the trial (26 January 1998 to 27 March 1998), and a layer mash (Mazuri, Brentwood, Missouri, USA) during the subsequent 90-day reproduction phase of the trial (28 March to 25 June 1998).
- Health (any disease or mortality observed): no data

PEN SIZE AND CONSTRUCTION MATERIALS (Mitchell et al. 2001a)
- Description: cages measuring 0.914 m long 3 0.914 m wide 3 0.457 m high in a minimally heated pole barn.
- Caging: housed randomly as male-female pairs

NO. OF BIRDS PER REPLICATE
- For negative control: Steel-dosed mallards (16 pairs); referred to as "control"
- For positive control: Lead (97% lead, 3% antimony) dosed mallards (6 male/female pairs)

NO. OF REPLICATES PER GROUP
- For negative control: Received 8 #4 pellets on days 0, 30, 60, 90, and 120
- For positive control: Received 8 # 4 pellets on day 0

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: The temperature, which was continuously monitored, was maintained above 0 degree C by a propane gas heater suspended from the ceiling in the middle of the room.
- Photoperiod: Photoperiod was controlled by a timer on incandescent lights and maintained at 8 hr light:16 hr dark through the first 60 days of the trial and increased in increments over six weeks to 18 hr light:6 hr dark during the last 90 days of the study.



Details on examinations and observations:
HEMATOLOGICAL EXAMINATIONS:
On day 7 of the trial, blood was collected from the brachial vein into two microhematocrit capillary tubes (Drummond Scientific, Broomall, Pennsylvania, USA), one 2-mL Vacutainer tube (Becton Dickinson and Company, Franklin Lakes, New Jersey, USA) containing EDTA and two 2 mL Vacutainer tubes containing sodium heparin. Microhematocrit tubes were centrifuged for 5 min.

Hematocrit (HCT) was measured using an IEC MB microcapillary reader (Needham, Massachusetts, USA). Cooled samples of whole-blood and samples of frozen plasma were shipped to the Division of Comparative Pathology, University of Miami School of Medicine (Miami, Florida, USA) for determination of hemoglobin (Hb) concentration, whole-blood delta aminolevulinic acid dehydratase (ALAD) activity, and plasma clinical chemistries.

On days 30, 60, 90, 120, and 150, blood was collected from all the ducks in the control and tungsten-polymer groups for determination of HCT and from eight pairs in the same treatment group for determination of plasma chemistries.


HISTOPATHOLOGY:
The right femur and portions of the liver, kidneys, and testes or ovary were frozen and shipped to CT&E Environmental Services (Ludington, Michigan, USA) for analysis of iron and tungsten.

Samples analyzed included: individual liver samples from eight males and eight females in the control and tungsten polymer groups and individual testis samples from the eight males in the control and tungsten-polymer groups; 16 (eight male and eight female) pooled kidney and femur samples, each consisting of tissues from two ducks in the control and tungsten-polymer groups and eight ovary samples each consisting of tissues from two female ducks in the control and tungsten- polymer groups.

Tissues were digested using EPA method 200.3 (US Environmental Protection Agency, 1991). Tungsten was analyzed by Inductively Coupled Argon
Emission Plasma Spectroscopy (ICAP) following SW-836 Method 6010, revision 2.0 (U.S. Environmental Protection Agency, 1996). A matrix spike was prepared and analyzed with each digestion batch. The ranges of percent recoveries of tungsten in the process spikes were 102 to 116% and 1 to 81%, respectively, and in the matrix spike, the ranges of percent recoveries of iron and tungsten were 79 to 186% and 78 to 98%, respectively.
Details on reproductive parameters:
no data
Reference substance (positive control):
yes
Remarks:
lead shots
Duration (if not bolus):
120 d
Dose descriptor:
other: NOAEL
Effect level:
1.21 other: g solubilized tungsten/kg bodyweight
Conc. / dose based on:
other: 120 days worth of gavage dosing of tungsten polymer shot.
Basis for effect:
other: Hematological Parameters
Remarks on result:
other: Exact tungsten levels not provided
Repellency factors (if applicable):
N/A
Mortality and sub-lethal effects:
Hematological parameters:
Ducks in the tungsten-polymer group had significantly higher Delta-aminolevulinic acid dehydratase (ALAD) activity than mallards in the Control groups. From day 30 to day 60, there was no evidence of significant differences in HCT among ducks in the control and tungsten-polymer groups. Between day 90 and day 150, there was a significant treatment by sex interaction for HCT. There was no evidence of significant differences among HCT of males, but HCT of tungsten-polymer dosed females (mean +/-SE = 40 +/- 0.8) was significantly lower than HCT of control (44 +/- 0.8).

Clinical Chemistry parameters:
There were a number of significant differences in plasma chemistry parameters at day 7. There was a significant treatment by sex interaction for creatine phosphokinase activity at day 7.

Between days 30 and 60, tungsten-polymer dosed ducks had significantly higher aspartate aminotransferase activity than control ducks (31.7, 23.56–42.58 [mean, 95% confidence interval] versus 17.9, 13.29–24.02). There was a significant treatment by sex interaction for lactate dehydrogenase activity. From day 90 through day 150, there was no evidence of significant differences in plasma chemistries.

Other findings (tissue residues):
There was a significant treatment by sex interaction for tungsten concentrations in the femur. Tungsten was detected in all of the femur samples from the females and in five of eight samples from tungsten-polymer-dosed females.

Tungsten was detected in the testes of two of eight tungsten polymer- dosed males. Tungsten-polymer dosed female mallards had significantly lower concentrations of iron in the ovary compared to controls. Tungsten was present in one of eight samples from tungsten-polymer dosed females.

Tungsten was detected in two of 16 kidney samples from the control group and seven of 16 kidney samples from the tungsten-polymer-dosed
ducks. There was a significant treatment by sex interaction for iron concentration in the kidney samples. Tungsten- polymer-dosed males had a significantly lower concentration of iron in the kidneys compared to control males. Tungsten-polymer-dosed females had significantly lower concentrations of iron compared to the controls.

Iron concentration in the liver of tungsten-polymer-dosed mallards was significantly lower compared to ducks in the other groups. Tungsten was detected in the liver of two of 16 tungsten-polymer dosed mallards.
Effects on reproduction:
no data
Results with reference substance (positive control):
Ducks dosed with lead had significantly lower HCT, Hb concentration, and wholeblood ALAD activity at day 7 compared to ducks in the control and tungsten-polymer groups.

Alkaline phosphatase activity was significantly lower in lead dosed ducks compared to ducks in the control and tungsten polymer groups. Uric acid concentration and activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were significantly higher in lead-dosed ducks compared to ducks in the control and tungsten polymer- dosed groups. Both lead-dosed males and females had significantly higher creatine phosphokinase activities compared to their counterparts in the other three groups.
Reported statistics and error estimates:
Statistical analyses were performed using SAS software (SAS; Statistical Analysis Systems, Release 6.12, Cary, North Carolina, USA). Hematocrit, Hb concentration, ALAD activity and plasma chemistries for ducks in all four treatment groups at day 7 were analyzed using a two-way analysis of variance (ANOVA) model involving the factors treatment and sex. Subsequent HCT and plasma chemistries for ducks in the control, tungsten-iron, and tungsten- polymer groups were analyzed by ANOVA involving the factors treatment and sex, with repeated measurements on ducks, over a third factor, days. All two-way interactions between treatment, sex, and days were also modeled. SASw PROC MIXED was used to model a first-order autoregressive correlation structure for repeated measurements over days within animals, as residuals involving measurements taken at adjacent time periods are more likely to have a higher correlation than measurements taken further apart in time. The post-day 7 HCT and plasma chemistries were analyzed separately over two different time periods (days 30 through 60 and days 90 through 150) due to differences in the reproductive status of the ducks. Concentrations of metal residues in tissues of the adults were also analyzed using a two-way ANOVA model. Residual plots were used to check for homogeneity of variance and for aberrant values. Residual plots for plasma chemistry parameters at days 7, 30, 60, 90, 120, and 150 and tissue metal residue concentrations of adults indicated increasing variability with higher responses; therefore those data were log transformed to stabilize variance.

Reported statistics and error estimates (Cont'd)

The reported means and 95% confidence intervals for treatment means of plasma chemistries and tissue metal residue concentrations

were back (anti-log) transformed to the scale of observation. Treatment group means were reported as the least square mean plus or minus the standard error. Treatment means were reported separately for each sex and/or day, if treatment by sex and/or treatment by day interactions, respectively, were statistically significant. Otherwise, reported treatment means and differences were based on pooling information over the sexes and/or days. To control for experimental Type 1 error rates, a Fisher’s protected least significant difference (LSD) was used to test comparisons between means based on the total number of pairwise comparisons. In the following sections, references to significant differences (whether higher or lower) across compared values indicate statistical differences

at P </- 0.05.

Conclusions:
Male and female mallards administered 40 #4 tungsten-polymer shot and maintained for 150 days were not adversely affected based on hematological parameters measured.
Executive summary:

No toxicity to birds data of sufficient quality are available for fused tungsten carbide (target substance). However, toxicity to birds data are available for tungsten metal (source substance), which will be used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
long-term toxicity to birds: reproduction test
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1998-01-26 to:1998-06-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, scientifically sound study with sufficient information presented on materials and methods to adequately evaluate results.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Metal
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
reference to other study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register. 1986. Nontoxic shot approval procedures for shot Federal Register, 51: 42098-42102
Principles of method if other than guideline:
The study design was based on a published protocol (Federal Register, 1986) and modified as requested by the USFWS.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Dose method:
gavage
Analytical monitoring:
no
Vehicle:
no
Details on preparation and analysis of diet:
Not relevant, dosages were not presented in the diet but via gavage. Ducks were dosed with pellets introduced into the proventriculus by means of a funnel and 21.6 cm long latex tube (0.953 cm I.D.) through the esophagus. About 5 ml of water helped flush shot into the proventriculus (Kelly et al, 1998).

Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: mallard/duck (game-farm)
- Source: obtained from Whistling Wings, Inc. (Hanover, Illinois, USA).
- Age at test initiation (mean and range, SD): 5 months
- Sexes used: female and male
- Cultural background:
- Disease free: no data
- Kept according to standard practices: no data
- Breeding population (eg reproductive history): Mallards were two generations removed from wild stock.
Limit test:
yes
Total exposure duration (if not bolus):
120 d
Post exposure observation period:
The last dosewas administered on day 120, but the trial continued until day 150.
No. of animals per sex per dose and/or stage:
Sixteen male/female pairs of adult mallards
Control animals:
other: Negative Control was steel-dosed mallards as considered by USFWS
Nominal and measured doses / concentrations:
The initial pellet weight for tungsten-polymer shots was reported to be 0.186 g (Mitchell et al, 2001a). Therefore, assuming the average erosion rate of 99% for the tungsten-polymer shots, and assuming equal corrosion of both the tungsten and nylon constituents in the shot, the ducks in this group were exposed to 0.176 g/pellet (0.186 g/pellet X 95.5% tungsten X 99% erroded). Eight pellets were administered per dose (5 dosing periods occurred over the 150 day trial), thus a total exposure of 1.41 g tungsten was received per dose. The average bodyweight of the mallards in the tungsten-polymer shot group, at the end of the study was 1.167 kg, resulting in a received dose of approximately 1.21 g solubilized tungsten/kg bodyweight.
Details on test conditions:
ACCLIMATION
- Acclimation period: The 26-day acclimation period was from 30 December 1997 to 25 January 1998.
- Acclimation conditions (same as test or not): no data
- Feeding: Provided feed and water ad libitum throughout the trial. The ducks were fed a pelleted duck grower ration (Purina Mills, St. Louis, Missouri,
USA) during the acclimation period, shelled corn during the first 60 days of the trial (26 January 1998 to 27 March 1998). On day 61 (28 March 1998) of the 150-day trial, all surviving mallards were switched from a shelled corn diet to a commercial layer ration (Mazuri Waterfowl Breeder, Brentwood, Missouri, USA) for the subsequent 90 days.
- Health (any disease or mortality observed): no data

PEN SIZE AND CONSTRUCTION MATERIALS: described in Mitchell et al, 2001a
- Description: cages measuring 0.914 m long 3 0.914 m wide 3 0.457 m high in a minimally heated pole barn.
- Caging: housed randomly as male-female pairs

NO. OF BIRDS PER REPLICATE
- For negative control: Steel-dosed mallards (16 pairs); referred to as "control"

NO. OF REPLICATES PER GROUP
- For negative control: Received 8 #4 pellets on days 0, 30, 60, 90, and 120

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: The temperature, which was continuously monitored, was maintained above 0 degree C by a propane gas heater suspended from the ceiling in the middle of the room.
- Photoperiod: Photoperiod was increased on a weekly basis over 6 wks from 21 April 1998 to 1 June 1998 to provide 18 hr light:6 hr dark.



Details on examinations and observations:
At 14 days of age, each duckling was weighed and blood was collected from the brachial vein into microhematocrit capillary tubes (32 3 0.8 mm) (Drummond Scientific, Broomall, Pennsylvania, USA) for determination of HCT. Ducklings from the first 21 eggs (except the 11th egg) from each hen, if available, were euthanized by cervical dislocation and necropsied. The brain, heart, liver, spleen, kidneys, and bursa were removed and weighed. Gonads were examined to determine sex. Samples of the liver and kidneys from eight male and eight female ducklings in each treatment were stored in 10% formalin-saline (10% formalin in 0.9% sodium chloride) to examine for histopathology.

Histology sections were assessed without knowledge of treatment. The right femur and the remaining portions of the liver and kidneys from each necropsied duckling were frozen and shipped to CT&E Environmental Services (Ludington, Michigan, USA) for analysis of metals. The samples analyzed included 16 (eight male and eight female) pooled liver, kidney, and femur samples, each consisting of tissues from three male or three female ducklings from the same hen in the control or tungsten- polymer groups. The ranges of percent recoveries of iron and tungsten in the process spikes were 104 to 113% and 1 to 63%, respectively and in the matrix spike the ranges of percent recoveries of iron and tungsten were 84 to 106% and 71 to 110%, respectively.
Details on reproductive parameters:
The following parameters were examined per parental pen per week:
- Eggs were collected twice daily from each pair of mallards throughout the 90 day reproduction phase.

- The 11th egg laid by each female was used for determination of shell thickness and for elemental analysis of shell and contents. Measurements of shell thickness were taken at six locations (two on the pointed end, two on the blunt end and two on the equator) on each egg with an Ames 25 ME Digimatic Outside Micrometer (Waltham, Massachusetts, USA) and the six measurements were averaged. Shells and egg contents were shipped to CT&E Environmental Services (Ludington, Michigan, USA) for analysis of metals. The ranges of percent recoveries of iron and tungsten in the process spikes were 106 to 111% and 16 to 43%, respectively, and in the matrix spike, the ranges of percent recoveries of iron and tungsten were 101 to 111% and 52 to 72%, respectively.

- Eggs, except the 11th egg from each hen, were set on a weekly basis and incubated with their blunt end up in a Petersime poultry incubator (Gettysburg, Ohio, USA) for up to 30 days. Conditions in the incubator were standard for commercial operations (99.0–99.5 F with wet bulb readings of 83–85 F to yield approximately 60% relative humidity). Eggs were automatically rotated every two hours. Embryo fertility and viability were determined by candling eggs on incubation days 7, 14 and 21. All viable eggs were transferred on day 23 to pedigree hatching baskets that were placed in a Sure-pip hatcher (Agro Environmental Systems Inc., Dallas, Georgia, USA). The temperature in the hatcher was 99.0 F with a wet-bulb reading of 89.0 F to yield approximately 70% relative humidity. Eggs were kept in the hatcher until hatching or until day 30 of incubation.

- Eggs not hatching were opened and the embryos were examined for deformities and their age determined.

- Ducklings were removed from the incubator (within 18 hr after hatching), weighed, and identified with a Swiftak identification tag (Heartland Animal Health, Inc., Fair Play, Missouri, USA). They were housed in heated floor pens (3.05 m W 3 2.29 mL) with water and starter mash (Purina Mills, St. Louis, Missouri, USA) provided ad libitum.


Reference substance (positive control):
no
Duration (if not bolus):
120 d
Dose descriptor:
other: NOAEL
Effect level:
1.21 other: g solubilized tungsten/kg bodyweight
Conc. / dose based on:
other: days worth of gavage dosing of tungsten polymer shot.
Basis for effect:
reproductive parameters
Remarks:
and offspring survivability over the first 14 days.
Remarks on result:
other: Exact tungsten levels not provided
Repellency factors (if applicable):
n/a
Mortality and sub-lethal effects:
- There was no difference in the survivability of duckling (the number of ducklings alive on day 14 of age divided by the number of hatchlings for each female. The control duckling survivability was 99% (96.31-100.00%) and the duckling survivability for tungsten-polymer was 96% (90.31-98.97%).

- There were no significant differences in hematocrit of ducklings in the control group compared to the tungsten-polymer shot group.

- Body weight over the 14-day period was similar for ducklings in the two groups. In the control body weight was 165 ± 2.1 grams and the tungsten-polymer shot was 167 ± 2.2 g.

- Relative (expressed as a percent of body weight) kidney weight of ducklings in the tungsten-polymer group was significantly higher compared to ducklings in the control group (mean [95% confidence interval]; 1.27 [1.245–1.290]; 1.21 [1.194–1.232], respectively). Relative weights of the liver, spleen, bursa, heart, and brain were equivalent across the groups.

- Liver samples from ducklings in the control and tungsten-polymer groups had mild to moderate diffuse hepatocellular vacuolation with the exception of samples from one female in the control group and one female in the tungsten-polymer group. No kidney lesions were observed.

- Tungsten was detected in four of 16 femur samples from the tungsten group. Tungsten also was detected in one of 16 kidney samples from the tungsten- polymer group. Two of 16 liver samples from the tungsten polymer group contained tungsten. There was no evidence of significant differences in tungsten concentrations in femur, liver, and kidney samples among the two groups.
Effects on reproduction:
- Tungsten-polymer-dosed females began laying eggs approximately seven days earlier than females in the control groups, which began laying
around day 92 of the study. Females in all two groups required 24 to 25 days to lay 21 eggs.

- There were two control female and three tungsten-polymer-dosed females that did not lay any eggs. Of those ducks that laid eggs, there were one control female and one tungsten-polymer-dosed female that did not lay at least 21 eggs. Percent egg production (total number of eggs per hen divided by 90 days) was similar among groups and ranged from 36% to 46% .

- Percentage of eggs hatching from fertile eggs had no evidence of differences among the control and tungsten-polymer groups. Iron was detected in two of 14 and two of 13 shells of eggs laid by control and tungsten-polymer-dosed females, respectively, and in the contents of all eggs analyzed.

- There was no evidence of significant differences in iron concentration of the eggshell or contents among the two groups. Tungsten was detected in the shells of three of 13 eggs laid by tungsten-polymer-dosed females. There was no evidence of significant differences in tungsten concentration in the eggshell or contents. Survivability of ducklings (the number of ducklings alive on day 14 of age divided by the number of hatchlings for each female) was equivalent for all two groups.

Reported statistics and error estimates:
All statistical analyses were performed using SAS software (SAS; Statistical Analysis Systems, Release 6.12, Cary, North Carolina, USA). Since all lead-dosed mallards died by day 25 of the 150-day trial, their data were not included in the statistical analyses. Body weights were analyzed by analysis of variance (ANOVA) involving the factors treatment and sex, with repeated measurements on ducks, when applicable, over a third factor, days. SAS PROC MIXED was used to model a first-order autoregressive correlation structure for repeated measurements over days within ducks, as residuals involving measurements taken at adjacent time periods are more likely to be highly correlated than measurements taken further apart in time.

Reported statistics and error estimates (Cont'd)

Where applicable, all two-way interactions between treatment, sex, and days were modeled. Body weights were analyzed separately over two time periods due to changes in reproductive status over these periods. Body weight differences from day 0 to day 60 were analyzed with mean weight differences compared among the three treatment groups. Next, body weight differences were analyzed over the time period that ducks were reproductively active (day 90 through day 150). Organ weights and percent shot erosion were also analyzed using an ANOVA involving the factors treatment and sex. Percent shot erosion and adult relative organ weights were percentage (p) data subjected to arcsine, square root transformation prior to statistical analysis. As standard errors are not readily back-transformed, the reported means and 95% confidence intervals for treatment means of percent shot erosion and adult relative organ weights were back-transformed to the scale of observation. Residual plots were used to check for homogeneity of variance and for aberrant values. Treatment group means were reported as the least squares mean plus or minus the standard error. Treatment means were reported separately for each sex and/or day, if treatment by sex and/or treatment by day interactions, respectively, were statistically significant. Otherwise, reported treatment means and mean differences were based on pooling information over the sexes and/or days. To control for experimental Type 1 error rates, a Fisher’s protected least significant difference (LSD) was used to test comparisons between means based on the total number of pairwise comparisons. In the following sections reference to significant differences (higher or lower) across compared values indicate statistical differences at P </= 0.05.

Conclusions:
Tungsten-polymer shot (approximately 1.21 g solubilized tungsten/kg bodyweight (based on body weights and shot erosion)) repeatedly administered to adult mallards over 120 days, during the 150 day trial did not adversely affect reproduction or their offspring. Offspring of tungsten dosed mallards had comparable survivability and growth over the first 14 days of age compared to the control ducklings.
Executive summary:

No toxicity to birds data of sufficient quality are available for fused tungsten carbide (target substance). However, toxicity to birds data are available for tungsten metal (source substance), which will be used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
long-term toxicity to birds
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1996-01-16 to: 1996-2-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, scientifically sound study with sufficient information presented on materials and methods to adequately evaluate results.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Metal
Target: Fused tungsten carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
other: Federal Register. 1986. Nontoxic shot approval procedures for shot. Federal Register 51: 42098-42102.
Principles of method if other than guideline:
The study design was based on a published protocol (Federal Register, 1986) and modified as requested by the USFWS.
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Dose method:
gavage
Analytical monitoring:
not specified
Vehicle:
no
Details on preparation and analysis of diet:
Not relevant, dosages were not presented in the diet but via gavage. Ducks were dosed with tungsten-polymer pellets introduced into the proventriculus by means of a funnel and 21.6 cm long latex tube (0.953 cm I.D.) through the esophagus. About 5 ml of water helped flush shot into the proventriculus.
Test organisms (species):
Anas platyrhynchos
Details on test organisms:
TEST ORGANISM
- Common name: game-farm mallards (ducks)
- Age at test initiation (mean and range, SD): 6 months old
Total exposure duration (if not bolus):
30 d
No. of animals per sex per dose and/or stage:
sixteen bird groups (sexes equal)
Control animals:
other: sham-dosed
Nominal and measured doses / concentrations:
Sixteen (eight males and eight females per group) ducks were dosed with tungsten-polymer via eight BB-size pellets. Exact tungsten concentrations were not provided. The amount of tungsten per tungsten-polymer shot dosage was, on average 4.2 g (4.43 g tungsten-polymer shot X 95.5 % tungsten). The rate of shot erosion (based on the average weight of the pellets recovered at day 30) was highest for the tungsten-polymer shot (80%), followed by tungsten-iron (55%), lead (50%), and steel shot (33%). Therefore, assuming an average erosion rate of 80% for the tungsten-polymer shots, and assuming equal corrosion of both the tungsten and nylon constituents in the shot, the ducks in this group were exposed to 3.4 g tungsten. The average bodyweight of the mallards in the tungsten-polymer shot group, at the end of the study was 1.109 kg, resulting in a received dose of 3.07 g solubilized tungsten/kg bodyweight.
Details on test conditions:
ACCLIMATION
- Acclimation period: 3 weeks
- Feeding: pelleted duck grower ration (Purina Mills, St. Louis, Missouri, USA) and water ad libitum.

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: 0.914 m long x 0.914 m wide x 0.457 m high
- Compliant to good husbandry practices: no data
- Suitable to avoid crowding stress: no data
- Caging: individual

NO. OF BIRDS PER REPLICATE
- For treated: Sixteen (eight males and eight females per group) ducks were dosed with tungsten-polymer and received eight BB-size pellets.

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: continuously monitored at above 0 degree C by a propane gas heater suspended from the ceiling in the middle of the room.
- Photoperiod: controlled by a timer on lights and was adjusted weekly to mimic the natural photoperiod for the experimental study.
Details on examinations and observations:
MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Observed all ducks twice daily to assess well-being. Noted in the daily log clinical signs, including inappetence, apparent weight loss, ataxia, lethargy, and discolored excreta. Daily examined excreta for expelled shot.

BODY WEIGHT
- Time schedule for examinations: On the first day of the dosing, each duck was weighed to the nearest gram. On day 30 of the trial, ducks still alive were weighed.

PATHOLOGY
- Dose groups that were examined: necropsy procedure included a gross examination of all body cavities and organs. Opened gizzards to inspect for cracked and discolored mucosa and to recover shot.

ORGAN WEIGHTS
- Dose groups that were examined: Collected and stored samples of kidneys and liver from each duck to examine for histopathology. The right femur and the remaining portions of the liver and kidneys were sent for analyses of metals.
- Organs: The brain, heart, liver, spleen, kidneys, and testes were removed weighed.

REGURGITATION
- Test material was regurgitated: not observed

OTHER: On day seven, shot retention was determined. Collected blood from the brachial vein into one 3 mL Vacutainer tube containing EDTA and two 3 mL Vacutainer tubes containing sodium heparin on days 15 and 30 of the trial. Samples were sent for determination of HCT, Hb concentration, whole-blood delta minolevulinc acid dehydratase (ALAD) activity, and plasma clinical chemistries. HCT was determined by drawing approximately 50 uL whole blood from the Vacutainer tube containing EDTA into a microhematocrit tube. Blood samples were collected on day 30 of the trial from the ducks still alive.
Details on reproductive parameters:
N/A
Reference substance (positive control):
no
Duration (if not bolus):
30 d
Dose descriptor:
other: NOAEL
Effect level:
>= 3.07 other: g solubilized tungsten/kg body weight
Conc. / dose based on:
other: BB sized pellets of tungsten polymer shot.
Basis for effect:
signs of toxicity
Remarks on result:
other: Exact tungsten levels not provided
Duration (if not bolus):
30 d
Dose descriptor:
LD50
Effect level:
> 3.07 other: g solubilized tungsten/kg bodyweight
Conc. / dose based on:
other: BB sized pellets of tungsten polymer shot.
Basis for effect:
mortality
Repellency factors (if applicable):
N/A
Mortality and sub-lethal effects:
MORTALITY: Mortality was not observed in ducks receiving tungsten-polymer shots.

BODY WEIGHT
- Results: During the trial, ducks receiving the control, steel shot, steel-iron shot and tungsten-polymer shot gained a slight amount of weight.


GROSS PATHOLOGY:
Four control ducks (one male and three females) had moderately fatty livers, whereas all ducks in the tungsten-polymer shot groups appeared normal.

Relative organ weights No significant differences between the control and tungsten-polymer relative organ weights (brain, heart, kidneys, liver, spleen, testes).

Histopathology of kidney and liver:
Three tungsten-polymer dosed ducks developed mild hepatic biliary stasis.

Metal residues in tissues:
Tungsten was detected in the femur of two ducks of the tungsten-polymer group.Tungsten was detected in the kidneys of two ducks dosed with tungsten-polymer shot. Tungsten was not detected in the control mallards.

HCT, Hb concentration, and ALAD activity:
No significant differences in HCT and Hb concentrations were detected among the treatment group at day 30, but both parameters decreased significantly between day 15 and day 30. Whole-blood ALAD activity did not vary statistically over time.
Effects on reproduction:
N/A
Results with reference substance (positive control):
N/A
Further details on results:
Average percent recovery of tungsten and lead were 96% and 98%, respectively.

Shot recovered and percent shot erosion:
Fluoroscopy of the mallards on day seven of the trial indicated that all ducks receiving shot had retained all eight pellets administered on day one. Most shot administered (75%) was recovered at necropsy regardless of type. No shot was found in the excreta of any ducks that we examined daily during the trial. The tungsten-polymer shot eroded the most compared to control. When recovered, the tungsten-polymer shot was flattened and disk-like.
Reported statistics and error estimates:
All statistical analyses were performed using SAS software (SAS; Statistical Analysis Systems, Release 6.12, Cary, North Carolina, USA). Differences between treatment group means were statistically significant based on a Type I error rate of 0.05. A Fisher's exact test was used to assess mortality associations with treatment. Body weights, plasma, and whole-blood parameters were analyzed under a three-way ANOVA involving the factors treatment and sex, with repeated measurements on animals over a third factor, days. SAS PROC MIXED was used to model a first-order autoregressive correlation structure for repeated measurements over days within animals, as residuals involving measurements taken at adjacent time periods are more likely to be closely related than measurements taken further apart in time. Organ weights, concentrations of metal residues in tissues, and percent shot erosion were analyzed under a two-way ANOVA model involving the factors treatment and sex. Treatment means were reported separately for each sex, and/or day, if treatment by sex and/or treatment by day interactions were statistically significant.

Reported statistics and error estimates (cont'd)

Otherwise reported treatment means and differences were based on pooling information over the sexes and/or days. To control for experimental error rates, a Bonferroni adjustment was used to test comparisons between means based on the total number of pairwise comparisons. Homogeneity of variance was assessed by residual plots. Residual plots for plasma chemistries indicated heterogeneity of variance; therefore those data were log-transformed and the heterogeneity of variance subsequently was alleviated. The reported means and 95% confidence intervals for treatment means of plasma chemistries were back (anti-log) transformed to the scale of observation. In the following sections reference to significant differences (whether higher or lower) across compared values indicate statistical differences at P 0.05, unless otherwise indicated.

Conclusions:
Results indicated that tungsten-polymer shot (8 shot/duck) orally administered to male and female mallards did not adversely affect them during a 30-day trial.
Executive summary:

No toxicity to birds data of sufficient quality are available for fused tungsten carbide (target substance). However, toxicity to birds data are available for tungsten metal (source substance), which will be used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Description of key information

No data are available for toxicity of fused tungsten carbide to birds. Therefore, read-across to tungsten metal data was used for this endpoint. An LD50 for mallards orally dosed with tungsten-polymer shot was determined.  Results indicated that tungsten-polymer shot orally administered to mallards did not adversely affect them during a 30-day trial and an LD50 of  >3.07 g tungsten/kg bw was identified (Kelly et al, 1998).  

In a separate study in which mallards were dosed with tungsten-nylon pellets once per month over a period 150 days (days 0, 30, 60, 90, 120 and 150),  the NOAEL determined was ≥ 1.21 g tungsten/kg bodyweight per month.  This study revealed that mallard ducks did not have deleterious health effects during the 150-day trial based on mortality, body weights, organ weights, and histology of the liver and kidneys (Mitchell et al, 2001a) or hematological effects (Mitchell et al, 2001b) compared to the control. There were no significant differences in egg production and fertility and hatchability of eggs from tungsten-polymer-dosed ducks compared to control ducks. There was no evidence of differences in percent survivability and body weight of ducklings from tungsten-iron and tungsten-polymer mallards compared to ducklings from control ducks (Mitchell et al, 2001c).  Ingested tungsten does not seem to adversely affect mallards after chronic exposures.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for birds:
1.21 g/kg food

Additional information

No toxicity to birds data of sufficient quality are available for fused tungsten carbide (target substance). However, toxicity to birds data are available for tungsten metal (source substance), which will be used for read-across. Due to similar water solubility and toxicity for the target substance compared to the source substance, the resulting read-across from the source substance to the target substance is appropriate. In addition, read-across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.