Potassium 1,1,2,2,3,3,4,4,4-nonafluorobutane-1-sulphonate

Toxicity to birds

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Jun - 19 Jul 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 205 (Avian Dietary Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: 41-2600-8442-5 Lot 2
- Purity: 97.3%
- Expiration date of the lot/batch: 17 Jan 2007
- Storage condition of test material: Ambient room temperature
- Stability under test conditions: Stable

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: Basal diet premix (5000 g, see Attachment for formulation) was amended with fresh corn oil (195 mL) using a commercial food mixer. Test substance was added to ca. 100 g of amended diet in a Waring blender (1 minute blending). Blended, treated feed was then added back to the main batch with ca. 5 min mixing, after which an addition mass of basal diet premix was added to bring total mass of premix plus test substance to 8820 g with an additional 10 min. mixing. Feed was prepared on an active ingredient basis, with dosing rates adjusted for factor purity.
- Type, identity and function of solvent/vehicle: None

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- How often was homogeneity and stability tested: Homogeneity tested on Day 0. Stability tested on Day 0 and 5.
- At what dose levels were samples of treated food analyzed for stability and concentration during the study: Homogeneity tested on six samples taken from dose levels 1000 ppm a.i. and 10,000 ppm a.i. on Day 0. Duplicate stability samples were taken from the remaining levels on Day 0 and at all levels on Day 5.
- Results of homogeneity analysis (range of values): Coefficient of variation, 6.46% at 1000 ppm ai, 6.65% at 10,000 ppm ai
- Concentration analysed (mg/kg feed): 938 ± 60.6 ppm ai at 1000 ppm, nominal, 9440 ± 627 ppm ai at 10,000 ppm nominal
- % of nominal: 94%
- Mixing procedure adequate and variance between nominal and actual dosage acceptable: yes
- Stability: Measured concentrations in feed samples taken on Day 5 ranged from 98.0% to 116% of concentrations in samples taken on Day 0.

Details on analytical methods
DETAILS ON PRETREATMENT
- Samples of the feed stock (10 g) were weighed and transferred to glass bottles. 100 mL of methanol was then added to the samples and shaken for a minimum of 30 min at approximately 250 rpm. Samples were then filtered through glass wool into a 200 mL volumetric flask, the filter and retained feed residue were rinsed three times with methanol, then the volume brought to 200 mL with methanol. An aliquot of each sample was then further filtered through a 0.45 µm filter into a scintillation vial. The aliquots were diluted volumetrically with 50% (v/v) methanol in NANOpure ® dilution water to bring the sample concentration into the calibration range.
IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method (e.g. HPLC, GC): HPLC-MS-MS
- Conditions (column, mobile phase, etc.): See below
HPLC:
Instrument: Hewlett-Packard Model 1100 Liquid Chromatograph
Keystone PRISM RP column, 50 x 2 mm, 3 μm particle size, with Keystone Javelin C18 guard column, 20 mm x 2 mm ID).
Oven temperature: 40°C
Flow rate: 0.200 mL/min
Injection volume: 5 μL
Mobile phase: 90% methanol: 10% water with 0.1% ammonium formate
Run time: 5 min
Detection: Perkin-Elmer API 100LC Mass Spectrometer with Perkin-Elmer TurboIonSpray ion source, operated in multiple ion reaction monitoring (MRM) mode, m/z 299.0 amu > 98.9 amu.
- Analytical standards: Test substance was weighed analytically and brought to 1.0 mg a.i./mL in methanol. Secondary stock were serially diluted in methanol. Calibration standards were diluted from secondary stock in 50% (v/v) methanol in NANOpure ® water.
- Detection limits (LOD, LOQ) (indicate method of determination /calculation): LOD = 0.5 ng on-column, based on the injection volume (5.00 µL) and the lowest standard concentration, 0.100 mg a.i./L. LOQ = 150 ppm a.i., based on the lowest matrix fortification level analyzed concurrently with the samples. The 0.100 mg a.i./L standard was equivalent to a calculated value of 40 ppm a.i. in the matrix blank extract. Measured values greater than or equal to the ppm a.i. equivalent were reported.
- Calibration standards ranged in concentration from 0.1 to 0.5 mg a.i./L. A typical calibration curve had a regression fit of r=0.995
- Extraction recovery: Matrix spikes (QC samples) were created by fortifying avian diet samples and were analyzed concurrently with the samples to determine the mean procedural recovery. Matrix spikes had a mean recovery of 95.0 % from samples (triplicate at each level) with spiking rates of 150 ppm a.i., 3000 ppm a.i., and 12000 ppm a.i.. Sample concentrations were not corrected for the mean procedural recovery of 95.0%.
- Method of confirmation of identity of measured compound: Mass spectroscopy in multiple ion reaction monitoring (MRM) mode, m/z 299.0 amu > 98.9 amu.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern bobwhite (quail)
- Source: Production flock at testing lab.
- Age at test initiation: Ten days
- Weight at test initiation: 17 - 24 g
- Sexes used / mixed or single sex: Sex could not be distinguished, birds used as such.
- Cultural background: All birds were from the same hatching, were pen-reared, and were phenotypically indistinguishable from wild birds
- Disease free: No apparent disease.
- Kept according to standard practices: Housing and husbandry practices were based on guidelines established by the US National Research Council

Limit test:

no

Total exposure duration (if not single dose):

5 d

Remarks:

Following exposure period all groups given untreated basal diet for three days. On day 8 half of the surviving treatment and control birds were euthanized. Remaining birds were maintained on basal ration until day 22 when they were euthanized.

Post exposure observation period:

3 or 17 days

No. of animals per sex per dose and/or stage:

12 per concentration level plus 30 control

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal feed concentrations: none, 1000 ppm a.i., 1780 ppm a.i., 3160 ppm a.i., 5620 ppm a.i., 10,000 ppm a.i.
Measured feed concentrations:

Details on test conditions:

ACCLIMATION
- Acclimation period: Ten days between hatch and initiation and test
- Acclimation conditions: Same as test
- Feeding: Ad libitum. Feed was same as amended basal diet, with water also containing a vitamin supplement during acclimation period (Durvet water soluble vitamin and electrolyte concentrate)
- Health: No mortality, birds appeared healthy
- Fasting period before study: No

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Thermostatically controled brooding pens were manufactured by Beacon Steel Company, Model B735Q. Each pen measured 72 x 90 x 23 cm. The external walls, ceiling and floor of the pens were constructed of galvanized wire mesh and galvanized sheeting.
- Compliant to good husbandry practices: Housing and husbandry practices were based on guidelines established by the National Research Council (USA)
- Caging: Five to six birds per cage

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: Thirty
- For treated: Twelve

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: Six replicate cages
- For treated: Two replicate cages, one cage euthanized on Day 8

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 39 ± 1 °C until day 12, 27.1 ± 1.7 °C thereafter.
- Relative humidity (%): 54 ± 11%
- Photoperiod: 16 h: 8h light:dark, ca. 180 lux illumination

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Typically at least twice daily
- Remarks: Half of birds euthanized day 8 for necropsy and tissue collection.

BODY WEIGHT
- Time schedule for examinations: Days 0, 5, 8, 15, 22

FOOD CONSUMPTION
- Time schedule for examinations: Periods Day 0-5, Day 6-8, Day 9-15, Day 16-22
- Remarks: Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over the specified interval. Accuracy may have been affected by the unavoidable feed waste by birds.

PATHOLOGY
- Dose groups that were examined: Animals which died during the study were subjected to necropsy. All surviving animals in all treatment groups were euthanized and subjected to gross necropsy either on Day 8 or Day 22
- Remarks: Only one animal died during the test.

ORGAN WEIGHTS
- Dose groups that were examined: All
- Organs: Liver only

Details on reproductive parameters:

Not applicable

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LC50

Effect level:

> 10 000 other: ppm a.i. (nominal)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

> 10 000 other: ppm a.i. (nominal)

Conc. / dose based on:

act. ingr.

Basis for effect:

organ weights

Remarks:

liver

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

3 160 other: ppm a.i. (nominal)

Conc. / dose based on:

act. ingr.

Basis for effect:

body weight

Repellency factors (if applicable):

There were no apparent treatment related effects on feed consumption. (see attachments for summary)

Mortality and sub-lethal effects:

There were no mortalities in the control group. One control bird was noted limping on Day 5 and 6 of the test, apparently due to an injury. Otherwise all control birds were normal in appearance and behavior throughout the test. In addition, there were no treatment related mortalities at the 1000, 1780, 3160, 5620 and 10000 ppm a.i. test concentrations. There were no overt signs of toxicity noted in any of the treatment groups. Toe picking, a cannibalistic form of aggression, was noted in all treatment groups, with two to five birds in each treatment group noted with toe, foot or ankle lesions due to pen-mate aggression. Lameness associated with foot lesions was noted in several birds and lethargy was noted in birds suffering extensive lesions. At the 1780 ppm a.i. test concentration, one bird was found dead on the morning of Day 3. Upon examination, the bird was noted with lesions on both feet and dried blood on the breast feathers. Necropsy revealed a pale spleen, liver and kidneys. The mortality was not considered treatment-related. One bird in the 1780 ppm a.i. treatment group and one bird in the 5620 ppm a.i. treatment group was noted with slight lateral head curl during the last week of the test. This condition is usually associated with head or neck injury and was not considered treatment related. With the exception of incidental observations associated with physical injury, all birds in all treatment groups were normal in appearance and behavior throughout the test.

Further details on results:

Body weight and feed consumption: Compared to the control group, no treatment-related effects were noted in the 1000, 1780 or 3160 ppm a.i. treatment groups throughout the test. A statistically significant (p<0.01) concentration responsive reduction in body weight gain was observed in the 5620 and 10000 ppm a.i. treatment groups during the exposure period that was considered treatment-related. There were no apparent treatment-related effects on feed consumption in any of the treatment groups.

Gross necropsy: Several birds in each treatment group were found to have lesions or conditions that were considered incidental to treatment. Necropsy results for all other birds were not remarkable.

Liver weight: Mean absolute liver weight was reduced at the 1000, 5620 and 10,000 ppm a.i. test concentrations at Day 8, but as a percentage of body weight, these differences were no longer apparent or statistically significant. For Day 22 absolute and relative liver weights, all test concentrations were comparable to the control group and not statistically significant. Compared to the control group, there were no apparent treatment-related effects on mean absolute liver weight at the 1780 or 3160 ppm a.i. test concentrations.

See attachments for summaries of mean feed consumption, mean body weight, and mean and relative liver weights.

Reported statistics and error estimates:

LC50 was not calculated due to a lack of mortality. For mean body weights, body weight change, absolute liver weights and and relative body weights, differences between the control and treatment groups were determined by Analysis of Variance (ANOVA). Bonferroni's t-Test was used to compare the five treatment group means with the control group means and assessed the statistical significance of the differences observed.

Validity criteria fulfilled:

yes

Remarks:

Mortality in the controls was less than 10% at the end of the test. The concentration of test substance was shown to be maintained in the diet of the birds. The lowest treatment group did not result in mortality or toxic effects.

Conclusions:

The 5-day LC50, dietary toxicity of PFBSK+ to Colinus virgianus (Northern Bobwhite) was determined to be greater than 10000 ppm a.i. (OECD TG 205).

Executive summary:

The acute dietary toxicity of PFBSK+ was determined for the bird Colinus virgianus (Northern Bobwhite) according to OECD TG 205. Concentrations (nominal) of PFBSK+ added to the basal diet were 0 (control), 1000, 1780, 3160, 5620, and 10,000 ppm a.i. with measured concentrations of <LOQ, 938, 1700, 2940, 5420, and 9440 ppm a.i. respectively. Birds were fed the treated diet for 5 days, then an untreated diet for 3 or 17 days, with half of the birds euthanized at 8 days, and the remaining birds at 22 days for gross necropsy and liver weight evaluation. One mortality was observed in in the 1780 ppm a.i. treatment group on Day 3, but the bird was observed to have suffered injuries consistent with pen-mate aggression and the mortality was not considered to be treatment-related. No other mortality, or signs of toxicity were noted in the control or treatment groups throughout the test. Several birds in each group were noted to have injuries or conditions consistent with pen-mate aggression. Food consumption was monitored over four continuous time periods over the test, and there were no apparent treatment related effects on feed consumption. A statistically significant (p<0.01) concentration responsive reduction in body weight gain was observed in the 5620 and 10,000 ppm a.i. treatment groups during the exposure period that was considered treatment-related. Gross necropsy revealed several birds in each treatment group that had lesions or conditions that were considered incidental to treatment; necropsy results for all other birds were not remarkable. Mean absolute liver weight was reduced at the 1000, 5620 and 10,000 ppm a.i. test concentrations at Day 8, but as a percentage of body weight, these differences were no longer apparent or statistically significant. For Day 22 absolute and relative liver weights, all test concentrations were comparable to the control group and not statistically significant. The NOEC for body weight was determined to be 3160 ppm a.i.; and the NOEC for liver weight was determined to be > 10,000 ppm a.i. The 5-day LC50 for dietary toxicity was determined to be > 10,000 ppm a.i. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

Reference 2

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Sept 2003 - 6 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA FIFRA Subdivision E., Subsection 71-4

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 206 (Avian Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E1062-86

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot #5
- Expiration date of the lot/batch: August 31, 2008
- Purity: 98.2%

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: Basal diet premix (see attachment: Diet and Supplement Formulations) was amended with 91 mL of corn oil using a commercial food mixer. Test substance was added to enough of the amended diet to cover the blender mixing blades in a Waring blender (1 minute blending). Blended, treated feed was then added back to the main batch with approximately 10 minutes mixing. Feed was prepared on an active ingredient basis, with dosing rates adjusted for factor purity. The basal diet contained approximately 1.1% calcium. An additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diets for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%). Offspring received basal diet without test substance and without the addition of 5% of supplemental limestone.
- Type, identity and function of solvent/vehicle: None

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- How often was homogeneity and stability tested: Homogeneity tested on Day 0. Stability tested on Day 7. Samples during Weeks 4, 8, 12, 16 and 20 were tested to measure/verify test concentrations.
- At what dose levels were samples of treated food analyzed for stability and concentration during the study: Homogeneity tested on six samples taken from each of the treated diets and one sample from the control diet. Control and treatment group diet samples were also collected for stability. In subsequent tests, a single sample was collected from the control and two samples were collected from each treatment group diet.
- Results of homogeneity analysis (range of values): Coefficient of variation, 4.91% at 100 ppm a.i., 4.00% at 300 ppm a.i., 1.82% at 900 ppm a.i.
- Concentration analysed (mg/kg feed): 105 ± 5.13 ppm a.i. at 100 ppm nominal; 299 ± 12.0 ppm a.i. at 300 ppm nominal; 880 ± 16.0 ppm a.i. at 900 ppm nominal
- % of nominal: 105%, 100%, and 98%
- Mixing procedure adequate and variance between nominal and actual dosage acceptable: yes
- Stability: Measured concentrations in feed samples taken on Day 7 averaged 96%, 105%, and 112% of concentrations in samples taken on Day 0 for 100, 300, and 900 ppm a.i., respectively.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern bobwhite
- Source: K&L Quail, 26 Thompson Flat Road, Oroville, CA 95965, USA
- Age at test initiation: 18 weeks
- Weight at test initiation: 171-220 g
- Sexes used / mixed or single sex: Mixed
- Cultural background: same hatch, approaching first breeding season, had not been used in prior testing
- Disease free: yes
- Kept according to standard practices: yes

Limit test:

no

Total exposure duration (if not single dose):

21 wk

Post exposure observation period:

6 weeks (final incubation, hatching, and 14-day off spring rearing period)

No. of animals per sex per dose and/or stage:

16

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal concentrations: 100, 300, 900 mg PFBS/kg feed
Measured concentrations (week 1, day 0): 105 ± 5.13 mg a.i./L, 299 ± 12.0 mg a.i./L, and 880 ± 16.0 mg a.i./L
Measured concentrations (weeks 4, 8, 12, 16, 20, day 0): 107 ± 7.74 mg a.i./L, 314 ± 5.97 mg a.i./L, and 961 ± 36.8 mg a.i./L

Details on test conditions:

ACCLIMATION
- Acclimation period: approximately 5 weeks
- Acclimation conditions: same as test
- Feeding: at least weekly
- Health (any disease or mortality observed): Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test.
- Fasting period before study: no

PEN SIZE AND CONSTRUCTION MATERIALS
- Description: The adult birds were housed indoors in batteries of pens manufactured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330) measuring approximately 27x51 cm with sloping floors resulting in ceiling height ranging from 20-25 cm. Hatchlings were reared in brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured 72 x 90 x 23 cm. The external walls and ceiling of either type of pen were constructed of galvanized wire mesh and galvanized sheeting. The flooring was galvanized wire mesh.
- Compliant to good husbandry practices: Housing and husbandry practices were based on guidelines established by the National Research Council (USA)
- Caging: pairs

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 16 breeding pairs
- For treated: 16 breeding pairs

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: 1
- For treated: 1

TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 23.2 ± 1.0 °C
- Brooder temperature: 38 °C
- Hatcher temperature: 37.2 ± 0.0 °C (relative humidity: 54 ± 0%)
- Relative humidity (%): 34 ± 17
- Photoperiod: 8 hours/day during acclimation and the first 7 weeks of the test; 17 hours/day starting at the beginning of week 8 until euthanization
- Ventilation: the room vented up to 15 room air volumes/hour and replaced with fresh air
- Other: All offspring received a water-soluble vitamin and electrolyte mix in their water

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: daily
- Remarks: observed for signs of toxicity or abnormal behavior

BODY WEIGHT
- Time schedule for examinations: At test initiation, weeks 2, 4, 6, 8 and at adult termination
- Remarks: body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production

FOOD CONSUMPTION
- Time schedule for examinations: weekly
- Remarks: the amount of feed wasted by the birds was not quantified since it was normally scattered and mixed with water and excreta

PATHOLOGY
- Dose groups that were examined: all
- Remarks: Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. All surviving birds were euthanized at the conclusion of the exposure period. Prior to euthanasia, blood samples were drawn from birds. The blood was separated and serum was collected for toxicokinetic analysis. Livers were also weighed and collected. A portion of the liver was collected for histopathological examination along with samples of kidney and gonad. All histopathological samples were fixed in 10% buffered formalin and shipped to EPL in Sterling, VA, USA for histopathology. Additionally, similar samples were collected from a single indiscriminately selected offspring from each pen (if available) at 16 days of age.

ORGAN WEIGHTS
- Dose groups that were examined: All
- Organs: Liver

OTHER:
Eggs were collected daily for all pens, when available, and coded by lot and pen. All eggs laid in a one-week interval were considered to be the same lot. The eggs were stored in a cold room until incubation. At the end of the weekly interval, all eggs were removed from the cold room, candled and counted and eggs selected by indiscriminate draw for egg shell thickness measurement.

Details on reproductive parameters:

The following parameters were examined per parental pen per week:
- Eggs laid
- Eggs cracked; eggs broken
- Egg abnormalities
- Eggshell thickness
- Embryos viable (at 10-12 and 21 days)
- Normal hatchlings
- Abnormal hatchlings
- Clinical signs of toxicity, abnormalities and mortality
- 14-day old surviving chicks
- Chick body weight at hatching and 14 days after hatching

Reference substance (positive control):

no

Key result

Duration (if not single dose):

21 wk

Dose descriptor:

NOEC

Effect level:

900 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

other: mortality, signs of toxicity, body weight, feed consumption, histopathology, reproductive parameters

Remarks:

nominal concentration

Repellency factors (if applicable):

There were no apparent treatment related effects on feed consumption (see attached table: Mean Feed Consumption).

Mortality and sub-lethal effects:

There were no treatment-related mortalities at any of the concentrations tested during the course of the test. There were three adult mortalities that were considered to be incidental to treatment that occured during the test, one in the control group and one each in the 100 and 300 ppm a.i. treatment groups. Additionally, no overt signs of toxicity were observed at any of the concentrations tested (see attached table: Mean Adult Body Weight). Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear, and were noted for the control group and all treatment groups.

Effects on reproduction:

There were no treatment-related effects upon any of the reproductive parameters at any of the concentrations tested (see attached table: summary of reproductive performance).

Further details on results:

There were no treatment-related effect upon feed consumption, egg shell thickness, offspring body weight, or adult or offspring liver weights at any of the concentrations tested. During histopathology, no lesions considered related to exposure to the test material were noted in the liver, kidney, ovary, and testes of the adults or their offspring. The few findings in various tissues of the adults and offspring from the control and treatment groups were considered to be incidental and unrelated to treatment.

Validity criteria fulfilled:

yes

Remarks:

In control, mortality <10%, # 14-day hatchling survivors >12, mean shell thickness 0.19 mm, and stable test substance concentration

Conclusions:

The NOEC (signs of toxicity, mortality, reproductive parameters) for PFBSK+ in northern bobwhite quail (Colinus virginianus) is 900 ppm a.i. (OECD 206).

Executive summary:

The effects upon the adult northern bobwhite quail (Colinus virginianus) of dietary exposure to PFBSK+ over a period of approximately 21 weeks was studied in this report. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to PFBSK+ on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated. No treatment related effect were observed at any concentration. The NOEC for PFBSK+ in northern bobwhite quail is 900 ppm a.i. (highest concentration tested) per the OECD 206 method under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification, & Labeling, and PBT Analysis.

Reference 3

Endpoint:

long-term toxicity to birds: reproduction test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Range-finding study for OECD 206

GLP compliance:

no

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: no

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- How often was homogeneity and stability tested: Homogeneity tested on Day 0. Stability under ambient conditions was tested on duplicate samples on Day 7. Triplicate samples collected on Week 6, Day 0 tested to measure/verify test concentrations.
- When and at what dose levels were samples of treated food analyzed for stability and concentration during the study: Homogeneity tested on six samples taken from each of the treated diets. Treatment group diet samples were also collected for stability.
- Results of homogeneity analysis (Week 1, Day 0): Coefficient of variation, 1.32% at 75 ppm a.i., 4.06% at 200 ppm a.i., 3.06% at 550 ppm a.i., 2.69% at 1500 ppm a.i.
- Nominal concentration: 75, 200, 550, 1500 ppm a.i.
- Concentration analysed (Week 1, Day 0): 75.6 ± 4.85 ppm a.i., 218 ± 8.87 ppm a.i., 561 ± 17.2 ppm a.i., 1530 ± 41.2 ppm a.i.
- % of nominal (Week 1, Day 0): 101%, 109%, 102%, 102%
- Mixing procedure adequate and variance between nominal and actual dosage acceptable: yes
- Stability: Measured concentrations in feed samples taken on Week 1, Day 7 averaged 128%, 116%, 115%, and 109% of concentrations in samples taken on Day 0 for 75, 200, 550, and 1500 ppm a.i., respectively.

Test organisms (species):

Colinus virginianus

Details on test organisms:

TEST ORGANISM
- Common name: Northern bobwhite

Limit test:

no

Total exposure duration (if not single dose):

6 wk

No. of animals per sex per dose and/or stage:

5

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal concentrations: 75, 200, 550, 1500 mg PFBS/kg feed
Measured concentrations (week 1, day 0): 75.6 ± 4.85 ppm a.i., 218 ± 8.87 ppm a.i., 561 ± 17.2 ppm a.i., 1530 ± 41.2 ppm a.i.
Measured concentrations (week 6, day 0): 71.7 ± 1.72 ppm a.i., 192 ± 3.21 ppm a.i., 519 ± 15.6 ppm a.i., 1410 ± 25.2 ppm a.i.

Details on test conditions:

ACCLIMATION
- Acclimation period: yes, details not available

NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: 5 breeding pairs
- For treated: 5 breeding pairs

NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: 1
- For treated: 1

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: daily
- Remarks: observed for signs of toxicity or abnormal behavior

BODY WEIGHT
- Time schedule for examinations: test initiation, week 2, week 4, test termination
- Remarks: none

FOOD CONSUMPTION
- Time schedule for examinations: weekly
- Remarks: due to the feed wastage by some birds, feed consumption was variable between pens

PATHOLOGY
- Dose groups that were examined: all
- Remarks: At the end of Week 6, all surviving birds were euthanized and subjected to gross necropsy. Effects on adult health, body weight gain, and feed consumption were evaluated. Egg, blood and liver samples were collected for possible analyses.

ORGAN WEIGHTS
- Dose groups that were examined: all
- Organs: Liver

Details on reproductive parameters:

The following parameters were examined per parental pen per week:
- Eggs laid

During the test, the number of eggs laid in each pen was recorded to evaluate egg production. Following enumeration, eggs laid during the first five weeks were disposed of by incineration. All eggs laid during the final week of exposure were stored refrigerated. Two eggs (when available) were indiscriminately chosen from each pen for possible analysis.

Reference substance (positive control):

no

Key result

Duration (if not single dose):

6 wk

Dose descriptor:

NOEC

Effect level:

200 mg/kg diet

Conc. / dose based on:

act. ingr.

Basis for effect:

reproductive parameters

Remarks:

egg production

Repellency factors (if applicable):

There were no apparent treatment related effects on feed consumption (see attached table: Mean Feed Consumption).

Mortality and sub-lethal effects:

No treatment-related mortalities or overt signs of toxicity were observed at any of the concentrations tested. The single incidental mortality occurred in the 1500 ppm a.i. treatment group; a hen that was noted with an apparent neck injury on Day 2 of the test was subsequently euthanized. Prior to euthanasia, the hen exhibited ventral head curl, wing droop, ataxia, a ruffled appearance and lethargy. At necropsy the bird was thin, with a body weight of 162 grams. The liver contained scattered white foci, the abdominal cavity showed evidence of egg yolk peritonitis, and the ovary was regressing. Since the bird was euthanized within three days of the experiment start and the mortality was clearly not treatment-related, the hen was replaced.

Additionally, no overt signs of toxicity were observed at the 75, 200, or 550 ppm a.i. test concentrations, or among females at the 1500 ppm a.i. test concentration. There was a slight but consistent reduction in adult male body weight at the 1500 ppm a.i. test concentration during each weight interval that may have been treatment-related (see attached table: Mean Adult Body Weight). Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear and/or pen mate aggression.

Effects on reproduction:

There were no apparent treatment-related effects upon egg production at the 75 and 200 ppm a.i. test concentrations. While there was a slight reduction in egg production at the 75 ppm a.i. concentration, the reduction was not dose-responsive and was due primarily to non-treatment related effects on two hens that showed signs of egg fragments in the abdominal cavity. However, there were reductions in mean egg production at the 550 and 1500 ppm a.i. test concentrations for which a treatment-related effect could not be precluded (see attached table: Egg Production Data by Week).

Further details on results:

There were no apparent treatment-related effects on liver weight at the 75, 200, or 550 ppm a.i. test concentrations, or among males at the 1500 ppm a.i. test concentration. There was a slight reduction in mean female liver weight at the 1500 ppm a.i. test concentration (see attached table: Mean Liver Weights). At the end of Week 6, all adult birds were euthanized and subjected to gross necropsy. All findings were considered to be incidental to treatment (see attached table: Summary of Gross Pathological Observations).

Reported statistics and error estimates:

Means and standard deviations for continuous variables were reported. All findings were reported based on visual inspection of the data. Statistical significance of findings was not evaluated.

Validity criteria fulfilled:

not applicable

Conclusions:

The NOEC (egg production) for PFBSK+ in northern bobwhite quail (Colinus virginianus) is 200 ppm a.i..

Executive summary:

The effects upon the adult northern bobwhite quail (Colinus virginianus) of dietary exposure to PFBSK+ over a period of 6 weeks was studied in this report. Effects on adult health, body weight, feed consumption, and egg production were evaluated at nominal concentrations of 75, 200, 550, and 1500 ppm a.i. There were slight reductions in mean egg production at the 550 and 1500 ppm a.i.test concentrations that may have been related to treatment. The NOEC for PFBSK+ in northern bobwhite quail is 200 ppm a.i.. It should be noted that the statistical significance of this finding was not evaluated. The study meets generally accepted scientific principles. It was a pilot study for a full test to be conducted under OECD 206, total population size was limited to five breeding pairs per dosing level, and the study was not GLP compliant. Therefore the study is considered reliable with restrictions and is not to be considered definitive.

Reference 4

Endpoint:

short-term toxicity to birds: dietary toxicity test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 6 - July 19, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 205 (Avian Dietary Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.2200 (Avian Dietary Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E857-87, "Standard Practice for Conducting SubDietary Toxicity Tests with Avian Species"

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:41-2600-8442-5, Lot 2
- Expiration date of the lot/batch: Jan 17, 2007
- Purity: 97.3%
- Storage condition of test material: Ambient room temperature
- Stability under test conditions: Stable

Dose method:

feed

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and analysis of diet:

DIET PREPARATION
- Description and nutrient analysis of basal diet provided in study report: yes
- Preparation of doses: Basal diet premix (5000 g, see Attachment 1 for formulation) was amended with corn oil (195 mL) using a commercial food mixer. Test substance was added to ca. 100g of amended diet in a Waring blender (1 minute blending). Blended, treated feed was then added back to the main batch with ca. 5 min mixing, after which an additional mass of basal diet premix was added to bring total mass of premix plus test substance to 8820 g with an additional 10 min. mixing. Feed was prepared on an active ingredient basis, with dosing rates adjusted for factor purity.
- Type, identity and function of solvent/vehicle: None

HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- How often was homogeneity and stability tested: Homogeneity tested on Day 0. Stability tested on Day 0 and 5.
- At what dose levels were samples of treated food analyzed for stability and concentration during the study: Homogeneity tested on six samples taken from dose levels 1000 ppm a.i. and 10,000 ppm a.i. on Day 0. Duplicate stability samples were taken from the remaining levels on Day 0 and at all levels on Day 5.
- Results of homogeneity analysis (range of values): Coefficient of variation, 6.46% at 1000 ppm a.i., 6.65% at 10,000 ppm a.i.
- Concentration analysed (mg/kg feed): 938 ± 60.6 ppm a.i. at 1000 ppm, nominal, 9440 ± 627 ppm a.i. at 10,000 ppm nominal
- % of nominal: 94%
- Mixing procedure adequate and variance between nominal and actual dosage acceptable: yes
- Stability: Measured concentrations in feed samples taken on Day 5 ranged from 92.9% to 108% of concentrations in samples taken on Day 0.

Test organisms (species):

Anas platyrhynchos

Details on test organisms:

TEST ORGANISM
- Common name: Mallard
- Source: Whistling Wings,113 Washington Street, Hanover, Illinois 61041, USA
- Age at test initiation: 8 days
- Weight at test initiation: 79-138g
- Sexes used / mixed or single sex: Sex could not be distinguished, birds used as such.
- Cultural background: All birds were from the same hatching, were pen-reared, and were phenotypically indistinguishable from wild birds
- Disease free: No apparent disease.
- Kept according to standard practices: Housing and husbandry practices were based on guidelines established by the US National Research Council

Limit test:

no

Total exposure duration (if not single dose):

5 d

Remarks:

Following exposure period, all groups given untreated basal diet for three days. On day 8 half of the surviving treatment and control birds were euthanized. Remaining birds were maintained on basal r ation until day 22 when they were euthanized.

Post exposure observation period:

3 or 17 days

No. of animals per sex per dose and/or stage:

12 per concentration level plus 30 control

Control animals:

yes, plain diet

Nominal and measured doses / concentrations:

Nominal feed concentrations: none, 1000 ppm a.i., 1780 ppm a.i., 3160 ppm a.i., 5620 ppm a.i., 10,000 ppm a.i.
Measured feed concentrations:

Details on test conditions:

ACCLIMATION
- Acclimation period: Six days
- Acclimation conditions: Same as test
- Feeding: Ad libitum. Feed was same as amended basal diet.
- Health: No mortality, birds appeared healthy
- Fasting period before study: No
PEN SIZE AND CONSTRUCTION MATERIALS
- Description: Thermostatically controled brooding pens were manufactured by Safeguard Products Inc., Model 5355. Each pen measured 62 x 92 x 25.5 cm. The external walls, ceiling and floor of the pens were constructed of vinyl-coated wire grid.
- Compliant to good husbandry practices: Housing and husbandry practices were based on guidelines established by the National Research Council (USA)
- Caging: Five to six birds per cage
NO. OF BIRDS PER STAGE OR REPLICATE
- For negative control: Thirty
- For treated: Twelve
NO. OF STAGES OR REPLICATES PER GROUP
- For negative control: Six replicate cages
- For treated: Two replicate cages, one cage euthanized on Day 8
TEST CONDITIONS (range, mean, SD as applicable)
- Temperature: 31 ± 1 °C through day 11, 24.9 ± 0.7 °C thereafter.
- Relative humidity (%): 74 ± 5% at ambient temperature
- Photoperiod: 16 h: 8h light:dark, ca. 196 lux illumination

Details on examinations and observations:

MORTALITY / CLINICAL SIGNS
- Time schedule for examinations: Typically at least twice daily
- Remarks: Half of birds euthanized day 8 for necropsy and tissue collection.
BODY WEIGHT
- Time schedule for examinations: Days 0, 5, 8, 15, 22
FOOD CONSUMPTION
- Time schedule for examinations: Periods Day 0-5, Day 6-8, Day 9-15, Day 16-22
- Remarks: Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over the specified interval. Accuracy may have been affected by the unavoidable feed waste by birds.
PATHOLOGY
- Dose groups that were examined: All animals in all treatment groups were euthanized and subjected to gross necropsy either on Day 8 or Day 22
- Remarks: No mortalities during the test.
ORGAN WEIGHTS
- Dose groups that were examined: All
- Organs: Liver only

Details on reproductive parameters:

Not applicable.

Key result

Duration (if not single dose):

5 d

Dose descriptor:

LC50

Effect level:

> 10 000 other: ppm a.i. (nominal)

Conc. / dose based on:

act. ingr.

Basis for effect:

mortality

Duration (if not single dose):

5 d

Dose descriptor:

NOEC

Effect level:

5 620 other: ppm a.i. (nominal)

Conc. / dose based on:

act. ingr.

Basis for effect:

body weight

Remarks:

(body weight gain)

Repellency factors (if applicable):

There were no apparent treatment related effects on feed consumption.

Mortality and sub-lethal effects:

All birds at all test concentrations and in the control were normal in appearance and behavior thoughout the test, and there were no mortalities.

Further details on results:

Body weight: Compared to the control group, no treatment-related effects were noted in the 1000, 1780, 3160, or 5620 ppm a.i. treatment groups throughout the test. A statistically significant (p<0.01) reduction in body weight gain was observed in the 10000 ppm a.i. treatment group during the exposure period that was considered treatment-related. Following the exposure period, body weight gain in the 10000 ppm a.i. treatment group during the post exposure period was not statistically different from the control group. When compared to the control group, there was a statistically significant (p<0.05) reduction in body weight gain among birds in the 3160 ppm a.i. treatment group from Day 15 to 22 of the post exposure period. However the reduction was not concentration responsive. Additionally, a similar reduction was not noted during the exposure period or during the subsequent post-exposure intervals (Day 5 to 8 and Day 8 to 15) at this treatment level. Therefore, this reduction was not considered treatment-related (See attachment for body weight summary).

Gross necropsy: Gross necropsy revealed one bird in the control group with a subcapsular hematoma on the right lobe of the liver. Another control bird was noted with a pale and mottled spleen. The necropsy results for all other birds were not remarkable.

Liver weight: Compared to the control group, there were no apparent treatment-related effects on mean absolute liver weight or mean relative liver weight (liver weight as a percentage of body weight) for any of the concentrations tested. Any differences between the control and each treatment were not statistically significant.

Reported statistics and error estimates:

LC50 was not calculated due to a lack of mortality. For mean body weights, body weight change, absolute liver weights and and relative body weights, differences between the control and treatment groups were determined by Analysis of Variance (ANOVA). Bonferroni's t-Test was used to compare the five treatment group means with the control group means and assessed the statistical significance of the differences observed.

Validity criteria fulfilled:

yes

Remarks:

Mortality in the controls was less than 10% at the end of the test. The concentration of test substance was shown to be maintained in the diet of the birds. The lowest treatment group did not result in mortality or toxic effects.

Conclusions:

The 5-day LC50, dietary toxicity of PFBSK+ to Anas platyrhynchos (Mallard) was determined to be greater than 10000 ppm a.i. (OECD TG 205).

Executive summary:

The acute dietary toxicity of PFBSK+ for the bird, Anas platyrhynchos (Mallard) was determined according to OECD TG 205. Concentrations (nominal) of PFBSK+ added to the basal diet were 0 (control), 1000, 1780, 3160, 5620, and 10,000 ppm a.i. with measured concentrations of <LOQ, 938, 1700, 2940, 5420, and 9440 ppm a.i. respectively. Birds were fed the treated diet for 5 days, then an untreated diet for 3 or 17 days, with half of the birds euthanized at 8 days, and the remaining birds at 22 days for gross necropsy and liver weight evaluation. All birds at all test concentrations and in the control were normal in appearance and behavior throughout the test, and there were no mortalities. Food consumption was monitored over four continuous time periods over the test, and there were no apparent treatment related effects on feed consumption. A statistically significant (p<0.01) concentration responsive reduction in body weight gain was observed in the 10,000 ppm a.i. treatment group during the exposure period that was considered treatment-related. Gross necropsy revealed abnormalities in two control birds, but the necropsy of all birds in the treatment groups was unremarkable. Mean absolute and relative liver weights were not statistically between birds in the treatment groups and control birds. The NOEC for body weight gain was determined to be 5620 ppm a.i. The 5-day LC50 for dietary toxicity was determined to be > 10,000 ppm a.i. The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.

Reference 5

Endpoint:

toxicity to birds, other

Remarks:

gene expression in isolated neuronal cell cultures

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Exposure of isolated embryonic neurons in cell culture, analysis of expression of thyroid hormone-responsive genes
- Short description of test conditions: neurons obtained from bird embryos, isolated and grown in standard cell culture medium
- Parameters analysed / observed: reverse transcription-Polymerase Chain Reaction used to monitor transcription of several genes after exposure.

GLP compliance:

no

Specific details on test material used for the study:

Obtained from Wellington Laboratories (Guelph, ON, Canada). Lot: 24318BB; MW: 338.19 g/mol. Ten other perfluoroalkyl acid substances were examined in the same experiment.

Dose method:

other: culture medium

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Test organisms (species):

other: Gallus domesticus, Larus argentatus

Details on test organisms:

TEST ORGANISM
Gallus domesticus
- Common name: white leghorn chicken
- Source: Canadian Food Inspection Agency (Ottawa, ON, Canada)
- Age at test initiation (mean and range, SD): 11 days' incubation (midincubation)
- Kept according to standard practices: Eggs incubated at 37 °C and 60% relative humidity until collection of embryos

Larus argentatus
- Common name: herring gull
- Source: wild-collected from Chantry Island, ON, Canada (44°29'22"N and 81°24'7"W)
- Age at test initiation (mean and range, SD): 14 days' incubation (midincubation)
- Kept according to standard practices: Eggs incubated at 37 °C and 60% relative humidity until collection of embryos

In both cases, embryos were euthanized by decapitation. Cerebral cortices were dissected from the embryos (24 G. domesticus, 15 L. argentus), pooled in isolation medium (2.5mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 128.5mM NaCl, 5.4mM KCl, 5.5mM D-glucose, 51.8mM sucrose, and 0.1% bovine serum albumin), and minced into small pieces using a scalpel. The mixture was incubated at room temperature for two hours and further dissociated by pipetting up and down against the glass bottom. The dissociated cells were vacuum filtered through a series of nylon sieves of decreasing pore sizes (~200, 120, 45, and 22 µm), and the filtrate was centrifuged for 13 min at 60 x g, 14°C. The resulting pellet was resuspended in isolation medium and vacuum filtered/centrifuged a second time. The cell pellet was resuspended in Neurobasal medium containing 0.5mM L-glutamine, 25 µM glutamate, and 2% B27 supplement (Invitrogen, Burlington, ON, Canada). Cells were plated at a density of 3.5 mg cells/ml in 48-well plates that were precoated with 3.3 µg/ml poly-D-lysine, and then incubated at 37 °Cand 5% CO2 for 24 h prior to exposure.

Limit test:

no

Total exposure duration (if not single dose):

24 h

Nominal and measured doses / concentrations:

Nominal: 0.01, 0.1, 1, 3, 10, and 50 µM (G. domesticus). 0.01, 0.1, 1, 3, and 10 µM (L. argentus).

Details on test conditions:

Cells were exposed in the microplates to which they had been seeded 24-hours prior. 1.75 µL of test substance dilution or 2.5 µL of reference substance dilution were added to initiate the test. Four replicates were done of each exposure level, and each replicate was done twice on separate plates to allow RNA extraction or cell viability tests. After 24-hours incubation at 37 °C, 5% CO2, medium was aspirated. Cells were either frozen at -80 °C for mRNA extraction or were used immediately for cell viability assays.

In the case of L. argentus cells, positive control exposure included one additional level and was examined 3, 6, 12, 18, 24, and 36 h rather than a single time.

Cell viability: A fluorescence activation was used to assess cell viability (Calcein AM is converted to a fluorescent form in viable cells). The aspirated plates were refilled with 200 µL of Calcein-AM solution (3 µL concentrate in phosphate-buffered saline-EDTA solution) and incubated for 45 minutes. Three wells were tested per substance or control. Ethanol (100%) served as control for loss of viability, with a DMSO vehicle control and an untreated control also tested. Fluorescence was measured with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Viability of HGEN was tested for the T3 treatment only at 3 and 24 hours, while viability of CEN was tested for all substances.

Gene expression: Total RNA from frozen plates was extracted using Qiagen RNeasy 96 kits (Qiagen, Mississauga, ON, Canada), with DNA removed using the Ambion DNA-free kit (Ambion, Austin, TX). Complementary DNA (cDNA) was synthesized using random hexamers and Superscript II reverse transcriptase (RT) (Invitrogen). Expression was quantified using quantitative real-time PCR with normalization to beta-actin. All PCR assays were performed using the Strategene Mx3000P or Mx3005P instruments (Strategene, La Jolla, CA) and TaqMan probes with Brilliant Quantitative-PCR Core Reagent kits (Stratagene). Standard curves were generated for all genes from a 1:2 dilution series of cDNA. Primer concentrations were optimized so that PCR reaction efficiencies for all target genes were within ±10% of the amplification efficiency of beta-actin. No-RT and no-template controls (NTCs) were included in all assays to verify the absence of contamination.
Genes examined:
beta-Actin
TH-receptor alpha and beta (TR-α and TR-ß)
Type II iodothyronine 5'-deiodinase (D2)
Type III iodothyronine 5'-deiodinase (D3)
Transthyretin (TTR)
Neurogranin (RC3)
Octamer motif–binding factor 1 (Oct-1)
Myelin basic protein (MBP)

Reference substance (positive control):

yes

Remarks:

Triiodothyronine (T3)

Duration (if not single dose):

24 h

Dose descriptor:

NOEC

Effect level:

10 other: µM

Conc. / dose based on:

test mat.

Remarks:

nominal

Basis for effect:

other: cell viability

Remarks on result:

other: cultured G. domesticus embryonic neurons

Results with reference substance (positive control):

T3 had no effect on cell viability up to 30 nM in CEN and up to 300 nM in HGEN. Most thyroid hormone-responsive receptors showed no change in expression on T3 exposure (see Table 1).

Further details on results:

Effects of PFBSK+ on gene expression are also shown in Table 1. Increases were at least two-fold relative to controls and were statistically significant. No clear pattern is evident. The reference substance T3 affected expression of five of the eight genes examined in G. domesticus cells and three of five genes in L. argentatus cells. PFBSK+ had effects on three G. domesticus genes and one L. argentatus gene, with only one in common with T3. The authors speculate that differences in the transcriptional responses between species to PFAAs in general may be related to variable levels of baseline contaminant exposure and/or unknown factors that determine species-specific differences in sensitivity. Based on a maximal possible uptake rate into neuronal cells of 100%, the lowest effective in vitro concentration (3 µM for Oct-1 mRNA expression) would be approximately three orders of magnitude greater than levels detected in herring gull eggs taken from the collection site used in this experiment (~340 µg/g vs. 0.192 µg/g ww). Given intergenic and interspecies inconsistency in effects on expression, and lacking information on the implications for avian toxicity, the relevance of this study cannot be assessed at this time.

Reported statistics and error estimates:

Cell viability data were analyzed between experimental and DMSO vehicle control groups using a one-way ANOVA followed by Bonferroni’s t-test for multiple comparisons versus control. Real-time RT-PCR data were analyzed using MxPro v3.00 software (Strategene). Relative quantity values were normalized to ß-actin, and fold changes were calculated relative to the DMSO-treated cells using the 2(-Delta Delta C(T)) method of Livak and Schmittgen (2001, Methods Vol. 25, pp. 402–408). Statistically significant differences between experimental and DMSO vehicle control groups were determined using a one-way ANOVA followed by Bonferroni’s t-test for multiple comparisons versus control. Changes were considered statistically different if p< 0.05.

Table, Significant changes in gene expression in embryonic neuron cell culture on exposure to T3 and PFBSK+

Gene		TR-α	TR-ß	D2	D3	TTR	RC3	Oct-1	MBP
Impact		Receptor	Receptor	TH availability	TH availability	TH transport	Signal transduction	Transcription factor activity	Myelination
Species	Compound
G. domesticus	T3	↑	↑	↑	─	↓	↑	─	─
G. domesticus	PFBSK+	─	─	─	↑	─	↑	─	─
L. argentatus	T3	↑	↑	─	n.d.¹	n.d.	↑	─	n.d.
L. argentatus	PFBSK+	─	─	─	n.d.	n.d.	─	↑	n.d.

1, n.d., not determined 

Validity criteria fulfilled:

not applicable

Conclusions:

PFBSK+ did not affect viability of Gallus domesticus embryonic neuron cell cultures at up to 50 µM. Differential impacts on G. domesticus and Larus argentatus gene expression were observed but their relevance to avian toxicity cannot be addressed at this time.

Executive summary:

Effects of PFBSK+ on gene expression were examined in isolated embryonic neuron cell cultures derived from Gallus domesticus (chicken) and Larus argentatus (herring gull). Triiodothryronine (T3) was used as positive control. PFBSK+ did not affect viability of the G. domesticus embryonic neuron cell cultures at up to 50 µM.  Inconsistent impacts of both T3 and PFBSK+ on G. domesticus and L. argentatus gene expression were observed. While some changes in expression were statistically significant, their relevance to avian toxicity cannot be addressed at this time.

The study was published in a peer-reviewed journal and uses generally accepted scientific principals. However, the direct application of the data to avian toxicity is unclear. With further research such information may be incorporated into adverse outcome pathways. At present, the study is given a Klimisch 3 score due to the suitability of the test in meeting the data requirement.

Description of key information

The 5-day LC50, dietary toxicity of PFBSK+ to Colinus virgianus (Northern Bobwhite quail) was determined to be greater than 10000 ppm a.i. (OECD TG 205). The 21-week NOEC (signs of toxicity, mortality, reproductive parameters) for PFBSK+ in northern bobwhite quail is 900 ppm a.i. (OECD TG 206).

Additional information

In the key acute study, the acute dietary toxicity of PFBSK+ was determined for Colinus virginianus (northern bobwhite quail) according to OECD TG 205. Concentrations (nominal) of PFBSK+ added to the basal diet were 0 (control), 1000, 1780, 3160, 5620, and 10,000 ppm a.i.. Birds were fed the treated diet for 5 days, then an untreated diet for 3 or 17 days, with half of the birds euthanized at 8 days, and the remaining birds at 22 days for gross necropsy and liver weight evaluation. Food consumption was monitored over four continuous time periods over the test, and there were no apparent treatment related effects on feed consumption. A statistically significant (p<0.01) concentration responsive reduction in body weight gain was observed in the 5620 and 10,000 ppm a.i. treatment groups during the exposure period. Gross necropsy revealed several birds in each treatment group that had lesions or conditions that were considered incidental to treatment. Mean absolute liver weight was reduced at the 1000, 5620 and 10,000 ppm a.i. test concentrations at Day 8, but as a percentage of body weight, these differences were no longer apparent or statistically significant. For Day 22 absolute and relative liver weights, all test concentrations were comparable to the control group and not statistically significant. The NOEC for body weight was determined to be 3160 ppm a.i.; and the NOEC for liver weight was determined to be > 10,000 ppm a.i. The 5-day LC50 for dietary toxicity was determined to be > 10,000 ppm a.i. A second study was done on Anas platyrhynchos (Mallard duck) according to OECD TG 205 and at the same test substance concentrations. All birds were normal in appearance and behavior throughout the test, and there were no mortalities. Food consumption was monitored over four continuous time periods over the test, and there were no apparent treatment related effects on feed consumption. A statistically significant (p<0.01) concentration responsive reduction in body weight gain was observed in the 10,000 ppm a.i. treatment group during the exposure period that was considered treatment-related. Gross necropsy of the birds in the treatment groups was unremarkable. Differences in absolute and relative liver weights were not statistically significant between birds in the treatment groups and control birds. The NOEC for body weight gain was determined to be 5620 ppm a.i. The 5-day LC50 for dietary toxicity was determined to be > 10,000 ppm a.i. Both tests were conducted according to an internationally accepted test guideline, were GLP compliant, and are considered reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis. Although both acute studies reported the same result, the key study was designated as such because more effects were seen in northern bobwhite quail.

In the key chronic study, the effects upon the adult northern bobwhite quail of dietary exposure to PFBSK+ over a period of approximately 21 weeks were studied. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to PFBSK+ on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated. No treatment related effects were observed at any concentration. The NOEC for PFBSK+ in northern bobwhite quail is 900 ppm a.i. (highest concentration tested) per the OECD 206 method under GLP. The study is deemed reliable without restriction and suitable for use in Risk Assessment, Classification, & Labeling, and PBT Analysis. A 6-week pilot study was conducted on this species prior to the key study. It is considered supporting but should not be considered definitive. Effects on adult health, body weight, feed consumption, and egg production were evaluated at nominal concentrations of 75, 200, 550, and 1500 ppm a.i. There were slight reductions in mean egg production at the 550 and 1500 ppm a.i.test concentrations that may have been related to treatment. However, it should be noted that the statistical significance of this finding was not evaluated. The study was a pilot study for a full test to be conducted under OECD 206, total population size was limited to five breeding pairs per dosing level, and the study was not GLP compliant; therefore the study is considered reliable with restrictions.

In the final available bird study, effects of PFBSK+ on gene expression were examined in isolated embryonic neuron cell cultures derived from Gallus domesticus (chicken) and Larus argentatus (herring gull). PFBSK+ did not affect viability of the G. domesticus embryonic neuron cell cultures at up to 50 μM. While some changes in gene expression were statistically significant, their relevance to avian toxicity and applicability to substance assessment cannot be addressed at this time. With further research such information may be incorporated into adverse outcome pathways.