3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

amphibian Xenopus laevis, larvae: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 January 2020 - August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is performed in accordance with GLP and to the relevant test guideline, with no deviations impacting the integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 231 (The Amphibian Metamorphosis Assay)

Version / remarks:

Adopted: 7 September 2009

Deviations:

yes

Remarks:

At test termination, tadpoles were fixed in Davidson's fixative for 24 h; whereas OECD 231 states that fixing duration should be at least 48 h. The deviation was deemed to result in good quality tissues for analysis and didn't affect the study quality.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium undecafluorohexanoate

EC Number:

220-881-7

EC Name:

Sodium undecafluorohexanoate

Cas Number:

2923-26-4

Molecular formula:

C6F11NaO2

IUPAC Name:

Sodium undecafluorohexanoate

Test material form:

solid

Details on test material:

Chemical name: Sodium perfluorohexanoate
Molecular formula: C6F11NaO2
Molecular weight: 336.03 g/mol
CAS number: 2923-26-4
Purity: 100 %
Expiry: 30 September 2021Storage conditions at test facility: 20 ± 5 °C, in the dark under inert gas argon
Colour: White/off-white

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 1.07, 10.7 and 107 mg test item/L
- Sampling method: At least one sample from the freshly prepared stock solutions and at least one sample from the test media of each replicate (aquaria) of all test concentrations and the control were taken prior to the start of the test (the equilibration phase). After the test started, duplicate samples from the test media of each replicate (aquaria) of all test concentrations and the control were taken at day 0, 7, 18 and 21 during exposure phase.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed unless analysis was performed directly after sampling. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Ratio of test item (sodium perfluorohexanoate) to perfluorohexanoate was calculated by using the molecular weights of both substances. 1.07, 10.7 and 107 mg test item/L (spacing factor 10), and a control were prepared, corresponding to 1.0, 10 and 100 mg perfluorohexanoate/L.
A concentrated stock solution of 10.7 g/L was prepared by dissolving 90.95 g or 53.5 g test item in 8.5 L or 5.0 L test water, respectively.This stock solution served also as application solution for the highest test item concentration (107 mg test item/L). The application solution for the target concentration of 10.7 mg test item/L was prepared by dissolving 750 or 500 mL concentrated stock solution ad 7500 or 5000 mL, respectively, resulting in 1070 mg test item/L. The application solution for the target concentration of 1.07 mg test item/L was prepared by dissolving 75 or 50 mL concentrated stock solution ad 7500 or 5000 mL, respectively, resulting in 107 mg test item/L.
To reach the target test concentrations of 1.07, 10.7 and 107 mg test item/L the application stock solutions of were dosed with 277 µL/min (± 10%) and mixed with 27.5 mL/min (± 10%) of test water, which were carried via peristaltic pumps. The application stock solutions and the test water were pumped in mixing vessels (one per replicate). In these vessels, the application stock solutions and the test water were continuously mixed by a magnetic stirrer. The mixing vessels and the aquaria were connected by a tube. The dosage accuracy of the peristaltic pumps for carrying the test water was checked on day -7, -3, -1, 4, 7, 11, 14, 17 and 20. The dosage accuracy of the peristaltic pumps for carrying the test item stock solutions was checked on day -7, -3, -1, 4, 7, 11, 14, 17 and 20. The stock solutions were renewed weekly.
- Eluate: N/A
- Differential loading: N/A
- Controls: Yes (reconstituted water, without addition of the test item).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A, no solvent used.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A, no vehicle used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
- Other relevant information:

Test organisms

Aquatic vertebrate type:

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: African clawed frog
- Strain: N/A
- Source: European Xenopus Resource Centre (EXRC), University of Portsmouth, King Henry Building, King Henry I Street, Portsmouth, PO1 2DY, UK.
- Life stage: Egg (procured already fertilised; rearing of eggs was conducted at ibacon test facility).
- Age at study initiation (mean and range, SD): Tadpoles at Nieuwkoop and Faber (NF) stage 51 (Nieuwkoop and Faber, 1994), equivalent to 16 days of age post fertilisation.
- Length at study initiation (length definition, mean, range and SD): In addition to the developmental stage selection, the whole body length was measured at day 0 for a sub-sample of 20 NF stage 51 tadpoles, to determine a mean whole body length. Tadpoles exhibiting grossly visible malformations or injuries were excluded from the assay. Body length (mean): 21.69 mm (SD 0.81 mm).
- Weight at study initiation (mean and range, SD): Not reported
- Method of breeding: N/A
- Pre-exposure reproductive information. N/A

ACCLIMATION
- Acclimation period: Fertilised Xenopus laevis eggs were first kept under semi static conditions. After 4 days embryos were transferred to flow through conditions.
- Acclimation conditions (same as test or not): After reaching NF stage 45/46, tadpoles were kept under conditions similar to test conditions.
- Type and amount of food during acclimation: Not reported
- Feeding frequency during acclimation: Not reported
- Health during acclimation (any mortality observed): Tadpoles did not receive any treatment for disease during rearing, in the pre-exposure period, or during the exposure period.

FEEDING DURING TEST
- Food type: Guideline-recommended solution of Sera Micron®.
- Amount: Not reported
- Frequency: Three times daily.

METHOD FOR PREPARATION AND COLLECTION OF FERTILISED EGGS - N/A, fertilised eggs obtained from a commercial breeding facility.

POST-HATCH FEEDING
- Start date: Not reported
- Type/source of feed: Sera Micron®
- Amount given: Not reported
- Frequency of feeding: Three times daily.

Study design

Test type:

flow-through

Water media type:

other: Reconstituted Water (ISO Medium)

Remarks:

See 'Any other materials and methods incl. tables' for explanation of ISO Medium.

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

N/A

Post exposure observation period:

N/A

Test conditions

Hardness:

160.2 - 178.0 mg CaCO3/L

Test temperature:

Water temperature: 21.7 - 23.0 °C punctual measurement, measured weekly in all test vessel; 22.3 - 23.4 °C continuous recording in control vessel.

pH:

7.1 - 7.5

Dissolved oxygen:

102 - 105 % of the air saturation value at test start
52 - 105 % of the air saturation value.

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal test concentration: 1.07, 10.7 and 107 mg test item/L; and a control, corresponding to 1.0, 10 and 100 mg perfluorohexanoate/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: Glass, 12 L in size, containing at least 8 L test medium.
- Aeration: N/A
- Type of flow-through: Peristaltic
- Renewal rate of test solution (frequency/flow rate):
Stock solution: 272.8 ± 21.4 µL/min (mean over test duration ± SD)
Dilution water: 26.7 ± 1.07 [ml/min] (mean over test duration ± SD)
Exchange rate: 5-fold per day (sum of flow rates of stock solution and test water)

- No. of organisms per vessel: 20 per replicate
- No. of vessels per concentration (replicates): 4 replicates per treatment group
- No. of vessels per control (replicates): 4 replicates
- Vehicle control performed: N/A, no vehicle used.
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: N/A
- Stocking density: 0.5 tadpoles / L test medium / day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium)
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: 43.6 to 65.4 mg/L HCO3-
- Ca/mg ratio: 4 : 1 (based on molarity)
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: The dissolved oxygen concentrations, pH and the water temperature were determined once a week in the test media of all test concentrations and the control. Additionally temperature was recorded continuously in the control vessels. The water hardness and the alkalinity were determined in the control and one replicate of the highest test concentration weekly.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 12 h light – 12 h dark (30 min dawn/dusk period was provided).
- Light intensity: 700 to 1040 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recorded effects were developmental stage (day 7 and 21); body weight (day 7 and 21); snout to vent length (SVL; day 7 and 21); hind limb length (HLL; day 7 and 21) thyroid histology (day 21) and mortality (daily) and abnormal behaviour (daily).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: N/A

RANGE FINDING STUDY
- Test concentrations: Nominal: 0.1, 1.0, 10 and 100 mg test item/L.
- Results used to determine the conditions for the definitive study:
For the determination of the test concentrations, a non-GLP range-finding test in a 96-hours flow-through design was performed to set the maximum tolerated concentration (MTC). Analytical dose verification confirmed correct dosage of these concentrations and the test item remained within the nominal range of 80 - 120% during the test.
The MTC is defined as the highest test concentration of the chemical which results in less than 10% mortality. Since no mortalities were observed in the range-finding test, no MTC was calculable. Therefore, the highest test concentration was set to 100 mg test item/L and a spacing factor of 10 was chosen.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

other: No effect

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: No statistically significant difference compared to control

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

other: No effect

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: No statistically significant difference compared to control

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

other: No effect

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks on result:

other: No statistically significant difference compared to control

Effect concentrations 4

Key result

Duration:

21 d

Dose descriptor:

other: No effect

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Remarks on result:

other: No statistically significant difference compared to control

Effect concentrations 5

Key result

Duration:

21 d

Dose descriptor:

other: No effect

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

thyroid gland histology

Remarks on result:

other: No histological findings

Effect concentrations 6

Key result

Duration:

21 d

Dose descriptor:

other: Effect observed

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

hind limb length

Remarks:

Normalised by snout to vent length

Remarks on result:

other: Statistically significant difference compared to control

Effect concentrations 7

Key result

Duration:

21 d

Dose descriptor:

other: Effect observed

Effect conc.:

107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

developmental stage

Remarks on result:

other: Statistically significant difference compared to control

Details on results:

-Overall mortality/survival: In the treatment groups, the mortality of tadpoles in each of the replicates was 0 % at the end of the exposure period. Thus, the validity criterion for mortality (≤ 10 %) in any of the treatment groups at the end of the study was met.
- Type of and number with behavioural abnormalities: Except for one animal at the test item concentration 1.07 mg/L, no abnormal behaviour (such as e.g. hyperventilation or uncoordinated swimming) was observed in any of the test item concentrations and controls during the study.
-Animal constitution: Several animals displayed scoliosis. Scoliosis mainly was observed in the tail region, slightly behind the pelvis. Since scoliosis was also observed in the control, an effect due test item can be clearly excluded. The scoliosis did not affect the tadpoles since the behaviour was comparable to those animals without tail flex.
-General toxicity: No test item related mortality or behavioral anomalies was observed in any treatment group. Also no delay in development or reductions in growth were observed. Therefore no indication to an effect was detectable and it can be concluded that an exposure to the test item caused no general toxicity to Xenopus laevis tadpoles up to 107 mg test item/L.
- Mortality in the controls: In the control, the mortality of tadpoles was 3.75%. Thus, the validity criterion for mortality (≤ 10 %) in the control at the end of the study was met.

- Developmental stage of Xenopus laevis tadpoles:
Median NF and Mean Median Stage on Day 7:
Control: NF 53.0 Mean of Replicate Median: NF 53.00
1.07 mg/L: NF 53.0 Mean of Replicate Median: NF 53.00
10.7 mg/L: NF 53.0 Mean of Replicate Median: NF 53.00
107/L: NF 53.0 Mean of Replicate Median: NF 53.00
No significant difference was found between the test item concentrations and the control.
Median NF and Mean Median Stage on Day 21:
Control: NF 58.0 Mean of Replicate Median: NF 57.50
1.07 mg/L: NF 58.0 Mean of Replicate Median: NF 58.00
10.7 mg/L: NF 58.0 Mean of Replicate Median: NF 58.25
107 mg/L: NF 58.0 Mean of Replicate Median: NF 58.25
A significant difference was found between the test item concentration of nominal 107 mg test item/L and the control (Multi-Quantal Jonckheere Terpstra Test procedure).
No severe alterations or asynchronous development was observed. No tadpole indicates a test item-related asynchronous development, since developmental stage could be identified by developmental landmarks with one exception in the control group. There was no indication to a delayed development in all test item groups.

- Hind limb length of Xenopus laevis tadpoles:
Mean Hind Limb Length (HLL) normalised with (SVL) on Day 7:
Control: 0.132 ± 0.009 mm
1.07 mg/L: 0.136 ± 0.005 mm
10.7 mg/L: 0.137 ± 0.001 mm
107 mg/L: NF 0.142 ± 0.002 mm
A statistical significant difference was found between the test item concentration of 107 mg test item/L and the control (Williams t-test, α = 0.05, two sided).
Mean Hind Limb Length (HLL) normalised with (SVL) on Day 21:
Control: 0.558 ± 0.037 mm
1.07 mg/L: 0.545 ± 0.026 mm
10.7 mg/L: 0.637 ± 0.077 mm
107 mg/L: 0.692 ± 0.062 mm
A statistical significant difference was found between the test item concentration of 107 mg test item/L and the control (Williams t-test, α = 0.05, two sided).

- Snout to vent length of Xenopus laevis tadpoles:
Mean Snout to Vent Length (SVL) on Day 7:
Control: 10.97 ± 0.43 mm
1.07 mg/L: 11.35 ± 0.39 mm
10.7 mg/L: 12.25 ± 0.78 mm
107 mg/L: 13.22 ± 0.79 mm
A statistical significant difference was found between the test item concentration of 10.7 and 107 mg test item/L and the control (Williams t-test, α = 0.05, two sided).
Mean Snout to Vent Length (SVL) on Day 21: After stage NF 60, tadpoles show a reduction in size and weight due to tissue resorption and reduction of absolute water content. Therefore, SVL data from organisms >NF 60 were excluded from data analysis.
Control: 24.22 ± 0.44 mm
1.07 mg/L: 24.94 ± 0.72 mm
10.7 mg/L: 24.41 ± 0.27 mm
107 mg/L: 24.02 ± 1.10 mm
No statistical significant difference was found between the test item concentrations and the control (Welsh t-test, α = 0.05, two sided).

- Wet body weight of Xenopus laevis tadpoles:
Wet Weight on Day 7: The mean wet weight of the tadpoles was:
Control: 132.1 ± 21.9 mg.
1.07 mg/L: 140.7 ± 20.7 mg
10.7 mg/L: 184.2 ± 32.5 mg
107 mg/L: 230.6 ± 41.8 mg.
A statistically significant difference was found between the test item concentrations of 10.7 and 107 mg test item/L and the control (Williams t-test, α = 0.05, two sided).
Wet Weight on Day 21: After stage NF 60, tadpoles show a reduction in size and weight due to tissue resorption and reduction of absolute water content. Therefore, wet weight data from organisms >NF 60 were excluded from data analysis.
The mean wet weight of tadpoles was:
Control: 1378.8 ± 104.2 mg
1.07 mg/L: 1495.7 ± 103.8 mg
10.7 mg/L: 1436.2 ± 102.8 mg
107 mg/L: 1418.5 ± 192.2 mg.
No significant difference was found between the test item concentrations and the controls (Dunnett’s t-test, α = 0.05, two sided).

Day 7 and day 21 snout vent length, hind limb length (normalised by snout-vent length) and developmental stage were evaluated to determine if advanced development occurred as a result of exposure to the test item.
On day 7 no advanced development regarding developmental stage was observed. However the tadpoles in the treatment groups of 10.7 and 107 mg test item /L showed a statistically significant increase regarding body growth (body mass and SVL). At 107 mg test item /L a statistically significant increase of normalized hind limb length was detected.
On day 21 the developmental stage was statistically significantly increased at 107 mg test item/L. Additionally the normalised hind limb length was statistically significantly increased at that treatment group.

- Thyroid gland histology of Xenopus laevis tadpoles:
There were randomly distributed few cases of thyroid gland and follicular epithelial atrophy including in controls.
No gross lesions were observed at necropsy. Under the conditions of this study, there were no changes in the thyroid glands that could be attributed to treatment with the test item. There were some minor findings of thyroid and follicular cell atrophy. However, all cases were distributed randomly throughout the groups including in controls.
No other findings were noted.

Analytical results: see 'any other information on results'.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

ToxRat Professional, v3.3.0, ToxRat Solutions GmbH was used for statistical anlysis.
Body weight, SVL and HLL all data were checked for normal distribution and variance homogeneity. Shapiro-Wilk’s Test (α = 0.01) was used to check for normal distribution. The check for variance homogeneity of the whole data set (control and treatment groups) was conducted by means of Levene’s Test (α = 0.01). Afterwards data were assessed by Trend Analysis by Contrasts to check for a significant linear or quadratic trend (α = 0.05).
In the case that the variance homogeneity check failed the Welch-t-test for non-homogenous variances with Bonferroni-Holm-adjustment was used for NOEC determination.
If a monotonic dose-response was determined, a Williams Multiple Sequential t-test Procedure was applied (α = 0.05) for comparison of treatments with control. Dunnett`s Multiple t-test Procedure was used in cases when a monotonic dose-response was not determined.
Statistical Evaluation for hind limb length (HLL) was performed with normalised HHL (ratio of HHL to SVL).
The statistical evaluation of the developmental stages on day 7 and day 21 was conducted by means of a Multi-Quantal Jonckheere Terpstra Test according to Green et al. (2018). In brief, the 20th to 80th percentiles of the developmental stages were calculated for each replicate, followed by a Jonckheere Terpstra Step Down Test (1). The p-value for the highest test item concentration was then recorded and the median of all p-values (from 20th to 80th percentile) was calculated. If the median of all p-values > 0.05, the NOEC was determined to be greater than the highest test item concentration (2). If the p-values < 0.05, the highest treatment group has been removed and the p-values were calculated for the reduced dataset. The steps (1) and (2) were repeated until the median p-values were greater than 0.05. 

Reference: Green et al., 2018, Statistical Analysis of Ecotoxicity Studies. 10.1002/9781119488798

Any other information on results incl. tables

Table 2. Summary of observed endpoints

Endpoints		Concentration (mg test item/L)
		1.07	10.7	107
Day 7	Developmental Stage	-	-	-
	Wet Weight	-	↑	↑
	Snout-Vent Length (SVL)	-	↑	↑
	Hind Limb Length (Normalised by SVL)	-	-	↑
Day 21	Survival	-	-	-
	Behaviour	-*	-	-
	Developmental Stage	-	-	↑
	Wet Weight	-	-	-
	Snout-Vent Length (SVL)	-	-	-
	Hind Limb Length (Normalised by SVL)	-	-	↑
	Thyroid Gland Histopathology	NF	NF	NF
- * only one individual of 20 at one observation point was found mainly on the bottom of the tank - Endpoint not impacted compared to the control ↑ Endpoint significantly increased NF – No histological findings

All reported biological results refer to nominal concentrations since the average test item concentration recoveries were within 100 ± 20% during the test.

 

Table 3. Summary of Analytical Results

Sample Description	% of	RSD
[mg test item/L]	nominal^1	[%]	n
Control	n.a.	n.a.	16
1.07	95	7	16
10.7	98	5	16
107	105	12	16

¹ Mean value of all measured samples per treatment group for whole exposure duration of 21 days
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

 

Table 4. Overview of all observed endpoints after 21 days of exposure to the test item

		Test Concentrations [mg/L]
		Control	1.07	10.7	107
Mean overall Mortality [%]*	mean	3.75	0.00	0.00	0.00
	SD	4.8	0.0	0.0	0.0
Number of tadpoles showing abnormal behaviour	#	0	1	0	0
	Type	-	MB	-	-
Mean tadpole weight at day 7 [mg]	mean	132	141	184	231
	SD	21.9	20.7	32.5	41.8
Mean tadpole weight at day 21 [mg]	mean	1379	1496	1436	1418
	SD	104.2	103.8	102.8	192.2
Median NF Stage on Day 7	median	53.0	53.0	53.0	53.0
Median NF Stage on Day 21	median	58.0	58.0	58.0	58.0
Mean SVL at Day 7 [mm]	mean	10.97	11.35	12.25	13.22
	SD	0.43	0.39	0.78	0.79
Mean SVL at Day 21 [mm]	mean	24.22	24.94	24.41	24.02
	SD	0.44	0.72	0.27	1.10
Mean HLL at Day 7 [mm]	mean	1.46	1.54	1.67	1.88
	SD	0.15	0.08	0.11	0.10
Mean HLL at Day 21 [mm]	mean	13.48	13.58	15.40	16.48
	SD	0.92	0.70	1.99	1.77
Mean HLL at Day 7 normalized with SVL [mm]	mean	0.132	0.136	0.137	0.142
	SD	0.01	0.01	0.00	0.00
Mean HLL at Day 21 normalized with SVL [mm]	mean	0.558	0.545	0.637	0.692
	SD	0.04	0.03	0.08	0.06
* mortality in the control was partly caused by accident during cleaning the tanks
Underlined values were statistically significant different compared to the control SVL: Snout to vent length, HLL: Hind limb length MB: Mainly on the bottom of the tank

 

Table 5. Tadpole developmental stages at test end (day 21)

Control                               1.07 mg/L                           10.7 mg/L                          107 mg/L
# of tadpoles	R1	R2	R3	R4	R1	R2	R3	R4	R1	R2	R3	R4	R1	R2	R3	R4
6	60	56	58	60	56	57	58	56	58	57	56	58	58	58	58	58
7	58	58	56	59	58	58	57	58	58	59	57	60	58	58	58	58
8	60	58	58	56	57	58	58	58	56	59	59	58	58	58	59	59
9	57	58	56	59	56	59	58	58	58	58	56	59	60	58	59	58
10	59	60	56	58	58	59	59	58	58	58	57	59	58	58	60	60
11	58	58	56	59	58	58	59	57	58	58	58	58	58	59	58	58
12	58	58	56	58	58	58	58	59	58	58	58	58	58	60	59	59
13	56	58	56	57	58	59	57	59	58	60	56	58	58	59	60	56
14	56	58	56	58	60	59	60	58	59	58	57	59	59	60	58	58
15	58	58	56	59	58	58	56	57	60	58	59	60	58	60	59	58
16	57	58	56	59	57	58	58	58	60	59	57	59	59	58	59	59
17	58	#	58	57	58	58	57	59	60	58	58	58	59	59	57	58
18	56	57	57	57	59	58	57	57	59	59	59	59	61	58	57	58
19	-	56	60	58	58	56	57	58	60	58	58	59	60	58	60	59
20	-	58	57	-	58	58	57	56	58	59	58	58	61	58	61	58
Mean	57.769	57.786	56.800	58.143	57.800	58.067	57.733	57.733	58.533	58.400	57.533	58.667	58.867	58.600	58.800	58.267
SD	1.363	0.975	1.207	1.099	1.014	0.799	1.033	0.961	1.125	0.737	1.060	0.724	1.125	0.828	1.146	0.884
Median	58.0	58.0	56.0	58.0	58.0	58.0	58.0	58.0	58.0	58.0	58.0	59.0	58.0	58.0	59.0	58.0
Mean*	57.62				57.83				58.28				58.63
SD*	0.576				0.159				0.512				0.269
Median	58.0				58.0				58.0				58.0
Mean of Median**	57.50				58.00				58.25				58.25
*calculated from the mean values
** calculated on replicate median values
# could not be determined, because the development was asynchronus
underlined: the development of the forelimbs was between 57 and 58; the development of hindlimbs indicate NF Stage 57
bold: individuals were used for histopathological evaluation
italics: individuals > stage NF 60 were excluded for the analysis of body weight  and SVL

 

Table 6. Tadpole hind limb length (normalized by snout-vent length) at test end (day 21)

Control                                  1.07 mg/L                           10.7 mg/L                          107  mg/L
# of tadpoles	R1	R2	R3	R4	R1	R2	R3	R4	R1	R2	R3	R4	R1	R2	R3	R4
1	0.765	0.472	0.544	0.760	0.512	0.438	0.569	0.375	0.531	0.426	0.471	0.569	0.605	0.688	0.648	0.593
2	0.539	0.583	0.441	0.679	0.553	0.419	0.559	0.557	0.489	0.793	0.369	0.630	0.662	0.624	0.590	0.764
3	0.870	0.520	0.576	0.521	0.521	0.499	0.595	0.444	0.440	0.911	0.340	0.756	0.754	0.548	0.793	0.774
4	0.475	0.524	0.398	0.814	0.513	0.598	0.485	0.526	0.612	0.751	0.574	0.771	0.886	0.636	0.706	0.539
5	0.693	0.912	0.393	0.656	0.583	0.452	0.634	0.588	0.509	0.661	0.650	0.834	0.926	0.550	0.738	0.770
6	0.544	0.578	0.488	0.695	0.522	0.624	0.716	0.415	0.711	0.518	0.480	0.503	0.782	0.718	0.536	-
7	0.627	0.559	0.489	0.461	0.563	0.708	0.549	0.780	0.469	0.740	0.567	0.509	0.530	0.960	0.755	0.769
8	0.376	0.517	0.389	0.399	0.558	0.726	0.362	0.686	0.475	0.951	0.335	0.587	0.609	0.729	0.826	0.496
9	0.380	0.557	0.398	0.530	0.789	0.680	0.608	0.497	0.700	0.558	0.475	0.470	0.737	0.873	0.622	0.509
10	0.600	0.546	0.437	0.771	0.550	0.615	0.562	0.358	0.714	0.695	0.700	0.910	0.653	1.010	0.774	0.460
11	0.609	0.515	0.573	0.714	0.486	0.571	0.623	0.586	0.676	0.777	0.404	0.725	0.743	0.606	0.644	0.702
12	0.644	0.588	0.515	0.365	0.591	0.640	0.536	0.578	0.931	0.501	0.549	0.605	0.717	0.774	0.508	0.475
13	0.450	0.534	0.628	0.305	0.677	0.433	0.405	0.522	0.754	0.694	0.798	0.688	1.059	0.706	0.545	0.650
14	-	0.264	0.851	0.598	0.619	0.301	0.420	0.572	0.984	0.538	0.625	0.898	0.923	0.700	0.691	0.641
15	-	0.562	0.519	-	0.649	0.506	0.414	0.293	0.735	1.000	0.549	0.652	0.924	0.553	0.590	0.597
Mean	0.582	0.549	0.509	0.591	0.579	0.547	0.536	0.518	0.649	0.701	0.526	0.674	0.767	0.712	0.664	0.624
SD	0.245	0.127	0.121	0.219	0.079	0.123	0.100	0.128	0.167	0.172	0.134	0.140	0.149	0.142	0.100	0.197
Mean*	0.558				0.545				0.637				0.692
SD*	0.037				0.026				0.077				0.062

*calculated from mean values

Table 7. Summary of thyroid gland histology reults

			Thyroid gland hyper-trophy	Thyroid gland atrophy	Follicular cell hyper-trophy	Follicular cell hyper-plasia	Follicular celll atrophy	Follicular lumen area increase	Follicular lumen area decrease
Control	Replicate	1	0	0	0	0	0	0	0
		2	0	1/2.0	0	0	1/1.0	0	0
		3	0	0	0	0	0	0	0
		4	0	0	0	0	0	0	0
0.1 mg/L	Replicate	1	0	0	0	0	0	0	0
		2	0	1/2.0	0	0	1/1.0	0	0
		3	0	0	0	0	0	0	0
		4	0	0	0	0	0	0	0
10 mg/L	Replicate	1	0	0	0	0	0	0	0
		2	0	0	0	0	0	0	0
		3	0	0	0	0	0	0	0
		4	0	0	0	0	0	0	0
100 mg/L	Replicate	1	0	0	0	0	0	0	0
		2	0	0	0	0	0	0	0
		3	0	0	0	0	0	0	0
		4	0	1/1.0	0	0	1/1.0	0	0

Values signify the  number of animals affected / mean grade (severity) of affected animals

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, there was a statistically significant increase compared to the control in normalised hind limb length and developmental stage (advanced development observed) at the top concentration of 107 mg test item/L at day 21.

Executive summary:

A test in accordance with OECD 231 (2009) in vivo Amphibian Metamorphosis Assay has been performed in order to assess the thyroid activity potential of the test item.

Fertilised Xenopus laevis (African clawed frog) eggs were obtained from a commercial breeding facility. After hatching, tadpoles were reared until they reached NF (Nieuwkoop & Faber, 1994) developmental stage 51. The organisms were then exposed in a flow-through test to aqueous test media containing the test item under defined conditions, for 21 days. There were 4 treatment groups (3 concentrations of test item and 1 negative control), with 4 replicates each containing 20 organisms. The test concentrations used were 1.07, 10.7 and 107 mg test item/L (spacing factor 10), and a control, corresponding to 1.0, 10 and 100 mg perfluorohexanoate/L.

Analytical verification of the test item concentration in test medium was performed on days 0, 4, 7, 18 and 21 during the course of the study. The quantification of perfluorohexanoate of the test item sodium perfluorohexanoate was performed using liquid chromatography with MS/MS detection. Mean recovery values of 95, 98 and 105 % were determined for the nominal test concentrations of 1.07, 10.7 and 107 mg test item/L, respectively. The overall mean recovery value was 99%.

The endpoints assessed in the study were developmental stage (day 7 and 21); body weight (day 7 and 21); snout to vent length (SVL; day 7 and 21); hind limb length (day 7 and 21) thyroid histology (day 21) as indicators of thyroidal activity of the test item. Furthermore, survival and behaviour were monitored daily.

At 1.07 mg test item/L, no statistically significant difference compared to the control was observed for any endpoint (developmental stage, body weight SVL and normalised HHL). At 10.7 mg test item/L, wet weight and SVL were statistically significantly increased compared to the control on day 7, whereas on day 21 no statistically significant difference compared to the control was observed for any endpoint. Body wet weight and SVL were statistically significantly increased on day 7, but not on day 21. A statistically significant increase of normalised HLL at 107 mg test item/L on day 7 and 21 were observed. At 107 mg test item/L, no statistically significant difference regarding developmental stage was detected on day 7. However, statistically significant advanced development on day 21 was observed. 

Under the conditions of the study, there were no changes in the thyroid glands that could be attributed to treatment with the test item. There were some minor findings of thyroid and follicular cell atrophy during the histopathological evaluation. However, all cases were distributed randomly throughout the groups including in controls. No test item related mortality or behavioural anomalies was observed in any treatment group. No delay in development or reductions in growth were observed. It was therefore concluded that there was no general toxicity triggered by the test item up to 107 mg test item/L.

All the performance criteria as outlined in the OECD 231 Test Guideline were met and therefore the test was concluded to be valid. Furthermore, the validity criterion for a test determined to be positive for thyroid activity (that mortality of no more than two tadpoles/replicate in the control group can occur) was also met in this test. 

Under the conditions of this study, there was a statistically significant increase compared to the control in normalised hind limb length and developmental stage (advanced development observed) at the top concentration of 107 mg test item/L at day 21.