Isopentyl p-methoxycinnamate

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:

from 2019-07-10 to 2020-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 4-methoxycinnamate

EC Number:

226-775-7

EC Name:

2-ethylhexyl 4-methoxycinnamate

Cas Number:

83834-59-7

Molecular formula:

C18H26O3

IUPAC Name:

2-​Propenoic acid, 3-​(4-​methoxyphenyl)​-​, 2-​ethylhexyl ester, (2E)​-

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test solutions (20 mL) were collected in duplicate, centrifuged (2795 x g or 5000 rpm for 30 min) and subsequently diluted with 20 mL acetonitrile (ACN). Samples were stored refrigerated (ca. 4 °C) in 40 mL glass vials. Samples were analysed for the concentration of the test item using liquid chromatography with a mass spectrometer detector (LC-MS/MS) in a multiple reaction monitoring mode (MRM). The duplicate sample was retained and stored at 4 °C (1 – 9 °C) to be used as necessary in the event of the need for re-analysis.

Test solutions

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

- Preparation of saturator columns:
Eight saturator columns were prepared, two for control groups and six for preparation of test item stock solutions. In general, columns were prepared by 125 g/arm pyrolytically cleaned glass wool into each arm of u-shaped glass columns (diameter = 1.54 cm, length = 152.4 cm). To further clean the glass wool, the glass wool was subsequently rinsed with acetone and the acetone purged from the system prior to test substance loading. For the columns used to prepare test substance stock solution, approximately 10 g of the test item was dissolved in 30 mL acetone and distributed equally to both arms of the column. A vacuum pump was used to pull the solution through the glass wool to provide an even coat. The acetone was then evacuated from the columns leaving the test item. Once the saturator columns were dried and the acetone evacuated, the columns were plumbed, and dechlorinated tap water pumped through them at a rate of 4 mL/min for 10 days to rinse the columns and remove any glass particles from the glass wool. When the rinsing phase was complete, the columns were configured in a recirculating loop with a flow rate of 25 mL/min to prepare the master stock.

- Determination of the practical water solubility:
Prior to in-life testing, water solubility was determined at test conditions.
When the columns ran for at least 4 weeks, samples were collected to determine saturation and practical solubility. Samples were collected, spaced by at least one day, from the stock solution tank in duplicate with one sample preserved and the duplicate sample centrifuged and preserved. Samples were collected and analysed until saturation was determined. After collection, samples were stored at 4 °C (1 - 9 °C). Once samples were collected and preserved, samples were analysed. Three primary factors were used to determine if saturation had been achieved: 1) in-life test conditions were maintained throughout assessment, 2) differences between three consecutive samples was ≤20 %, and 3) differences between centrifuged and uncentrifuged samples was ≤20 %. When each of the three criteria were met, the mean concentration of the three consecutive samples was considered saturation or the practical soluble concentration, and the in-life study commenced.

Test organisms

Aquatic vertebrate type:

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
- Common name: African Clawed Frog
- Source: Male and female frogs were originally purchased from Xenopus I, Dexter, MI, USA. Larvae used for this study were obtained from an in-house culture.
- Life stage: NF 51 larvae
- Method of breeding: At least 3 pair of adult frogs were injected with human chorionic gonadotropin (hCG) to induce reproduction initially. A second breeding of three pair following the initial breeding was performed and used as a potential backup for the study. Breeding performed in line with OECD 231. Fertilized egg collection was performed as described in ASTM E1439-12 (2019) and OECD 231. All tadpoles used as test organisms were derived from the same clutch (spawn).

FEEDING OF ADULTS
- Type and amount of food: Fin fish Starter 50-15 slow sinking food
- Feeding frequency: ad libitum daily

HANDLING
- Housing: In-house culture in which groups of males and females are housed separately in 50 L tubs plumbed with individual flow through dechlorinated tap water.

FEEDING DURING TEST
- Food type: Sera Micron throughout the pre-exposure period (after NF stage 45/46) and during the entire test period of 21 days
- Amount: 600 mg Sera Micron® per tank per day (300 mg twice daily) for the first 4 days of exposure, then the amount was increased exponentially
- Frequency: twice daily on week days, once per day at twice the weekday volume on weekends

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
In order to guarantee at least 600 stage 51 acceptable larvae at test setup, clutch sizes of ca. 1500 embryos are recommended. In addition, at least three clutches were collected to evaluate the quality of the spawns and determine which produce the highest quality larvae for the initiation of the study. Criteria for selection was based on the proportion of normal-appearing larvae at NF stage 51 within the specific developmental window. Embryos were maintained at 22 ± 1°C for 4 days to allow for hatching and development to NF stage 45/46, at which time they were divided into groups of approximately 200 and maintained in tanks containing 50 L of dilution water with a population density of 4 larvae/L at a constant flow rate (50 mL/minute) and water temperature (22 ± 1 °C) until they reached developmental NF stage 51.
- Selection of test animals: When a sufficient number of the normal-appearing pre-exposure population reached developmental stage 51 (14 to 17 d post-fertilization), larvae were transferred to a pooling tank containing dilution water. Larvae used in the present study required 14 d to reach NF stage 51. All larvae used in the in-life study were from the same clutch of offspring. Individual larvae were removed from the pooling tank by scooping with a small strainer. Animals were carefully handled during this transfer in order to minimize handling stress and to avoid any injury. The developmental stage of the animals was then determined by using a binocular dissection microscope. The primary developmental landmark for selecting stage 51 organisms is hind limb morphology. Animals that met the stage criteria were transferred to a holding tank containing 100 % dilution water.

Study design

Test type:

flow-through

Water media type:

freshwater

Remarks:

dechlorinated (charcoal-filtered) tap water

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

76 - 112 mg/L as CaCO3

Test temperature:

22.5 - 23.0 °C

pH:

6.8 - 7.6

Dissolved oxygen:

6.0 - 8.2 mg/L

Conductivity:

501 - 508 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 5.50, 16.5 and 50 µg/L
Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tanks in a flow-through system
- Material, size, headspace, fill volume: The system had water-contact components of glass (aquaria), stainless steel (diluter housing and water bath), and Teflon (tubing responsible for test material delivery). Exposure tanks were glass aquaria (with approximate measurements of 22.5 x 14.0 x 16.5 cm deep) equipped with standpipes that result in an actual tank volume of 4.0 L and minimum water depth of 10 to 15 cm.
- Aeration: none
- Type of flow-through: flow-through diluter system - Benoit Mini-Diluter
- Renewal rate of test solution (frequency/flow rate): 25 mL/min which provided a complete volume replacement every 2.7 h
- No. of organisms per vessel: 20 larvae
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Stocking density: 5 larvae/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (charcoal-filtered) tap water prepared at the laboratory using 4-filter system - a multimedia filter to remove suspended solids in the feed water; a 10” pre-treatment filter (5 µm) to remove any additional solids; a 0.1 m³ activated virgin carbon treatment filter to remove chlorine, ammonia, and higher molecular weight organics; and a 5 µm polishing filter to remove any carbon particles from the carbon treatment phase.
- Total organic carbon: 0 - 1.3 mg/L
- Alkalinity: 36 - 76 mg/L as CaCO3
- Iodide: 7.6 - 7.9 µg/L
- Ammonia: <0.06 µg/L
- Chlorine: <0.05 µg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: twice a month

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 12 h light : 12 h dark
- Light intensity: 612 - 1151 lux

EFFECT PARAMETERS MEASURED
- Mortality - daily
- Snout-vent length (SVL), Hind limb length (HLL), developmental stage, wet body weight - on day 7 and 21 (the end of the test)
- Thyroid histopathology - on day 21 (the end of the test)

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

hind limb length

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

snout-vent length

Effect concentrations 4

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Effect concentrations 5

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

thyroid gland histology

Effect concentrations 6

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

developmental stage

Effect concentrations 7

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 44.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Details on results:

- Overall mortality/survival: No mortality was observed.
- Type of and number with morphological abnormalities: none
- Type of and number with behavioural abnormalities: none
- Type and number of developmental / reproductive effects: none
- Type and magnitude of hormonal changes: none
- Other biological observations: none observed
- Effect concentrations exceeding solubility of substance in test medium: no
- Mortality in the controls: none observed
- Incidents in the course of the test which might have influenced the results: none observed
- Developmental stage of Xenopus laevis tadpoles : median stage in the control and the treatments at day 7 was NF 54 and at day 21 was NF 60
- Hind limb length of Xenopus laevis tadpoles: no significant difference with the control
- Snout to vent length of Xenopus laevis tadpoles: no significant difference with the control
- Wet body weight of Xenopus laevis tadpoles: no significant difference with the control
- Thyroid gland histology of Xenopus laevis tadpoles: no significant difference with the control

Any other information on results incl. tables

Determination of Practical Water Solubility

The difference between each of the three consecutive samples collected from the saturators relative to the mean ranged from 2.1 - 7.1 %. The difference between centrifuged and uncentrifuged samples for the three consecutive samples ranged from 2.8 - 6.3 %. The mean concentration based on the three consecutive centrifuged samples was 48.1 µg/L test item. Based on these results, the target concentration was set at 50.0 µg/L test item.

 

Range-Finding Test

Based on the non-GLP range-finding study, the maximum tolerable concentration (MTC) was determined to be 50 µg/L. The test concentration series was 1x, 0.33x, 0.11x, and 0.04x, where x was the MTC value. Therefore, the test concentrations were 50.0, 16.5, and 5.50 µg/L.

 

Water Quality Measurements and Test System Performance

Iodine (I^-) levels in the dilution water were measured on Study Day (SD) 0 and 21 (in-life conclusion) of the current study and contained 7.9 (± 0.17) and 7.6 (± 0.19) µg/L I-, respectively, which fell within the acceptable range. All physicochemical water quality parameters in study were within acceptable ranges.

 

Confirmation of Test Concentrations

Target test item concentrations selected for the Amphibian Metamorphosis Assay study were 0.0 (control), 5.50, 16.5 and 50 µg/L. Dilution water control samples collected during the study contained< LOD (LOD = 0.912 μg/L). Since time interval between sample collection events was consistent throughout the study, the mean measured concentration represented a reasonable estimation of exposure concentration. The mean measured concentration represented the average of each data point from SD 0, 7, 14, and 21 for each replicate (A-D) of the control and each treatment per facility SOP. IQRs (Interquartile range) determined for the 0.0 (control), 5.50, 16.5, 50.0 µg/L test item treatments were 0.46 - 0.46, 4.05 - 8.25, 9.89 - 25.6, and 28.36 - 62.10 µg/L test item, respectively. Based on IQR analysis of the control and each treatment group, one outlier was identified, original sample 5.50 µg/L replicate A (11.4 µg/L test item). In this case, results of the duplicate sample of 5.50 µg/L replicate A (5.62 µg/L test item) was used in the analysis. The corresponding mean measured concentrations in the definitive study were 5.97, 18.1, and 44.2 μg/L test item, respectively. The coefficient of variation (CV) [(Standard deviation/mean)100] was based on the standard deviation of the four replicate means (n = 4) for the control and the four replicate means (n = 4) for each treatment per facility SOP. The CVs of the intra-replicate means of the measured test concentrations for the 5.50, 16.5, and 50.0 µg/L test item treatments were 3.0 %, 4.5 % and 5.9 %, respectively; which were acceptable based on the criteria established in the test guidance and the study plan.

 

Mortality

No mortality was observed during the study. Since all larvae survived during the study, no statistical analyses were performed.

 

Development

Developmental Stage

Six (10 %), 6 (10 %), 8 (13.3 %), and 9 (15 %) specimens in the control, 5.50, 16.5, and 50.0 μg/L test item treatments were identified as late stage (>NF stage 60), respectively. Since the occurrence per treatment was <20 %, these organisms were excluded from analysis of growth endpoints (SVL and body weight). The median developmental stage on exposure SD 7 was 54 for the control and each treatment, as well as all replicates of all treatments. At test conclusion (day 21), the median developmental stage for the control was 60 in each replicate. The median developmental stage for the 0.0, 5.50, 16.5 and 50.0 μg/L test item treatments were each 60. The IQR and the number of different stages occurring between the 10th and 90th percentiles in the control on SD 7 were 0 and 1, respectively. At the conclusion of the study, the IQR and the number of different stages occurring between the 10th and 90th percentiles in the control on SD 7 were 0 and 1, respectively. The IQR for each of the OMC treatments at SD 7 and 21 was 0. The median developmental stages attained at SD 7 and 21 were not significantly different from the control (Mann-Whitney U, p = 1.000). No signs of asynchronous development were noted in the control or the test item treatments during the conduct of the study.

 

Hind Limb Development

SVL results were used to normalize hind limb length. On SD 7, the mean HLLs were 1.5 mm in the control and 1.6 mm in each of the test item treatments. On SD 21, the mean HLLs were 12.9 mm in the control and 13.7, 14.2, and 13.5 mm in the 5.50, 16.5, and 50.0 μg/L treatments. On SD 7, the mean normalized HLLs were each 0.1 in the control and each test item treatment. At test termination (SD 21), the mean normalized HLLs were 0.6 in the control and 5.50 μg/L treatment; and 0.7 in the 16.5 and 50.0 μg/L treatments. HLL and normalized HLLs in the test item treatments were not significantly different from the control at SD 7 (ANOVA, p = 0.937 and 0.909, respectively). HLL and normalized HLLs in the test item treatments were not significantly different from the control at SD 21 (ANOVA, p = 0.839 and 0.726, respectively).

Growth

Snout-Vent Length (SVL)

SVL, one of two measures of larval growth, ranged from 13.2 mm in the control and 50.0 μg/L test item treatments to 13.7 mm in the 5.50 μg/L test item treatment on exposure day 7. At exposure day 21, SVL ranged from 21.2 mm in the control to 22.7 mm in the 5.50 μg/L test item treatment. SVLs measured in the test item treatments on SD 7 and 21 were not significantly different from the control (ANOVA, p = 0.761 and 0.251, respectively).

 

Body Weight

Body weight, the second measure of larval growth, ranged from 0.353 g in the control to 0.380 g in the 5.50 μg/L test item treatment on SD 7. Body weight ranged from 1.482 g in the 16.5 μg/L treatment to 1.831 g in the 5.50 μg/L treatment on SD 21. Body weights measured in the OMC treatments on SD 7 and 21 were not significantly different from the control (ANOVA, p = 0.099 and 0.089, respectively).

 

Thyroid Gland Histopathology

The prevalence of mild follicular cell hypertrophy and hyperplasia were consistent between control and test item-exposed X. laevis and treatment-related effects were not noted. There were no treatment-related histopathologic findings in the thyroids of tadpoles exposed to the test item. The lack of treatment-related effects in this study is consistent with the absence of group-wise differences in median NF stage scores. The occurrence of mild follicular cell hypertrophy and hyperplasia in larvae nearing metamorphic climax is not atypical and the rationale for this response is that anuran metamorphosis is considered to be a thyroid-dependent process; therefore, basal levels of follicular cell hypertrophy and hyperplasia are anticipated findings in control frogs at the developmental stage at which they were sacrificed in the study (i.e., median Stage 60). Larvae preparing for metamorphic climax (NF stage 61) require a large surge of thyroid hormone to initiate the final cascade of metamorphic processes, including resorption of the tail. This process significantly taxes the thyroid during the assay, which results in follicular hypertrophy and in some cases hyperplasia. This stress diminishes at stage 62 as metamorphic climax proceeds.

 

Clinical Signs of Toxicity

Clinical signs of toxicity were not observed during the conduct of the present study.

Histopathological Report

Whole frog specimens were submitted for gross trimming, histologic processing, and pathologic evaluation. The specimens were transected through the head and neck region at the anterior margin of the forelimbs. The head/neck samples were then decalcified using a commercial formic acid/EDTA solution prior to routine processing for paraffin embedding. The processed specimens were embedded so that the cut surface of the posterior margin (neck side) of each sample was microtomed first. For each block, excess paraffin was trimmed away until at least one of the thyroid glands was reached (approximately 500 microns into the tissue). At least five step sections, each 5 - 7 microns thick, were obtained at approximately 30 micron intervals through the central portion of the thyroid gland, and two serial sections acquired at each level were placed on each slide. The sections were stained with hematoxylin and eosin and mounted with a glass coverslip using an appropriate permanent mounting medium.

All sections were evaluated using brightfield microscopy. Histopathologic findings were scored for severity according to the following grading system:

Severity grading scheme for follicular cell hypertrophy

Grade	Descriptor	Criteria
0	Non-remarkable	Fewer than 20 % of the cells exhibit hypertrophy.
1	Mild	30 - 50 % of follicular cells exhibit hypertrophy.
2	Moderate	60 - 80 % of follicular cells exhibit hypertrophy.
3	Severe	Over 80 % of follicular cells exhibit hypertrophy.

 

Severity grading scheme for follicular cell hyperplasia

Grade	Descriptor	Criteria
0	Non-remarkable	Focal or diffuse crowding of follicular cells affecting less than 20 % of the tissue.
1	Mild	Focal or diffuse crowding of follicular cells affecting 30 – 50 % of the tissue, and/or single or multiple papillary infoldings of follicular cell layer.
2	Moderate	60 – 80 % of the follicles exhibit focal hyperplasia characterised by pseudostratified or stratified follicular epithelium – papillary infolding may be present.
3	Severe	Over 80 % of follicles exhibit extensive hyperplasia with stratification 2 - 3 cell layers thick - papillary infolding may be present

 

There were no treatment-related histopathologic findings in this study. As anticipated, a proportion of control frogs exhibited baseline levels of thyroid follicular cell hypertrophy (mild to moderate) and/or follicular cell hyperplasia (mild). The severity of follicular cell hypertrophy was slightly greater in teat item-treated frogs relative to the untreated control frogs, but these were low magnitude group-wise differences and they did not display a monotonic dose-response pattern (the highest overall severity of follicular cell hyperplasia occurred in the 5.50 μg/L group). Additionally, the prevalence of follicular cell hypertrophy was actually greatest (albeit by a narrow margin) in the negative control group. There was a slight dose-dependent increase in the prevalence of mild follicular cell hyperplasia in frogs of the 16.5 and 50 μg/L dose groups when compared to controls, but the magnitude of the difference was too small (e.g.,10/20 frogs affected in the 50 μg/L group vs. 7/20 frogs in the control group) to conclude that this was a treatment effect.

Prevalence and Severity of Thyroid Findings

Test Item Treatment Group (target concentration)	0.0 µg/L	5.50 µg/L	16.5 µg/L	50.0 µg/L
Number Examined	20	20	20	20
Follicular cell hypertrophy	19	18	17	18
mild	19	13	15	15
moderate	0	5	2	3
Follicular cell hyperplasia	7	7	9	10
mild	7	7	9	10

 

The lack of treatment-related effects in this study is consistent with the absence of group-wise differences in median NF stage scores – median NF stage score in the control and all treatment groups was 60. Anuran metamorphosis is considered to be a thyroid-dependent process; therefore, basal levels of follicular cell hypertrophy and hyperplasia are anticipated findings in control frogs at or around the developmental stages (i.e., metamorphic climax) at which they are generally sacrificed in AMA studies. For reasons that are not yet completely clear, the rapid elevation in TSH that is associated with metamorphic climax occurs despite a concomitant rise in circulating thyroid hormones (TH), which would otherwise be expected to suppress pituitary TSH production via the classic hypothalamus-pituitary-thyroid (HPT) negative feedback mechanism. Following metamorphic climax (e.g., Nieuwkoop and Faber (NF) Stage 66), levels of TSH and TH decrease, at which point the histological appearance of the thyroid glands becomes more quiescent. The stimulus for both follicular cell hypertrophy and hyperplasia is increased circulating levels of TSH, concentrations of which are highest in the X. laevis pituitary between NF Stages 58 - 62.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Results from the present study indicated that larvae exposed to the test item did not alter developmental rate based on developmental stage obtained at the conclusion of in-life exposure (SD 21). Asynchronous development was not noted in the control or treatments during the conduct of the study. Further, no effect of test item treatment on HLL (including SVL-normalized HLL), SVL, and body weight were observed. Finally, the observations of mild follicular cell hypertrophy and hyperplasia were consistent between control and test item-exposed X. laevis and treatment-related effects were not indicated. Based on the decision criteria in the TG 231, the test item did not affect amphibian metamorphosis or the thyroid axis directly at the concentrations tested based on the endpoints measured. The test item did not affect larval growth (SVL and body weight) at the concentrations tested based on the endpoints measured. The highest tested concentration represented the limit of solubility under test conditions.

Executive summary:

In accordance with OECD Test Guideline 231 an amphibian metamorphosis assay (AMA) was performed with the test substance in which Nieuwkoop and Faber (NF) stage 51 Xenopus laevis larvae were exposed to different concentrations of the test substance for 21-days. The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. According to OECD 231, the general experimental design entailed exposing tadpoles to three (3) different concentrations of the test chemical (n = 4 replicates per concentration) and dilution water control (n = 4 replicates). Larval density at test initiation was 20 tadpoles per test tank (i.e., replicate for all treatment groups). The treatment tanks were randomly assigned to a position in the exposure system in order to account for possible variations in temperature and light intensity. The primary endpoints were hind limb length, body length (snout to vent [SVL]), developmental stage, wet body weight, thyroid histology, and daily mortality.

Prior to in-life testing, practical water solubility was determined at test conditions for the AMA. The route of exposure was aqueous, which is the most appropriate method for aquatic organisms. A flow-through diluter system (Benoit Mini-Diluter) was used. The Benoit mini-diluter, is based on the relationship of volume and concentration, V1C1=V2C2. The system was capable of supporting 3 exposure concentrations and a control, with up to 4 replicates per treatment. The flow rate to each tank was 25 mL/min which provided a complete volume replacement every 2.7 h. Fluorescent lighting was used to provide a photoperiod of 12 h light and 12 h dark at an intensity that ranged from 600 to 2000 lux (lumens/m²) at the water surface. Water temperature was maintained at 22 ± 1 °C, pH maintained between 6.5 to 8.5, and the dissolved oxygen (DO) concentration > 3.5 mg/L (> 40% of the air saturation) in each test tank. Test solution from each replicate tank at each concentration was sampled for chemical analysis on study day (SD) 0, once per week during in-life study, and on in-life SD 21 at test termination. Thus, during the in-life study 4 sets of samples were analysed.

On SD 0, healthy and normal appearing tadpoles of the stock population were pooled and the developmental stage was determined using a binocular dissection microscope. No anaesthesia was used. Animals were carefully handled during this transfer to minimize handling stress and to avoid injury. On SD 7, 5 randomly chosen tadpoles per replicate were removed from each test tank and humanely euthanized in 150 to 200 mg/L MS-222, appropriately buffered with sodium bicarbonate to achieve pH 7. Tadpoles were rinsed in water and blotted dry, followed by body weight determination. Hind limb length and SVL, along with developmental stage were determined for each tadpole.

At test termination (SD 21), the remaining tadpoles were removed from the test tanks and humanely euthanized using the same procedure from SD 7. Tadpoles were rinsed in water and blotted dry, followed by body weight determination. Developmental stage, hind limb length, wet body weight and SVL were measured for each tadpole. All larvae were placed in Davidson’s fixative for 48 to 72 hours as whole-body samples for histological assessments. Larvae were then rinsed in dechlorinated tap water and preserved in 10 % (w/v) neutral buffered formalin (NBF). For histopathology, a total of 5 tadpoles were sampled from each replicate tank. Since follicular cell height is stage-dependent, the most appropriate sampling approach for histological analyses was to use stage-matched individuals, when possible. Animals selected for histopathology (n=5 from each replicate) were matched to the median stage of the controls (pooled replicates) whenever possible. Since replicate tanks with more than five larvae at the appropriate stage existed, 5 larvae were randomly selected.

Results of practical water solubility analyses of centrifuged samples from the recirculating saturator columns indicated that the mean concentration based on the three consecutive centrifuged samples was 48.1 µg/L test item. The target test item concentrations selected for the Amphibian Metamorphosis Assay study were 0.0 (control), 5.50, 16.5, and 50 µg/L. Dilution water control samples collected during the study contained < LOD test item (LOD = 0.912 μg/L). The corresponding mean measured concentrations in the definitive study were 5.97, 18.1, and 44.2 μg/L test item, respectively. No mortality was observed during the study. Significant differences between the median developmental stage of the test item treatments on SD 7 or at the conclusion of the study (SD 21) relative to the control were not observed. The occurrence of late stage specimens (>NF 60) was <20 % in the control and each treatment. Late stage specimens were omitted from formal analysis of SVL and body weight. Non-normalized and SVL-normalized HLL in the treatments were not significantly different from the control at SD 7 or SD 21. Asynchronous development was not noted in the control or test item treatments during the conduct of the study. SVL and body weight in the treatments were not significantly different from the control at SD 7 and at SD 21.

The prevalence of mild follicular cell hypertrophy and hyperplasia were consistent between control and test item-exposed X. laevis and treatment-related effects were not noted. The lack of treatment-related effects in this study is consistent with the absence of group-wise differences in median NF stage scores. No significant effects on behaviour or signs of overt toxicity were noted.

In conclusion, results from the present study indicated that larvae exposed to the test item did not alter developmental rate based on developmental stage obtained at the conclusion of in-life exposure (SD 21). Asynchronous development was not noted in the control or treatments during the conduct of the study. Using the decision criteria in the OECD 231, the test item did not affect amphibian metamorphosis or the thyroid axis directly at the concentrations tested based on the endpoints measured. Further, the test item did not affect larval growth (SVL and body weight) at the concentrations tested. The highest tested concentration represented the limit of solubility under test conditions.