N-cyclohexylcyclohexanamine 2-[1-[[[(1R)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl]methyl]cyclopropyl]acetate

Long-term toxicity to fish

Administrative data

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[1-[[1-[3-[2-[(7-Chloro-2-quinolyl)]vinyl]phenyl]-3-[2-(1-hydroxy-1-methyl-ethyl)phenyl]-propyl]sulfanylmethyl]cyclopropyl]acetic acid sodium salt

EC Number:

604-813-7

Cas Number:

151767-02-1

IUPAC Name:

2-[1-[[1-[3-[2-[(7-Chloro-2-quinolyl)]vinyl]phenyl]-3-[2-(1-hydroxy-1-methyl-ethyl)phenyl]-propyl]sulfanylmethyl]cyclopropyl]acetic acid sodium salt

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: montelukast sodium
Synonyms: MK-0476, active ingredient of SINGULAIR®
Lot No.: XTA-719
CAS No.: 151767-02-1
Purity: 100.4 (tested as 100%)
Recertification Date: 1 July 2013
Upon receipt at Springborn Smithers, the test substance (SSL No. 4647) was stored at room temperature in the original container in a dark ventilated cabinet

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Prior to the start of the definitive exposure, samples from one replicate of each treatment level and control solution were collected and analyzed for montelukast. In addition, a sample of the diluter stock solution was analyzed twice prior to test initiation. Results of the pretest analyses were used to judge whether sufficient quantities of montelukast were being delivered and maintained in the exposure aquaria in order to initiate the early life-stage exposure.
During the in-life phase of the definitive study, water samples were removed from alternating replicates (A, B, C and D) of each treatment level and the control ontest day 0, 4 (hatch), 8, 15, 22 and 32. All test samples were removed from the approximate midpoint of each aquarium using a pipette. A sample of the stock solution was also analyzed at each sampling interval during the definitive study period. In addition, three quality control (QC) samples were prepared at each sampling interval and remained with the exposure solution samples throughout the analytical process. These QC samples were prepared in dilution water at nominal concentrations similar to the exposure concentration range. Results of the analyses of the QC samples were used to judge the precision and the quality control maintained during the analysis of exposure solution samples

Test solutions

Vehicle:

no

Details on test solutions:

A 25 mg/mL diluter stock solution was prepared prior to test initiation and as needed throughout the definitive exposure by placing, for example, approximately 0.2500 g of test substance in a 10 mL volumetric flask and diluting to volume with dilution water. The resultant solution was observed to be clear and yellow in color following preparation.

Prior to test initiation, a Harvard syringe pump was calibrated to deliver 0.0241 mL/cycle of the stock solution (25 mg/mL) into the diluter's chemical mixing chamber, which also received 1.94 L of dilution water per cycle. The mixing chamber was positioned over a magnetic stir plate and was partially submerged within an ultrasonic water bath to aid in mixing the stock solution with the dilution water. The solution contained in the mixing chamber constituted the highest nominal test concentration (0.31 mg/L) and was sequentially diluted (50%) to provide the remaining nominal exposure concentrations (0.16, 0.078, 0.039 and 0.019 mg/L).
A set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no montelukast

Test organisms

Test organisms (species):

Pimephales promelas

Details on test organisms:

Fathead minnow eggs were obtained from brood stock maintained at Springborn Smithers for more than 25 years. The brood stock originated from the U.S. Environmental Research Laboratory, Duluth, Minnesota. Fathead minnow from reputable commercial suppliers have been periodically added to the Springborn Smithersgene pool. No outbreak of disease has occurred and no medications have been used to treat the brood stock in the last fifteen years. The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

32-day duration (28 days post-hatch)

Post exposure observation period:

Dead and live embryos were counted daily until hatching was complete and dead eggs were
removed during this observation period. Day of hatch was considered to be exposure day 4,
when no more than 10% unhatched viable embryos remained in any control or treatment level
egg incubation cup. Calculations of hatching success of organisms were based on the number of
live, dead or deformed larvae per incubation cup after hatching was complete (day 4) compared
to the number of embryos per cup on test day 0.
The 28-day post-hatch larval exposure was initiated on the day of hatch (test day 4). On test
day 4, the surviving larvae present in each incubation cup were thinned to 15 organisms per
replicate/60 organisms per treatment level or control and placed into each respective exposure
aquarium. During the post-hatch exposure period, dead larvae were removed when observed and
behavior and appearance of the larval fish were observed and recorded daily. Larval survival
was estimated daily throughout the post-hatch period. At 28 days post-hatch exposure (test termination), the percentage of larval survival was
determined.

Test conditions

Hardness:

CaCO3 of 66 to 84 mg/L

Test temperature:

23 to 25 ºC throughout the exposure

pH:

pH range of 6.0 to 7.2

Dissolved oxygen:

5.0-8.7 mg/L

Conductivity:

330 to 390 µmhos/cm

Nominal and measured concentrations:

NOMINAL TEST CONCENTRATIONS: 0.019, 0.039, 0.078, 0.16 and 0.31 mg/L
MEAN MEASURED CONCENTRATIONS: 0.019, 0.041, 0.073, 0.14 and 0.27 mg/L

Details on test conditions:

The exposure of fathead minnow embryos and larvae to montelukast was initiated when the embryo incubation cups were distributed to each of the test aquaria The eggs were impartially distributed to the egg incubation cups in the following manner: Twenty-four labeled incubation
egg cups containing control water were placed in a water bath maintained at 25 ± 2ºC. A Carolina dish containing the eggs was placed in the same water bath. The eggs were impartially placed in the egg cups five at a time until each cup contained 30 eggs. To initiate the study, the incubation cups, each containing 30 eggs, were then suspended in the respective exposure aquaria (one cup per replicate vessel). At study initiation, the embryos were < 24 hours old.
Mortality of less than 1% was observed in the brood stock during the 48-hour period prior to exposure initiation

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: percent live normal fry, total length and dry weight

Effect concentrations 2

Key result

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Most sensitive iinidcators were percent live normal fry, total length and dry weight

Details on results:

An 18-day (14 days post-hatch) preliminary test was conducted exposing 30 fathead minnow embryos per replicate (< 24 hours old) to concentrations of 0.019, 0.039, 0.078, 0.16 and 0.31 mg/L and a dilution water control. Based on these results nominal concentrations of 0.019,0.039, 0.078, 0.16 and 0.31 mg/L were selected for the definitive exposure.

The diluter system which prepared and delivered the test solutions to the exposure aquaria functioned properly during the pretest period and throughout the definitive study. Analysis of the exposure solutions during the pretest period established that the appropriate concentrations of montelukast (ranging from 87 to 103% of nominal concentrations) were maintained in the exposure solutions and that the delivery apparatus maintained the expected concentration gradient (approximately 50% dilution factor) of the test substance. Analysis of the stock solution during the pretest period resulted in a measured concentration range of 92 to 140% of nominal. No undissolved test substance (e.g., precipitate) was observed in the diluter system (e.g., mixing chamber and chemical cells) or exposure solutions.

Analysis of the QC samples resulted in measured concentrations which were consistent with the predetermined recovery range and ranged from 90.5 to 114% (N = 18) of the nominal fortified levels (0.00500 to 0.350 mg/L). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of exposure solutions.

At the completion of hatch (day 4), hatching success in the control averaged 86%. Embryo hatching success in the 0.019, 0.041, 0.073, 0.14 and 0.27 mg/L treatment levels was 79, 79, 76, 74 and 83%, respectively. Williams' Test determined no significant difference in embryo hatching success among organisms exposed to any treatment level tested compared to the hatching success of the control organisms (86%).
At the completion of the hatching period (day 4), the percent of normal larvae in the control averaged 100%. The percent of normal larvae in the 0.019, 0.041, 0.073, 0.14 and 0.27 mg/L treatment levels averaged 96, 99, 98, 81 and 82%, respectively. Steel’s Many-One Rank Test determined a significant difference in the percent of normal larvae among organisms exposed to the 0.14 and 0.27 mg/L treatment levels compared to the percent of normal larvae in the control
(100%).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations and the most sensitive indicators of toxicity in this study (i.e., percent life normal fry, total length and dry weight), the No Observed-Effect Concentration (NOEC) was determined to be 0.073 mg/L. The Lowest-Observed-Effect Concentration (LOEC) for montelukast and fathead minnow was determined to be 0.14 mg/L.

Executive summary:

The objective of this study was to determine the No-Observed-Effect Concentration (NOEC) of montelukast to fathead minnow (Pimephales promelas) embryos and larvae under flow-through conditions. The endpoints evaluated were embryo hatching success, defined as percentage of embryos that produce live normal fry at hatch, survival at test termination, and larval growth (total length and dry weight).