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Short term toxicity to fish:

1: The objective of this test was to estimate the toxicity of hydroxypropylated .beta.-cyclodextrin to Zebrafish (Brachydanio rerio) under static conditions following the procedures outlined in ABC Protocol No. EEC C1 for ABC Study Number 43997. The test was designed to yield LC50 values following 24, 48, 72, and 96 hours of exposure and a no-observed effect concentration (NOEC), if possible. The definitive test was conducted from June 23, 1997, to June 27, 1997.

The definitive study was conducted as a limit test using the nominal test concentration of 1000 mg/L of hydroxypropylated .beta.-cyclodextrin along with a control. Twenty fish (ten fish in each of the two replicates) were exposed to the control and test concentration. Samples for analytical confirmation were collected from each test replicate at 0 and 96 hours during the definitive test. The mean measured test concentration for samples analyzed at 0 and 96 hours was 1131 mg/L, representing 113% of the 1000 mg/L nominal test concentration.

Water quality parameters of temperature, dissolved oxygen, and pH were measured throughout the study and were within acceptable limits. The temperature of the test solutions ranged from 22.4 to 23.4°C and the dissolved oxygen levels ranged from 5.1 to 8.2 mg/L. The pH of the test solutions ranged from 7.8 to 8.4.

All fish in the control and 1000 mg/L nominal test concentration were normal during the study with no observed abnormal effects or mortality. The 24-, 48-, 72-, and 96-hour LC50 values for hydroxypropylated .beta.-cyclodextrin to Zebrafish (Brachydanio rerio) were all estimated to be greater than the mean measured test concentration of 1131 mg/L. The 96-hour NOEC was determined to be >= 1131 mg/L which was based on the lack of abnormal effects and mortality at this concentration. A slope of the 96-hour dose-response line could not be calculated since the study was conducted as a limit test.All results were based on the mean measured concentration.

 

2: The acute toxicity of hydroxypropylated .beta.-cyclodextrin in the Bluegill sunfish (Lepomis macrochirus) was investigated in a static fish test.

Nominal test concentrations were 1, 10, 100 and 1000 mg hydroxypropylated .beta.-cyclodextrin/L.

The exposure period was 96 hours. The behavior and the mortality of the Bluegill sunfish (Lepomis macrochirus) were recorded daily.

Oxygen concentration, temperature and pH remained within the range to provide normal growth and development of the Bluegill sunfish (Lepomis macrochirus).

The LC50 - 96 h of hydroxypropylated .beta.-cyclodextrin in the Bluegill sunfish (Lepomis macrochirus) could not be determined as the highest nominal concentration i.e. 1000 mg/l did not cause mortality.

Long term toxicity to fish:

Prolonged (28 days) juvenile growth test in Carp with hydroxypropylated .beta.-cyclodextrin.

The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals 215: "Fish, juvenile growth test - 28 days", accepted January 21, 2000.

The main test was performed with carp exposed to a range of hydroxypropylated .beta.-cyclodextrin target concentrations of 10, 18, 32, 56 and 100 mg/L,in a flow-through system. Sixteen fish were exposed per concentration and to untreated test medium (blank-control). Stock solutions were dosed via a computer-controlled system and entered a mixing chamber separately from the tap water supply (flow-rates 0.3 to 6 L/h). In addition, all test solutions were refreshed four times during the first 13 days of exposure to ensure optimal quality and cleanness of the test solutions. The functioning of the system was checked daily during the 28-day test period.

Fish were fed daily with Trouvit at 4% of the body weight per vessel. After 14 days of exposure the ration was recalculated based on the fish weights then determined. Sampling for analysis was performed weekly in all test solutions. No specific analytical method was available for measuring hydroxypropylated .beta.-cyclodextrin concentrations. Consequently, the Total Organic Carbon (TOC) content was analyzed as a measure for the actual exposure concentrations. Fish were daily observed for possible mortality, while three times a week fish were observed for clinical signs. Specific growth rates were based on the body weights measured at the start of the study and after 14 and 28 days of exposure.

TOC-analysis of the samples taken during the 28-day test period from the target concentrations of 32 mg/Land higher revealed that recoveries for organic carbon were at more than 80% compared to the expected carbon content. Results further showed that measured TOC concentrations were maintained within 80-120% of the mean measured values at these target concentrations. The TOC-results at the target concentrations of 10 and 18 mg/L were difficult to interpret. This was a consequence of the limited accuracy at TOC levels below 10 mg/L, i.e. close to the limit of detection of the equipment, which resulted in a high variation rate.

The overall conclusion was that measured TOC-concentrations were in agreement with what was expected based on the nominal carbon content of hydroxypropylated .beta.-cyclodextrin. Further results will therefore be based on the target concentrations.

The mean weight of the fish in the control groups had increased by an average of > 150% above their initial weight over 28 days. Oxygen concentration was generally found to be > 6 mg O2/L for all measurements. The few occasions with lower oxygen concentrations were only short lasting and did not affect the fish. During the study, temperature was maintained between 20.0 and 22.3°C and variation between the different test chambers was always within 1°C at anyone time during the test.

TOC-analyses showed that measured TOC-concentrations were in agreement with what was expected based on the nominal carbon content of hydroxypropylated .beta.-cyclodextrin. A reference test with pentachlorophenol showed normal sensitivity of the supplied carp.

 

Short term toxicity to aquatic invertebrates:

1:  The test article, hydroxypropylated .beta.-cyclodextrin , was tested in Daphnia magna equivalent to the OECD Guideline Study 202.

The primary objective of this study was to estimate the acute toxicity of hydroxypropylated .beta.-cyclodextrin to Daphnia magna under static conditions. The test was designed to yield EC50 values following 24 and 48 hours of exposure and a no-observed effect concentration (NOEC), if possible. The definitive test was conducted from June 10, 1997, to June 12, 1997.

The definitive study was conducted as a limit test using the nominal test concentration of 1000 mg/L of hydroxypropylated .beta.-cyclodextrinalong with a control. Twenty Daphnia magna (five daphnids in each of the four replicates) were exposed to the control and test concentration. Samples for analytical confirmation were collected from each parent solution at 0 hour and from each replicate at 48 hours of the definitive test. The mean measured test concentration for samples analyzed at 0 and 48 hours was 1084 mg/L, representing 108% of the 1000 mg/L nominal test concentration.

Water quality parameters of temperature, dissolved oxygen, and pH were measured 0 and 48 hours of the definitive study and were within acceptable limits. The temperature of the test solutions ranged from 20.9 to 21.3°C and the dissolved oxygen levels ranged from 7.7 to 8.2 mg/L. The pH of the test solutions ranged from 8.2 to 8.4.

All daphnids in the control and 1000 mg/L nominal test concentration were normal during the study with no observed abnormal effects or immobilization. The 24- and 48-hour EC50 values for hydroxypropylated .beta.-cyclodextrin to Daphnia magna were all estimated to be greater than the mean measured test concentration of 1084 mg/L. The 48-hour NOEC was determined to be >=1084 mg/L, which was based on the lack of abnormal effects and immobilization at this concentration. A slope of the 48-hour dose-response line could not be calculated since the study was conducted as a limit test.All results were based on the mean measured concentration.

 

2: The acute toxicity of hydroxypropylated .beta.-cyclodextrin in the water-flea Daphnia magna was investigated following the FDA handbook "Environmental Assessment Technical Assistance Handbook PB 87 -175345"; Document 4.08: "Daphnia Acute Toxicity" (1).

Nominal test substance concentrations in the final test were 10, 32, 100, 320 and 1000 mg hydroxypropylated .beta.-cyclodextrin per liter.

The exposure period was 48 hours and the number of immobile daphnids was recorded daily.

Oxygen concentration, pH and temperature remained within the range to provide normal growth and development of daphnids.

Based on the nominal concentrations of hydroxypropylated .beta.-cyclodextrin in the test vessels the EC50 - 48 h of hydroxypropylated .beta.-cyclodextrin in the daphnia magna was higher than 1000 mg/l, since no mortality occurred.

 

3: The 48 hours daphnia acute toxicity test was conducted with a mixture of hydroxypropylated .alpha.-cyclodextrin and hydroxypropylated .beta.-cyclodextrin at 20°C in synthetic water. The preliminary test was conducted at nominal concentrations of 0.1, 1, 10, 100 and 1,000 ppm for 48 hours. After 48 hours, the test animals were observed for mortality or any adverse effects. The inspection of test containers showed all animals alive without any adverse effects. To further confirm the preliminary results, a limit test was conducted at the highest nominal concentration used in the preliminary test (1,000 ppm) using 20 animals and four replicates both for control and test material. The results of the limit test also showed no mortalities or adverse effects, which confirmed the results of preliminary test. Based on these experiments, the toxicity of the hydroxypropylated .alpha.-cyclodextrin and hydroxypropylated .beta.-cyclodextrin mixture is greater than 1,000 ppm.

 

4: The primary objective of this study was to evaluate the chronic toxicity of hydroxypropylated .beta.-cyclodextrin to Daphnia magna under static-renewal according to OECD Guideline 202. The test was designed to yield a no-observed effect concentration (NOEC) and a lowest-observed effect concentration (LOEC) based on the statistical analysis of survival, growth, and reproduction.

An estimate of the maximum acceptable toxicant concentration (MATC) was calculated using the NOEC and LOEC. If possible, a 7-, 14-, and 21-day EC50 based on adult immobilization and 95 % confidence limits were to be calculated.

The definitive study was initiated on July 21, 1997, and lasted 21 days before it was terminated on August 11, 1997. The nominal exposure concentrations were 62.5, 125, 250, 500, and 1000 mg/L of hydroxypropylated .beta.-cyclodextrinalong with a control. The test was conducted in 32 oz. clear glass containers containing 400 mL of the test solution. There were four replicates (A, B, C, and D) per test concentration and each initially contained 10 Daphnia magna (<24 hours old). The daphnids were exposed to newly prepared test (and control) solutions every Monday, Wednesday, and Friday, throughout the definitive test. The daphnids were fed and observed daily. After 21 days, survival, time to first brood, young per adult reproduction day, length, and weight were statistically compared to the control to evaluate the toxicity of hydroxypropylated .beta.-cyclodextrin to Daphnia magna.

The terms "new" and "old" will appear throughout this report. The term "new" is defined as newly prepared parent test solutions and control having contained no daphnids or food. The term "old" is defined as approximately 48- or 72-hour old test solutions and control that had contained daphnids and/or food.

Water quality parameters of temperature, dissolved oxygen concentration and pH were measured in "new" and "old" solutions. The temperature of the "new" control and test solutions ranged from 20.7 to 22.8°C and the temperature of the "old" control and test solutions ranged from 20.0 to 20.9°C. The pH values of the "new" control and test solutions ranged from 8.1 to 8.5 and the pH values of the "old" control and test solutions ranged from 7.9 to 8.6. Dissolved oxygen concentrations for the control and test solutions ranged from 7.5 to 8.4 mg/L in the "new" solutions and ranged from 6.2 to 8.8 mg/L in the "old" solutions. These dissolved oxygen values represented 91 and 100% saturation at 23°C and 22°C, respectively, for the "new" solutions, and 73 and 101% saturation at 21°C and 20°C, respectively, for "old" solutions. Each solution during the definitive test was gently aerated. The dissolved oxygen saturation (100 %) at temperatures of 20, 21, 22. and 23 °C corrected for local altitudinal pressure. are 8.7. 8.5, 8.4, 8.2 mg/L, respectively. All water quality parameters were considered to be within acceptable limits.

The parameters of survival, time to first brood, young per adult reproduction day (YAD), and growth (length and weight) were statistically compared to the control. The mean measured test concentrations of 251, 513 and 1074 mg/L were shown to be significantly different(p< 0.05) as compared to the control for the survival parameter. For all other parameters no exposure concentration was shown to be significantly different as compared to the control.

The NOEC and LOEC were determined to be 127 and 251 mg/L, respectively, as indicated by the statistical analysis. The MATC was based on these concentrations and was calculated to be 179 mg/L for hydroxypropylated .beta.-cyclodextrin to Daphnia magna. The day-7, -14, and -21 EC50 values based on immobility/mortality estimated to be >1074, > 1074, and 446 mg/L, respectively.

 

Toxicity to aquatic algae and cyanobacteria:

1: The influence of various concentrations of hydroxypropylated .beta.-cyclodextrin on the growth of the unicellular green algaSelenastrum aipricomutumwas investigated according to the OECD guideline No. 201 "Alga, Growth Inhibition Test".

The exposure period was 3 days.

First a preliminary test was performed to determine the concentration range for the confirmatory test. The following concentrations were used in the preliminary test 1, 10, 100 and 1000 mg hydroxypropylated .beta.-cyclodextrin /l and a control.

Based on the nominal concentrations the EC5O (mean effective concentration) after 72 hours with respect to average specific growth rate, was higher than 1000 mg hydroxypropylated .beta.-cyclodextrin /l.

 

2: The test article, hydroxypropylated .beta.-cyclodextrin , was tested in Selenastrum capricornutum according to OECD Guideline 201.

The primary objective of this test was to evaluate the inhibition or enhancement effect of hydroxypropylated .beta.- cyclodextr into Selenastrum capriconutum Printz under static conditions following the procedures outlined in ABC Protocol No. OECD 201. The test was designed to yield EC50 (EbC50 and/or ErC50) values following 24, 48, 72, and 96 hours of exposure and a 96-hour no-observed effect concentration (NOEC).The definitive study was conducted from June 2, 1997, to June 6, 1997.

The 96-hour acute toxicity test was conducted by exposing Selenastrum capriconutum Printz to the single nominal exposure concentration of 1000 mg/L hydroxypropylated .beta.-cyclodextrin, along with a control. The test was conducted in 250-mL Erlenmeyer flasks containing 100 mL of the test solution. There were four control replicates (A, B, C, and D) of the control solution and six replicates (A, B, C, D, E, and F) of the 1000 mg/L test solution. Each A, B, C, and D replicate received 1.0 mL of algal inoculum containing approximately 1.0 x 106 cells/mL, resulting in approximately 1.0 x 104 cells/mL for each flask. The D replicates were used only for water quality measurements and collection of analytical samples at 72 hours. The1000 mg/L replicates E and F were· used as the abiotic (no algae present) replicates and were used for collection of 72- and 96-hour analytical samples.

Water quality parameters of temperature and pH were measured at 0, 72, and 96 hours of the study. The temperature of the test solutions ranged from 24 to 25°C. The pH values of the test solutions ranged from 7.5 to 9.9.

Samples of the control and 1000 mg/L test solutions were collected at test initiation (0 hour), 72 hours, and at test termination (96 hours) for analytical confirmation. The measured concentration at test initiation (0 hour) was 1248 mg/L for the 1000 mg/L nominal test concentration, which represents 125% of the nominal test concentration. At 72 hours, the measured concentration was 1115 mg/L for the 1000 mg/L nominal test concentration, which represents 112% of the nominal test concentration. The measured concentration at test termination (96 hours) was 1096 mg/L for the 1000mg/Lnominal test concentration, which represents 110% of the nominal test concentration. The 0- to 72-hour mean measured concentration was 1182mg/L.The 0- to 96-hour mean measured concentration was 1153 mg/L.

The 72- and 96-hour 1000 mg/L abiotic solutions were measured to be 1153 and 1129 mg/L, respectively, which represents 115 and 113 %, respectively, of the 1000 mg/L nominal concentration. All results were based on both the 0- to 72-hour mean measured concentration and 0- to 96-hour mean measured concentration.

Using the 0- to 72-hour mean measured concentration, the 24-, 48-, 72-hour EbC50 and ErC50 values for hydroxypropylated .beta.-cyclodextrinwere estimated to be > 1182mg/L.Using the 0- to 96-hour mean measured concentration, the 96-hour EbC50 and ErC50 values forhydroxypropylated .beta.-cyclodextrinwere estimated to be > 1153 mg/L. Based on the area under the growth curve parameter, the 72- and 96-hour no-observed effect concentrations were estimated to be >= 1182 mg/L (0- to 72-hour mean measured concentration) and < 1153 mg/L (0- to 96-hour mean measured concentration) respectively. Based on the growth rate parameter, the 72- and 96-hour no-observed effect concentrations were estimated to be > 1182 mg/L (0- to 72-hour mean measured concentration) and > 1153 mg/L (0- to 96-hour mean measured concentration), respectively.

 

Toxicity to microorganisms:

1: The influence of hydroxypropylated .beta.-cyclodextrinon the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984 and EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L133, adopted May 30, 1988.

Hydroxypropylated .beta.-cyclodextrinis white powder that was easily soluble in milli-Q water. A stock solution was prepared in milli-Q water at a concentration of 0.5 g/l. The pH of the clear stock solution was 7.6. A concentration of 100 mg/I, corresponding with 100 ml of the 0.5 g/l stock solution in 500 ml final volume, was tested in duplicate.

No significant inhibition in respiration rate of the sludge was recorded at 100 mg hydroxypropylated .beta.-cyclodextrinper litre. The duplicate measurement confirmed the result of the first measurement. Values of -7% and -5% inhibition were recorded (negative values indicate stimulation in respiration rate of the sludge). Hence, the EC50 for hydroxypropylated .beta.-cyclodextrinexceeded 100 mg/l and no further testing was necessary.

The respiration rates of the controls were within 15% of each other. The EC50 of the reference substance, 3,5 -dichlorophenol, was 15 mg/l. Therefore, the test was considered to be valid.

In conclusion, under the conditions of this present test,hydroxypropylated .beta.-cyclodextrinwas not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.

 

2: The test substance hydroxypropylated .beta.-cyclodextrin had no inhibition effect on the activated sludge after 3 hours.The highest inhibition that was noted was 8.8 %.

 

3: According to FDA-guidelines “Environmental Assessment Technical Assistance Document 4.02; Microbial Growth Inhibition”, the in vitro activity of hydroxypropylated .beta.-cyclodextrin against a number of micro-organisms that carry out important, ecosystem functions was investigated. Since the compound is highly soluble in water, a sterile solution in this solvent is used for the experiment. The Minimum Inhibitory Concentration (MIC-value) is situated above 1,000 ppm.

 

4: According to FDA-guidelines “Environmental Assessment Technical Assistance Document 4.02; Microbial Growth Inhibition”, the in vitro activity of hydroxypropylated .beta.-cyclodextrin against a number of micro-organisms that carry out important, ecosystem functions was investigated. Since the compound is highly soluble in water, a sterile solution in this solvent is used for the experiment. The Minimum Inhibitory Concentration (MIC-value) is situated above 1,000 ppm.

 

5: According to FDA-guidelines "Environmental Assessment Technical Assistance Document 4.02; Microbial Growth Inhibition", the in vitro activity of hydroxypropylated .beta.-cyclodextrin against a number of micro-organisms that carry out important, ecosystem functions was investigated. Since the compound is highly soluble in water, a sterile solution in this solvent is used for the experiment. The Minimum Inhibitory Concentration (MIC-value) is situated above 1,000 ppm.

 

6: According to FDA-guidelines “Environmental Assessment Technical Assistance Document 4.02; Microbial Growth Inhibition”, the in vitro activity of hydroxypropylated .beta.-cyclodextrin against a number of micro-organisms that carry out important, ecosystem functions was investigated. Since the compound is highly soluble in water, a sterile solution in this solvent is used for the experiment. The Minimum Inhibitory Concentration (MIC-value) is situated above 1,000 ppm.

 

7:According to FDA-guidelines “Environmental Assessment Technical Assistance Document 4.02; Microbial Growth Inhibition”, the in vitro activity of hydroxypropylated .beta.-cyclodextrin against a number of micro-organisms that carry out important, ecosystem functions was investigated. Since the compound is highly soluble in water, a sterile solution in this solvent is used for the experiment. The Minimum Inhibitory Concentration (MIC-value) is situated above 1,000 ppm.