## Case Study 2:



# Fragrance Materials Daniel Salvito The Research Institute for Fragrance Materials

Henry Krueger Wildlife International Mention of trade names, products, or services does not convey official EPA approval, endorsement, or recommendation. 1• Discrete organic compounds, enantiomeric mixtures, and natural oils • Structurally diverse • Physical -chemical properties cover a wide range • Presently over 3000 materials listed as fragrance materials • More than 80% of the materials in use have volumes of use <10 tonnes 

# What Are Fragrance Materials?

2

## Fragrance Materials- Case Study Example 3,5,5 – Trimethylhexyl acetate CAS 58430 - 94-7 Material Description Log Kow Water Sol (mg/L) VP (mm Hg) Hydrolysis (halflife) 3,5,5-Trimethylhexyl acetate CAS 58430-94 -7 Branched Chain Hydrocarbon Acetate 4.6 57 0.00706 4 d (pH 4 @ 8 mg/L) to 650 d (pH 7 @ 0.8 mg/L) 3Material Biodegradation Data 3,5,5-Trimethylhexyl acetate CAS 58430-94-7 OECD 301B (28 d): 74.5% OECD 301F ( 28 d ): 80% OECD 301D ( 28 d ): 27% 

## Biodegradation Data

4

### • Acute studies for 3,5,5 -Trimethyl hexyl acetate sufficient for PNEC refinement • Testing issues identified through acute studies 

### • Material volatility in test systems • Possibility of degradate formation 

## Testing Plan/Testing Issues

5

# Testing Difficult Test Substances Concept

• Well known toxicology and physiology
• Well known chemical and physical properties in water
• Known test concentrations 

## Reality

• Missing Data and Data gaps

• Unknown or poorly understood

• Range finding can be challenging

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## Selecting Test Concentrations

• How high do we need to test? • What are the limiting factors in achieving nominal concentrations in aquatic tests? • Is it necessary for test concentrations to be between 80% and 120% of nominal? • Your phys- chem data and models may not be as good as you think they are 7

## Limitations on Number of “Effective”

Test Concentrations

Low solubility common. Test concentrations limited by LOQ and limit of solubility. Limits on “closeness” of test concentrations are imposed by difficulty in maintaining stable test concentrations analytical variability Above limit of solubility Below the limit of quantitation

0 2 4 6 8 10 12

0 2 4 6 8 10 Concentration (μg/L ) Response

8

## What are the Challenges in Selecting Test Concentrations ? Meaningful Range of Test Concentrations Toxicity Chemical/Physical Properties Delivery of Test Material
9

## Selection of Test Concentrations for

Aquatic Testing Guideline Recommendations for limits of testing: 

### 1. 100 mg/L Pesticides 2. 1000 mg/L Industrial Chemicals 3. Water solubility limit 4. 1/3 the LC50 ≈ Maximum Tolerated Dose Recommend using 1/10 th 

### the LC50 as a better estimate of the Maximum Tolerated Dose based on acute to chronic ratios 10

## Choose your Weapon
• Static - no replacement, only option for algae 

### • Static Renewal - solutions replaced at some frequency

(daily, every few days) 

### • Flow-Through – continual replacement of test solutions 

### • Closed Bottle Testing – used for volatile test materials 

### • Water Accommodated Fractions - used for materials with low solubility

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## Complications • Mixtures – what are we testing? 

## • Formulations – do co -solvents alter the formulation? 

## • Testing near Solubility – stock volumes 

## • Insoluble Materials - how low do we need to go?

12

## Large Volumes for Chronic Tests

Key to Delivery is Mixing a Concentrated Stock in Dilution Water to make test solutions in short periods of time 13

## Volumes of Test Solutions Required Volumes of Test Solutions per Treatment (L) per Day (Tank Volumes x Number of Reps)

Fish Daphnia Acute ELS Acute Life Cycle Initial volumes 30 32 22 44 5 turnovers 150 160 110 220 10 turnovers 300 320 220 440 14

## Volumes of Stock Solutions Required Dilution Factors and Daily Volumes (L) of Stocks Delivered to Tanks for Daphnia LC Test (5 test levels) [Solvent] [Solvent] Dilution Factors 0.1 mL/L 0.02 mL/L 0 10X 100X 1000X 10,000X 50,000X Initial volumes 85 8.5 0.85 0.085 0.0085 0.0017 5 turnovers 425 42.5 4.25 .425 0.0425 0.0085 10 turnovers 850 85 8.5 0.85 0.085 0.017 15

# Stability of Stock Solutions
• Hydrolysis, Photolysis, Volatility, Adsorption • Frequency of Stock Renewals • Daily • Three times per week (MWF) • Once per week • Impacts Volumes that need to be prepared • Can affect Measured Concentrations of test Solutions

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## Is there a Role for Solvents ??

• Obviously they should be avoided and used as a last resort • Not all solvents are the same (TEG, DMF preferred to DSMO and Alcohols) • Result in stable stock solutions that greatly facilitate mixing • Useful when testing: • near solubility • unstable materials that readily hydrolyze • materials with extremely low solubility 170

10 20 30 40 50 60 70 80 90 100 110 120 130

0 24 48 72 96 % Recovery
Time 

# Acceptable Chemistry Results Ideal Difficult

18

## Case Study

• Physical / Chemistry Data • Log Kow = 3.34 • Water Solubility = 57 mg/L • Vapor Pressure = 0.00706 mm Hg • Aquatic Toxicity Information • Limited 

## 3,5,5 -Trimethylhexylacetate Branched Chain Hydrocarbon Acetate

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# Where do we Start?

Stability Adsorption Degradation (Hydrolysis and Photolysis) Volatility

Functional Solubility

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### • Shake Flask Solubility was determined to be 40 ppm 

### • 100 ppm solutions prepared in well water in 2 sealed French square bottles sampled in triplicate at 1 , 4, 24, 48 and 72 hours at 20°C ± 1 °C. 

### • Concentrations stable thru 72 hours



## Functional Solubility 

# What you can achieve in test system Prepared without a solvent

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## Volatility Suspected

• Actions Taken : • Went to preparing test solutions with a solvent to shorten mixing time in water or algal media to prevent hydrolysis occurring during stock preparation
• Decided on Closed bottle testing
• Preconditioning of Glassware

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## Algal Study • Must be run as a static test • Closed bottle test requires: • Sealed 300 mL glass bottles • 2 marbles added to filled bottles • Dosed, sealed, and placed on shaker 

### tables • Addition of sodium bicarbonate as a source of CO2 

### • Addition of abiotic control to determine adsorption or uptake by algae
230.51 1.2 1.9 3 3.8 5.7

1 2.6 6.4 16 40 40

0.1 1 10 100

L1 L2 L3 L4 L5 Abiotic Geometric Mean Nominal 

## Algae Measured Test Concentrations

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## Algal EC50 (mg/L)

Exposure Duration Area Under

the Growth Curve Growth

Rate Yield 

### 72 hours 1.3

0.69 to 2.6 

### >3.8 1.3 0.69 to 2.6 

### 96 hours 2.1

1.2 to 3.5 

### >3.8 >3.8

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## Daphnia Acute Study

Exploratory range -finding done in closed bottle static and flow-through test systems, flow thru better due to loss at 48 hours in static closed bottle system Functional Solubility in the test system determined to be 20 ppm rather than 40 ppm Covered t est chambers and added mini mixers to mixing cup 260.29 0.44 1.3 3.5 5.4

1.3 2.5 5 10 20

0.1 1 10 100

L1 L2 L3 L4 L5 Geometric Mean Nominal 

## Daphnia Measured Concentrations

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## Daphnia Results 48 - Hour EC50 : > 5.4 mg/L No - Immobility Concentration : > 5.4 mg/L No -Observed - Effect Concentration: > 5.4 mg/L

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## Fathead Minnow Results

Exploratory range -finding done in closed bottle static renewal and flow-through test systems, flow thru better due to loss at 48 hours in static closed bottle system Functional Solubility in the test system determined to be 20 ppm rather than 40 

### ppm Covered test chambers and added mini 

### mixers to mixing cup
290.97 2.2 4 6.3 11

2.5 5 10 20 40

0.1 1 10 100

L1 L2 L3 L4 L5 Mean Measrued Nominal 

## Fathead Minnow Measured Concentrations

30

## Fathead Minnow Results 96 - Hour LC50: 7.7 mg/L 95 % Confidence Interval : 6.3 – 11 mg/L No - Mortality Concentration: 4.0 mg/L No -Observed - Effect Concentration : 4.0 mg/L

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## Summary Data 3,5,5-Trimethylhexyl acetate LC50 EC50 Fish (OECD 203: Fathead minnow) 7.7 mg/L Daphnia (OECD 202) > 5.4 mg/L Algae Inhibition Study (OECD 201) 1.3 mg/L

32• Understand physico -chemical properties of the

chemical • Confirm with Functional Water Solubility and Stability • Determine if solvents are needed • Decide which test system to use based on substance’s physico -chemical properties such as volatility and degradate formation



## Conclusion

33• Barbara Lewis - Givaudan Fragrances Corp. • Anne Kim - US EPA 

## Other Contributors

34