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)

3
Material 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

6
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 1/10th 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

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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

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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

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0

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

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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

19
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

21
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

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0.51

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 test chambers and added mini mixers 

to mixing cup

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0.29

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 

27
Daphnia Results

48-Hour EC50: > 5.4 mg/L

No-Immobility Concentration: > 5.4 mg/L

No-Observed-Effect Concentration: > 5.4 mg/L

28
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

29
0.97

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

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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

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• 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

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• Barbara Lewis - Givaudan Fragrances Corp.

• Anne Kim - US EPA

Other Contributors

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