IR 3535 Interim specification WHO/IS/TC/667/2001

TECHNICAL MATERIAL

IR 3535
ETHYL BUTYLACETYLAMINOPROPIONATE

INFORMATION
Common name
Ethyl butylacetylaminopropionate (INCI)
Synonyms
®
IR3535 , AI 3-70763, EBAAP, ethyl 3-(N-butylacetamido)propionate,
Merck 3535, OMS 3065
Chemical names
IUPAC: 3-(N-acetyl-N-butyl)aminopropionic acid ethyl ester
CA: beta-alanine, N-acetyl-N-butyl-, ethyl ester
CAS Registry number
52304-36-6
CIPAC number
667
Structural formula
O

OC2 H 5
N

Empirical formula
C11H21NO3
Relative molecular mass
215,3

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TECHNICAL MATERIAL

ETHYL BUTYLACETYLAMINOPROPIONATE
TECHNICAL MATERIAL
Interim specification: WHO/IS/TC/667 (2001)
This specification, which is PART ONE of this publication, is based on an
evaluation of data submitted by the manufacturer whose name is listed in the
evaluation report (WHO/667/2001). It should be applicable to relevant products
of this manufacturer but it is not an endorsement of those products, nor a
guarantee that they comply with the specification. The specification may not be
appropriate for the products of other manufacturers. The evaluation report
(WHO/667/2001), as PART TWO, forms an integral part of this publication.

1 Description (Note 1)
The material shall consist of ethyl butylacetylaminopropionate together with
related manufacturing impurities, in the form of a colourless to slightly
yellowish and almost odourless liquid, free from visible extraneous matter
and added modifying agents.

2 Active ingredient
2.1 ldentity tests (Note 1)
The active ingredient shall comply with an identity test and, where the
identity remains in doubt, shall comply with at least one additional test.
2.2 Ethyl butylacetylaminopropionate content (Note 1)
The ethyl butylacetylaminopropionate content shall be declared (not less
than 980 g/kg) and, when determined, the mean measured content shall
not be lower than the declared minimum content.

3 Physical properties
3.1 pH range (MT 75)
pH range: 4.0 to 6.0

Note 1 Methods for sampling and analysis

1 Sampling. Thoroughly mix the contents of the container, remove an aliquot (not less
than 50 ml) and place it in a new, clean, brown glass bottle fitted with teflon screw cap.
Characteristics described by the specification should be assessed from this aliquot.
2 Identity tests
2.1 Gas Chromatography. Use the method described below. The retention time of
ethyl butylacetylaminopropionate in the sample should not deviate by more than ±1.5%
from that of the reference standard.
2.2 Infra-red spectroscopy. The capillary layer IR spectrum should be comparable with
that of a standard spectrum of ethyl butylacetylaminopropionate, with characteristic bands
-1
at 2960, 2933, 1734 and 1648 cm .

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20
2.3 Refractive index. nD 1.452-1.455
3 Determination of ethyl butylacetylaminopropionate content
OUTLINE OF METHOD
Ethyl butylacetylaminopropionate is separated from other components by gas
chromatography, following direct injection of the liquid via a split injector, and quantified
using a flame ionization detector. An autosampler and electronic integration are used to
produce area percent data.
The method assumes that area percent of response is directly related to concentration in
the TC. However, the method does not detect the presence of water, other inorganic
compounds or involatile compounds and it will produce inaccurate results if such
chemicals are present in significant quantities. If the results are of doubtful accuracy, they
should be checked using a method based on internal or external standardisation and
calibration with ethyl butylacetylaminopropionate reference standard.

APPARATUS
Gas chromatograph with split/splitless capillary inlet and flame ionisation detector (FID).
Electronic integrator capable of determining area percent values.
Autosampler, CTC Analytics A200SE, or equivalent capable of injecting 0.1 to 1 µl.
Capillary column, 25 m length, 0.32 mm i.d., film thickness 1.2 µm, fused silica with
CP-Sil 5 CB coating, or equivalent.

REAGENTS
Ethyl butylacetylaminopropionate reference standard.
Ethanol.
Solutions for linearity check. Accurately prepare solutions of 1% w/w and 10% w/w ethyl
butylacetylaminopropionate in ethanol.

PROCEDURE
(a) Operating conditions (typical)

Carrier Gas (helium) linear flow 69 cm/sec

Hydrogen 30 ml/min
Air 600 ml/min
Make-up gas (helium) 25 ml/min
o
Injector (splitless) temperature 290 C, split ratio 1 : 13
Injection volume 0.4 µl
Column oven temperatures
o
initial 70 C for 5 min
o
programme rate 10 C/min
o
final 280 C for 0 min
o
Detector temperature 300 C
Retention time ethyl butylacetylaminopropionate 19.5 to 20 min

(b) System suitability test

Make replicate injections of 0.10, 0.3, 0.5 and 1.0 µl volumes of undiluted ethyl
butylacetylaminopropionate and of 0.5 µl volumes of the 1% and 10% solutions. The
correlation coefficient (r) of the regression line should be 0.99 or higher. A coefficient of
variation of <0.05% should be obtainable with injections of 0.4 µl undiluted ethyl
butylacetylaminopropionate. Ensure that retention time is consistent within ±1.5%.
Modify the typical operating conditions to comply with these requirements, if necessary.

(c) Determination
Inject the liquid ethyl butylacetylaminopropionate TC and determine the peak areas as
percentages of the total peak area.

(d) Calculations
Determine the area percent due to ethyl butylacetylaminopropionate, which corresponds
to the percentage in the TC.

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TECHNICAL MATERIAL

ETHYL BUTYLACETYLAMINOPROPIONATE (WHO/667/2001)

EVALUATION REPORT

Explanation
The data for ethyl butylacetylaminopropionate were evaluated in support of WHO
specification.
The active ingredient is not under patent.
Ethyl butylacetylaminopropionate was reviewed by US EPA in 1999 (Reg. No.
70759) and has been evaluated for efficacy by the WHOPES programme (ref. 1).
It has also been evaluated by the WHO/PCS (ref. 1).
The draft specification and the supporting data were provided by Merck KGaA,
Germany, in 2000.
Uses
Ethyl butylacetylaminopropionate is an insect repellent for application to human
skin and clothing in public health applications, to repel biting arthropods such as
mosquitoes, flies and ticks (ref. 1).
Identity
INCI name: ethyl butylacetylaminopropionate
Synonyms: IR3535®, AI 3-70763, EBAAP,
ethyl 3-(N-butylacetamido)propionate, Merck 3535, OMS 3065
Chemical names
IUPAC: 3-(N-acetyl-N-butyl)aminopropionic acid ethyl ester
CA: beta-alanine, N-acetyl-N-butyl-, ethyl ester
CAS No: 52304-36-6
CIPAC number
667
Structural formula:
O

OC2 H 5
N

Molecular formula: C11H21NO3

Relative molecular mass: 215,3
Identity tests: GC (relative retention time), IR, refractive index

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Physico-chemical properties of pure ethyl butylacetylaminopropionate

(Table 1)
(1)
Parameter Value(s) and conditions Purity % Method reference
Vapour pressure: 0.15 Pa at 20°C not known OECD no.104; EEC
A.4; EPA D § 63-9
Melting point and Melting point: below -20 °C not reported Merck method, no
temperature of Decomposition temperature of reference given
decomposition: TC: 141°C
Solubility in water: 70 ± 3 g/l at 20°C not reported OECD no.105; EEC
non-buffered A.6; EPA D § 63-8
Solubility in organic n-heptane, >1000 g/l not reported EPA D § 63-8
solvents dichloromethane, >1000 g/l
ethyl acetate, >1000 g/l
p-xylene, >1000 g/l
acetone, >1000 g/l
methanol, 865 g/l
all at room temperature
Octanol / water log POW = 1.7 at 23°C not known OECD no.117; EEC
partition coefficient: non-buffered A.8; EPA D § 63-11
Hydrolysis In 3 months, 5% w/w solutions in not reported Not reported.
characteristics: unbuffered water (pH 4.7) Measurements made
showed <0.2% degradation in a by HPLC with external
refrigerator (+2 to +6°C), about standardisation.
10% degradation at room
temperature (20-25°C), and
about 80% degradation at 40°C.
Hydrolysis is acid catalysed.
Photolysis No UV absorption > 250 nm - -
characteristics:
Dissociation None. 5% solution in non- - -
characteristics: buffered water was of pH 4.7
Flash point 159°C not known DIN EN 22719; EEC
A.9; EPA D § 63-15
Boiling point Estimated metastable boiling not known EPA D § 63-6; EEC
point, slightly below 300°C A.2
Mass per millilitre 998 g/l at 20 ± 0.5°C not known EEC A.3; OECD 109;
EPA D § 63-7
(1)
DIN= Deutsche Industrie-Norm, a standard of the Deutsche Institut für Normung; EEC= a
standard of the European Economic Community; EN= a standard of the Comité Europeén de
Normalisation; OECD=a standard of the Organization for Economic Cooperation and
Development; and EPA=a standard of the [United States] Environmental Protection Agency.

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Chemical composition and properties of ethyl butylacetylaminopropionate

technical material (TC) (Table 2)
Manufacturing process, maximum limits for
impurities ≥ 1 g/kg, 5 batch analysis data.
Declared minimum [a.i.] content:
Relevant impurities ≥ 1 g/kg and maximum
limits for them:
Relevant impurities < 1 g/kg and maximum
limits for them:
Stabilisers or other additives and maximum
limits for them:
Melting or boiling temperature range
Confidential information supplied and held on file by
WHO. Mass balances were 99.6-101.5% and
percentages of unknowns were 0.16-0.21% (ref. 2).
980 g/kg
None
None
None
Estimated metastable boiling point, slightly below
300°C. Not stable above about 141°C.

Toxicological summaries
Notes.
(i) The proposer confirmed that the toxicological and ecotoxicological data included in the
summary below were derived from ethyl butylacetylaminopropionate having impurity profiles
similar to those referred to in the table above.
(ii) The conclusions expressed in the summary below are those of the proposer, unless otherwise
specified.

Table 3. Toxicology profile of ethyl butylacetylaminopropionate technical

material, based on acute toxicity, irritation and sensitization.
Species Test Duration and conditions Result
or guideline adopted
Wistar rats (5 males oral 87/176/EEC LD50 > 5000 mg/kg bw
and 5 females)
Sprague-Dawley rats (5 oral Undiluted test material LD50 = 14.0 ml/kg
males and 5 females applied at doses of 7.9,
per dose group) 10.0, 12.6, 15.9 and
20.0 ml/kg
Wistar rats (6 males oral Undiluted test material LD50 = 24 ml/kg
per dose level) was applied by stomach
tube; dosing intervals
between 20-30 ml/kg
were 2 ml/kg.
Mongrel dogs (1 male oral Undiluted material by All animals survived;
and 1 female per dose gavage at dose levels doses of 2 g/kg bw or
group) of 1, 2, 4 and 8 g/kg bw more induced vomiting
followed by 10 ml water after 30 to 60 minutes
followed by salivation
after 20 to 30 minutes.
Sprague-Dawley rats (5 dermal Undiluted test material Up to a dose of 10.00
males and 5 females was applied to the ml/kg bw no systemic
per dose level) shaven dorsal skin reactions were
(6-hour exposure; observed; erythemas at

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recovery period 14 all dose levels, one rat

days); the dose levels had an erythema of
were 6.35, 7.9 and grade 2.
10.00 ml/kg.*
NMRI mice (5 males dermal Undiluted test material Up to a dose of 10.00
and 5 females per dose was applied to the ml/kg bw no systemic
level) shaven dorsal skin reactions were
(6-hour exposure; observed; erythemas
recovery period 14 were observed at all
days); the dose levels dose levels.
were 6.35, 7.9 and
10.00 ml/kg.*
Beagle dogs (1 male dermal Undiluted test material Up to a dose of 10.00
and 1 female per dose was applied to the ml/kg bw no systemic
level) shaven dorsal skin reactions were
(6-hour exposure; observed; local
recovery period 14 erythemas were
days); the dose levels observed at all dose
were 6.35, 7.9 and levels.
10.00 ml/kg.*
Rat (5 males and 5 inhalation EPA Guideline No. 81- LC50 > 5.1 mg/L
females) 3; OECD Guideline 403
New Zealand White skin irritation A 10% solution (in 50% No reactions of local or
rabbits (3 males and 3 aqueous ethanol) was systemic intolerance.
females) applied to the shaven
intact and scarified
dorsal skin; exposure
over 24 hours and 14
days recovery period.
Human volunteers (30) skin irritation Closed epicutaneous No skin reactions were
irritation test using a observed.
solution of 15% a.i. in
aqueous alcohol.
Human volunteers (10) skin irritation Repetitive exposure No toxic or allergic
test using a solution of reactions of the skin.
15% a.i. in aqueous
alcohol; 3 weeks/ twice
a week (induction
phase); 12 days break;
then application for a
seventh time
(challenge).
Himalayan white phototoxic A 10 % solution (+ 2% No erythema and
spotted albino guinea potential DMSO) in ethanol oedema were observed
pigs (10) applied to shaven at the UV-exposed and
flanks unexposed sites.
Himalayan white photoallergenic A 10% solution in No erythema and
spotted albino guinea potential ethanol applied to oedema were observed
pigs (10) shaven nuchal area. at the UV-exposed and
unexposed sites.
New Zealand White photochemical 25% solution in 95% No photochemical
rabbits (6) skin study (aqueous) ethanol were reactions were
applied to intact skin observed.
and exposed to UV
light.
Rabbits (males and eye irritation EPA Guideline No. 81-4 Undiluted ethyl
females) Purity 98.9% (GC butylacetylamino-
assay). propionate is classified
as irritant to the eye.
Rabbit (5) eye irritation 10% solution in olive oil Erythema and swelling

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Purity of a.i. not were observed which

recorded. completely receded
after 24 and 72 hours
respectively.
Rabbits (two groups eye irritation Single treatment with Intolerance reactions
of 3) undiluted test material. were observed which
In one group the eyes receded faster in the
were rinsed in the other group where the eyes
group the eyes were were rinsed.
not rinsed.
Purity of a.i. not
recorded.
Albino rabbits (2 groups eye irritation 15% aqueous alcoholic Intolerance reactions
of 4 animals each) solution. In one group were observed which
the eyes were rinsed in receded faster in the
the other group the group where the eyes
eyes were not rinsed. were rinsed.
Purity of a.i. not
recorded.
Albino rabbits (6) eye irritation 20% liquid in isopropyl After 48 hours complete
palmitate. recession of the mild
Purity of a.i. not irritations.
recorded.
New Zealand White eye irritation 0.1 ml technical grade Mild injury to cornea
rabbits (6) ethyl butylacetylamino- and conjunctiva.
propionate was applied
to one eye of each
rabbit.
Purity of a.i. not
recorded.
Hartley guinea pigs skin sensitisation EPA Guideline No. 81-6 No sensitization
(male and female, total (Buehler technique) potential.
of 43)
Hartley guinea pigs (10) skin sensitization
Intra-dermal injection of No sensitization
0.1 ml of a 0.1 % potential.
suspension in
propylene glycol/saline
* The proposer confirmed that the dosage levels for dermal exposure of mice, rats and dogs
were identical on a body weight basis.

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Table 4. Toxicology profile of ethyl butylacetylaminopropionate technical

material based on repeated administration (subacute to chronic)
Species Test Duration and conditions Result
or guideline adopted
Sprague- 4-week oral feeding 900 and 2700 mg/kg bw LOEL assumed to be
Dawley rats study daily. >2700 mg/kg bw
(15 males and
15 females
per dose
group)
Beagle dogs 4-week oral feeding 100 and 1800 mg/kg bw Lowest toxic dose
(3 males and study daily by gavage in 1% assumed to be
3 females per aqueous methyl- >1800 mg/kg bw, although
dose group) hydroxyethyl cellulose some test material may
gel. have been lost by vomiting
at this dose. Vomiting
started 30 min after dosing
and occurred on 2-7
days/week.
New Zealand 4-week oral feeding 500 and 1500 mg/kg bw Lowest toxic dose
White rabbits study daily by gavage in 1% assumed to be between
(3 males and aqueous carboxy- 500 and 1500 mg/kg bw
3 females per ethylcellulose gel. per day. 1500 mg/kg led
dose group) to deeper breathing and
unrest for a short period of
time after dosing. Food
consumption and body
weight gain were
significantly reduced. No
other effects were
observed.
New Zealand 2-week oral feeding EPA Guideline No. 83-3 Slight inhibition of food
White rabbits study consumption and body
weight gain were observed
at a dose level of 600
mg/kg/day.
New Zealand 4-week dermal 1/10 of body surface LOEL assumed to be
White rabbits application treated with 3.33% >33.33% concentration .
(3 males and solution, or 10.00 and
3 females per 33.33%
dose group) solution/suspensions, in
1% aqueous
methylhydroxyethyl-
cellulose gel.
Wistar rats 90-day sub-chronic 100, 1000, and 3000 NOEL = 3000 mg/kg/day
(10 males and dermal study mg/kg, daily, by
10 females occlusive dermal
per dose application in a W/O
group ) cream over 13 weeks
Wistar rats 2-generation toxicity EPA guideline No. 83-4; NOAEL = 300 mg/kg/day
(25 males and study 100, 300 and 1000 LOAEL = 1000 mg/kg/day.
25 females) mg/kg daily in water by
gavage.

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Species Test Duration and conditions Result

or guideline adopted
Sprague- Embryo-foetal toxicity 1800 mg/kg bw by Lowest toxic dose for
th
Dawley rats – oral (pilot study) gavage daily, from 6 to dams assumed to be
th
(20 + 20 as 15 day of pregnancy, in about 1800 mg/kg bw and
control) 1% aqueous methyl for foetuses above that
hydroxyethyl cellulose dose. There were no
gel. indications of
teratogenicity.
New Zealand Embryo-foetal toxicity 1500 mg/kg bw by Lowest toxic dose for
th
White rabbits – oral (pilot study) gavage daily, from 6 to dams assumed to be
th
(10 + 10 as the 18 day of slightly below 1500 mg/kg
control) pregnancy, in 1% bw and for foetuses above
aqueous methyl that dose. There were no
hydroxyethyl cellulose indications of
gel. teratogenicity.
New Zealand Dose-range finding 50, 100, 300, 600 and No test-related deaths or
White rabbits developmental toxicity 1000 mg/kg per day, in abortions at any dose
(6 per dose study 1% aqueous carboxy level. No external
group) methylcellulose gel, by malformations or
gavage in single daily developmental variations
doses from days 7-19 of observed in foetuses.
gestation. Body weight gain and food
consumption were
inhibited at 600 and 1000
mg/kg. No other effects
were observed.
New Zealand Developmental 100, 300 and 600 The NOAEL for maternal
White rabbits toxicity study mg/kg/day. toxicity was considered to
(20 per dose EPA Guideline No. 83-3 be 300 mg/kg/day.
group) The NOAEL for
developmental toxicity was
considered to be 600
mg/kg/day.
Himalayan Developmental 100, 300 and 1000 The high and mid dose
rabbits (15 toxicity study mg/kg per day in water level produced significant
+15 as by gavage in a single maternal toxicity and were
control) daily dose from days 6 to embryotoxic. With respect
19 of gestation. to the marginal findings
attributable to 0.1 ml/kg,
this dose is considered to
be a LOEL.

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Table 5. Mutagenicity profile of ethyl butylacetylaminopropionate technical

material, based on in vitro and in vivo tests
Species Test Conditions
Bacteria Ames test (in vitro) 150, 300, 600, 1200,
2500 and 5000 µg/plate
using Salmonella
typhimurium TA 100, TA
98, TA 1535, TA 1537,
TA 1538 and Escherichia
coli WP2 uvrA.
Bacteria Ames test (in vitro) EPA Guidelines No. 84-1,
84-2; 5-5000 µg/plate
tested using Salmonella
typhimurium TA 98, TA
100, TA 102, TA 1535,
TA 1537and Escherichia
coli WP2 uvrA pkM101.
CHO cells Chromosomal EPA Guideline No. 84-2;
aberration test (in vitro) cultures of CHO cells
treated with 250, 500,
750, 1000, 1500, 2000,
2500 and 3000 µg/ml for
approx. 28 h without
metabolic activation and
500, 750, 1000, 1500,
2000, 2500, 3000 4000
and 5000 µg/ml for 3 h
with metabolic activation.
CHO cells HGPRT test (in vitro) 0 – 4.2 µl/ml without
metabolic activation and
0 – 8.0 µl/ml with
metabolic activation were
tested.
V 79 cells HGPRT test (in vitro) EPA Guideline No. 84-2;
Various concentrations
up to 5000 µg/ml tested.
Mouse (5 Micronucleus test (in EPA Guideline No. 84-2;
males and 5 vivo) dose levels of 475, 950
females) and 1900 mg/kg were
tested.
Result
No mutagenic activity
with and without addition
of S-9 as the
metabolizing system.
No mutagenic activity
with and without addition
of S-9 as the
metabolizing system.
No chromosomal
aberrations in CHO cells
without metabolic
activation. With
metabolic activation the
test was positive at the
two highest dose levels
but these doses also
produced cytotoxic
effects.
No mutations were
induced at the HGPRT
locus in CHO cells.
No mutations observed
in the presence or
absence of an
exogenous metabolizing
system.
Up to 1900 mg/kg – a
dose equivalent to
approx. 73 % of the LD50
- no micronuclei in the
polychromatic
erythrocytes were
induced.

No data were available on the ecotoxicology profile of ethyl butylacetylamino-

propionate. The Proposer noted that national authorities do not request such
data, because this active ingredient is unlikely to pollute the environment by
direct use.
WHO/PCS has proposed Class “unlikely to present acute hazard in normal use“
for ethyl butylacetylaminopropionate, pending final approval of this classification

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in 2003. The compound has been assessed according to EU Directives and

allocated the following risk phrases:
Xi Irritant
R 36 Irritating to eyes.
S 26 In case of contact with eyes, rinse immediately with plenty of water
and seek medical advice.

Formulations
The main formulation types are lotions, creams, milks, gels, sprays, roll-ons and
powders.

Methods of analysis and testing

The analytical method for identification and determination of ethyl
butylacetylaminopropionate in the TC is based on gas chromatography (GC)
using a flame ionization detector (FID). The content of ethyl
butylacetylaminopropionate is determined from the proportion of its peak area to
the total area of peaks integrated in the chromatogram (referred to by the
proposer as the “100%” method).
This method cannot be used for the analysis of formulations but a suitable
method for this purpose (Merck 1/11887) was presented. It is based upon
reversed-phase HPLC, UV absorption detection at 220 nm and external
standardisation.
Another method, based on GC-FID but involving internal standardization with
methyl undecanoate, was also used by the proposer for the determination of both
the active ingredient and the impurity profiles but the performance of this method
was not satisfactory in a peer validation study.
The material accountability study was performed according to the US
Environmental Protection Agency’s, Pesticide Assessment Guidelines,
Subdivision D; Series 62.
Alternative methods for identification of the active ingredient are by means of the
IR spectrum or refractive index.

Physical properties
Test methods used to determine the physical properties of technical active
ingredient were OECD, EPA, EU and DIN.

Containers and packaging

The active ingredient and its formulations should be stored in high density
polyethylene containers.

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Expression of the active ingredient

The active ingredient is expressed as ethyl butylacetylaminopropionate.

Appraisal
Ethyl butylacetylaminopropionate is an active ingredient that is not under patent
and has not previously been the subject of WHO specifications. Its INCI
common name is less convenient for general use than the proprietary name
®
IR3535 . The repellent has been evaluated by WHOPES for efficacy (ref. 1) but
reports on efficacy were not used for the present evaluation.
It is moderately volatile, moderately soluble in water, very soluble in a wide range
of organic solvents but it is not classed as fat soluble. It has no acidic or basic
characteristics. A 5% aqueous solution had a pH of 4.7 but it is not clear whether
this was due to dissolved CO2 or to acidic impurities. Ethyl
butylacetylaminopropionate does not absorb UV light at wavelengths >250 nm
and therefore photolytic degradation should not occur. Degradation by hydrolysis
of a 5% solution in non-buffered water (pH 4.7), over a 3-month period was too
low to measure in a refrigerator, rising to 10% at room temperature and 80% at
40°C. The data indicate that hydrolysis is acid catalysed. Although hydrolysis is
slow, even at 40°C, the clause restricting the pH range in the TC is therefore
justified.
Confidential information on the manufacturing process, and the impurities
present at or above 1 g/kg, was provided by the proposer. Mass balances were
high, in the range 996-1015 g/kg, and the proportion of unknowns was very low,
about 2 g/kg.
The Proposer initially suggested that 4 impurities (2 by-products of synthesis,
water and lead (Pb)) should be regarded as relevant impurities. At the levels at
which they were proposed for control, there was no evidence to show that these
impurities would confer any adverse toxicological, ecotoxicological, product
stability, efficacy or taint/odour properties to the TC or formulations. There are
no specific toxicological and ecotoxicological data available for the 2 by-products
of synthesis identified. The minimum purity of the TC is high (980 g/kg) and the
WHO/PCS opinion confirmed that there was no evidence to suggest that the
process impurities are more toxic than the active ingredient Thus no relevant
impurities are specified.
Data provided on the toxicity of ethyl butylacetylaminopropionate indicate that the
only demonstrated hazards relate to eye and skin irritation. The associated risks
were consequently evaluated by WHO/PCS. The skin irritation observed in
animal experiments was mild and had not been observed in humans. As eye
exposure can be prevented, the irritation risks were considered acceptable.
No long-term toxicity or carcinogenicity studies were reported. The WHO/PCS
considered that the consistently negative findings in genotoxicity testing, together
with the apparently innocuous chemical structure, make it unlikely that ethyl
butylacetylaminopropionate is carcinogenic to humans. Nonetheless,
considering the direct and potentially long-term dermal exposure resulting from

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intended use of the chemical, WHO/PCS indicated that it would be advisable for
the Proposer to conduct a long-term carcinogenicity assay in rodents.
The overall WHO/PCS Secretariat view, supported by registration acceptance
decisions in the USA, Australia and elsewhere, was that the intended use of ethyl
butylacetylaminopropionate as an insect repellent does not pose undue risks to
the users.
Physical and toxicological test methods followed internationally recognised
protocols.
No data were provided on the ecotoxicity of ethyl butylacetylaminopropionate.
The Proposer stated that the active ingredient is unlikely to pose risks to the
environment in normal use. The WHO/PCS concurred with this view, as the
intended use is unlikely to lead to release of the chemical in the environment.
The analytical method for determination of the active ingredient in the TC (the
“100%” method”) is not published and has not been validated by collaborative
study. Its accuracy for the analysis of the TC could be doubtful in some cases
because, for example, water or non-volatile impurities are not detected and
therefore are not included in the 100% measured. Although these impurities are
not considered relevant according to the usual definition, they would require
specification clauses and limits if the “100%” method became the referee method
supporting a full specification. Although the data provided showed that the
“100%” method is very precise, a method is required which is inherently more
amenable to assessment of accuracy.
WHO full specifications must be supported by methods proven to give
comparable results in different laboratories, to avoid disputes over the results.
However, WHO interim specifications may be supported by methods which have
not been validated to the same extent. Therefore, although the “100%” method
is lacking inter-laboratory validation and its accuracy will be in doubt if significant
concentrations of water or non-volatile impurities occur in the TC, it is considered
adequate for support of an interim specification.
Details of the analytical method are provided in Note 1 of the TC specification.
To ensure that users of the interim specification are aware of the limitations of
the method, they are identified in the “Outline of method” section of that Note.
The primary test for identity is based on GC relative retention time and is
satisfactory for the purpose. A supporting identity test, based on IR, is
acceptable for the technical grade active ingredient, and it may be acceptable for
formulations following extraction of the active ingredient. A third test for identity
is measurement of refractive index, which may be of restricted value for the
characterisation of formulations.

Recommendations

The draft specification for ethyl butylacetylaminopropionate TC, attached, should

be adopted as a WHO interim specification.

A collaboratively studied, robust and accurate method for determination of the

active ingredient content must be provided before the interim specification is
upgraded to WHO full specification status.

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IR 3535 Interim specification WHO/IS/TC/667/2001
TECHNICAL MATERIAL

The Proposer should consider undertaking a long-term carcinogenicity study, to

support the PCS preliminary conclusion that ethyl butylacetylaminopropionate is
not a carcinogen.

References
1. World Health Organization (2001). Report of the fourth WHOPES Working
Group meeting. Geneva, WHO document WHO/CDS/WHOPES/2001.2
2. Brekelmans M. J. C. (1997). Determination of the purity and impurities of
insect repellent 3535 (TGAI). Notox BV confidential report to Merck KGaA on
project 183746.

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