2-Ethylhexyl trans-4-methoxycinnamate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restrictions. Restriction: documentation describes clearly methodology and results on the vapour phase, but is less clear regarding particles. Due to the low volatility of 2-Ethylhexyl 4-methoxycinnamate, particle analysis is most important. Though not easy to find, particle results are contained in the report, which is considered to be valid.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 4-methoxycinnamate

EC Number:

226-775-7

EC Name:

2-ethylhexyl 4-methoxycinnamate

Cas Number:

83834-59-7

Molecular formula:

C18H26O3

IUPAC Name:

2-​Propenoic acid, 3-​(4-​methoxyphenyl)​-​, 2-​ethylhexyl ester, (2E)​-

Details on test material:

- Name of test material (as cited in study report): HR 92/660 523 (Neo Heliopan, Typ AV)
- Purity: 99.2%
- Lot/batch No.: 2040059
- Expiration date of the lot/batch:
- Stability under test conditions: given; descrbed in a separate study report (BAYER AG; Rüngeler, 1992)
- Physical state: liquid

- Analytical purity: not reported
- Composition of test material, percentage of components:
The composition of two different samples tested were as follows:

Sample I:
HR 92/660 523: 5.0%
Ethanol: 45.0%
Ethane: 0.2%
Propane: 8.4%
iso-butane: 7.2%
n-butane: 34.2%

Sample II:
HR 92/660 523: 2.0%
Ethanol: 18.0%
Ethane: 0.32%
Propane: 13.4.4%
iso-butane: 11.52%
n-butane: 54.72%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen, Germany
- Age at study initiation: 2 to 3 months
- Weight at study initiation: 160 to 220 g
- Fasting: no
- Housing: singly in macrolon cages type II
- Diet: rodent diet "Altromin 1324" ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40 - 60 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a spray tube containing the test material was placed into a water bath (20 °C). Every 10.5 seconds test material was sprayed for 0.5 seconds into a mixing chamber where it was diluted with air (20 L/min). The resulting atmospherer was guided to the inhalation chamber (stainless steel, volume 50 L). The animals were kept in plexi glass tubes with the head protruding into the inhalation chamber. Electronic measurement and regulation devices allowed to adjust the exposure concentration.
- Exposure chamber volume: 50 L
- Method of holding animals in test chamber: plexi glass tubes allowing head-nose inhalation
- Source and rate of air: air was compressed to 8 - 10 bar and cleaned from water, dust and oil. At reduced pressure, the air was guided into the mixing chamber at a rate of 20 L/min
- Method of conditioning air: water, dust and oil were removed
- System of generating particulates/aerosols: the apparatus is described above.
- Method of particle size determination:
1) TSI Laser-Velocimeter APS 3300 in the breathing zone (results not reported)
2) Impact cascade (BERNER-Kaskadenimpaktor)
- Treatment of exhaust air: the air was guided through an aerosol filters which was incinerated at the end of the exposure period.
- Temperature, humidity, pressure in air chamber: 24.6 °C; mean humidity 15.9 ± 2.7 (control), and 25 - 35 % (test material, samples I and II; cf. report, pages 47 - 49).

TEST ATMOSPHERE
- Brief description of analytical method used:
1) nominal concentration was calculated from the weight of material sprayed into the mixing chamber divided by the air volume
2) vapour: air samples were taken from the breathing zone and analysed for the volatile components (propane, butane) using GC-FID
3) Particles:
a) Particles were absorbed to Florisil in the breathing zone of the animals. After elution with ethanol, 2-Ethylhexyl 4-methoxycinnamate was determined by GC-FID using a calibration curve
b) particle size distribution:
- TSI Laser-Velocimeter APS 3300 in the breathing zone (results not reported, due to technical problems)
- Impact cascade (BERNER-Kaskadenimpaktor). Description of statistical evaluation: report, page 23.

VEHICLE
- Composition of vehicle: ethanol, propellents (propane, butane etc. Cf. report, page 11)
- Concentration of test material in vehicle: 5 % (sample I), and 2 % (sample II)
- Justification of choice of vehicle: low volatility of 2-Ethylhexyl 4-methoxycinnamate

TEST ATMOSPHERE
- Particle size distribution: log normal (report, pages 44 -46)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
sample I: 8.29 ± 3.47 µm (report, page 43)
sample II: 3.78 ± 2.36 µm (report, page 45)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Active ingredient (a.i., report page 40)
Sample I: 497 mg/m³
Sample II: 524 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations: at least daily; weighing: once before treatment, and on days 3, 7 and 14 post treatment
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs; body weight; organ weights; functional tests (reflexes (visual placing, muscle tone, cornea, pupil and pinna reflexes, startle response (sound), touch-escape response, tail-pinch response, righting reflex) and grip strength)

Statistics:

Generally calculation of means ± standard deviation. Several different statistical tests to analyse differences between treatment groups.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC0

Effect level:

0.511 mg/L air

Exp. duration:

4 h

Remarks on result:

other: 2-Ethylhexyl 4-methoxycinnamate

Effect levels 2

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 0.511 mg/L air

Exp. duration:

4 h

Remarks on result:

other: 2-Ethylhexyl 4-methoxycinnamate

Mortality:

There was no mortality (0/20), all rats survived.

Clinical signs:

other:

Body weight:

A slight reduction of body weight gain was seen in treated animals compared to controls, however without gaining a level of statistical significance.

Gross pathology:

There were no changes noted in control an dtreated animals.

Other findings:

- Other observations:
Reflexes and grip strength were not affected in treated animals.

Any other information on results incl. tables

It should be noted that 2-Ethylhexyl 4-methoxycinnamate (the active ingredient )was contained in a spray can, together with solvents and propellents in proportions listed above.

The reported concentrations represent in most instances the vapour concentrations of the volatile components, which were analysed using GC-FID. The resulting propane and butane concentrations were then used to calculate LC50 -values for the two samples, i.e. >41.8 mg/m³ (sample I) and >43.3 mg/m³ (sample II).

The volatility of the active ingredient, 2-Ethylhexyl 4-methoxycinnamate, is however low. This compound is predominantly present in the particle fraction.The particles were adsorbed to Florisil in the breathing zone of the test animals. HR 92/660 523 was then eluted from Florisil with ethanol and quantified by GC-FID, using a calibration curve of HR 92/660 523 (purity: 99.15 %). The concentration of the active ingredient was 497 and 524 mg/m³ in samples I and II, respectively (cf. report page 40). The mean concentration is therefore 511 mg/m³.

It should be noted that particle size distribution was influenced by the solvent composition. Particles with a Mass Median Aerodynimc Diameter (MMAD) of </= 3.0 µgm were considered to be respirable. Based on the results of the particle analysis, the dose of 2-Ethylhexyl 4-methoxycinnamate was 0.104 mg/L with sample 1, and 0.21 mg/L with sample 2.

   

		Sample 1	Sample 2
MMAD ± geometric SD (µm)		8.29 ± 3.47	3.78 ± 2.36
Concentration (mg/m³)		497	524
Respirability (% = 3 µm)	Mass related	21 % (measured)	40 % (measured)
	Number related	100 % (extrapolated)	99 % (extrapolated)

 

Applicant's summary and conclusion

Executive summary:

In an acute inhalation toxicity study (Bayer AG, 1993), groups of young adult Wistar rats (male and female,5/sex) were exposed by inhalation route to HR 92/660 523 (2-Ethylhexyl 4-methoxycinnamate) contained in spray can at 2 and 5 %, respectively, along with considerable amounts of ethanol as solvent, and hydrocarbons as propellents. The rats were exposed for 4 hours to head only at a mean concentration of  0.511 mg 2-Ethylhexyl 4-methoxycinnamate /L. The animals then were observed for 14 days.

 

There were no findings in any test animal regarding mortality, clinical signs, changes in functional tests (reflexes and grip strength), or gross pathology except from a slightly reduced body weight gain in exposed animals, but without gaining a level of statistical significance. Therefore, the LC₀ was 0.511 mg/L in male and female rats.

 

Conclusion:

This study design is regarded to be suitable for testing the acute inhalation toxicity of low volatility compounds like 2-Ethylhexyl 4-methoxycinnamate. The study was conducted similar to OECD Test Guideline No. 403 and under GLP conditions. The acute LC_{0(4 h)} was 0.511 mg/L in male and female Wistar rats.