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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method

Test material

Constituent 1
Reference substance name:
136210-32-7
EC Number:
603-947-3
Cas Number:
136210-32-7
IUPAC Name:
136210-32-7
Constituent 2
Chemical structure
Reference substance name:
Bis(4-(1,2-bis(ethoxycarbonyl)ethylamino)-3-methylcyclohexyl)methane
EC Number:
412-060-9
EC Name:
Bis(4-(1,2-bis(ethoxycarbonyl)ethylamino)-3-methylcyclohexyl)methane
Cas Number:
136210-32-7
Molecular formula:
C31H54N2O8
IUPAC Name:
bis(4-(1,2-bis(ethoxycarbonyl)ethylamino)-3-methylcyclohexyl)methane

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Strain: Hsd Cpb:WU(SPF)
- Age at study initiation: animals of the weight class used are 2-3 months old
- Housing: singly
- Diet and water: ad libitum
- Acclimation period: at least 5 days


Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure chamber volume: internal volume 3.8 L
- Mode of exposure: directed-flow nose-only exposure
- Generation of atmospheres: Atmospheres of the test substance were generated under dynamic conditions using a Braun-infusion pump and a modified binary nozzle. The test substance was nebulized using conditioned (dry, oil free) compressed air. The respective concentration was achieved by spraying different volumes of liquid per unit of time.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric and analytical. Two methods described for analytical determination:
1. Quantification of the test material: test material adsorbed on Florisil. After complete desorption with n-heptane quantification by gas chromatography using a flame ionization detector.
2. Quantification of n-butyl acetate: test material adsorbed in a N,N-dimethyl formamide solution. After complete desorption aliquots were transfered in capsuled vials. Quantification of the n-butyl acetate via headspace sampling technique by gas chromatography using a flame ionization detector.

VEHICLE:
- No vehicle used; test material contains approx. 10 % n-butyl acetate

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: analysed using a Berner-Type Aeras low-pressure critical orifice cascade impactor.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): approx. 1.5 µm (GSD approx. 1.8)
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
1000, 5000 mg/m³ (target concentrations)
1436, 4224 mg/m³ (analytical concentration)
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: Body weights measured before exposure, on days 3, 7 and weekly thereafter. Appearance and behavior of each rat examined carefully several times on the day of exposure and at least once daily thereafter, except on weekend (once daily)
- Necropsy of survivors performed: yes
- Other examinations performed: examinations of reflexes, rectal temperature (directly after cessation of exposure)
- Control animals: exposed to conditioned air
Statistics:
Calculation of LC50: according to Rosiello et. al. (1977), modified by Pauluhn (1983). Method is based on the maximum-likelihood method of Bliss (1938).

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4 224 mg/m³ air
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
other: NO(A)EL
Effect level:
ca. 1 436 mg/m³ air
Exp. duration:
4 h
Remarks on result:
other: basis for effect level: clinical signs
Mortality:
No mortality was observed after exposure to concentrations up to and including 4224 mg/m³.
Clinical signs:
other: Signs were transient and resolved within the first postexposure week. The major signs observed are as follows: bradypnea, labored and unregular breathing pattern, bristled and ungroomed hair-coat, nostrils reddened, motility reduced, hindlimbs unable to s
Body weight:
Comparison between control animals with those in the groups exposed did reveal some isolated statistically significant effects on body weight gains. However, these changes are not considered to be of any toxicological relevance.
Gross pathology:
A conclusive, concentration-dependent increased incidence of macroscopic findings could not be ascertained.
Other findings:
No effects on reflexes after exposure were seen. Rats exposed to the test substance experienced a concentration-dependent decrease in body temperature.

Any other information on results incl. tables

Study was performed with Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester which is a structural analogue to Aspartic acid, N,N'-(methylenedi-4,1-cyclohexanediyl)bis-, 1,1',4,4'-tetraethyl ester. Both substances are diethyl esters of aspartic acid linked to a dicyclohexylmethyldiamine moiety. The difference between these two substances is merely the presence of two methyl groups connected to the cyclohexane rings. This structural analogy was confirmed by the Member State responsible for the notification of both substances under the NONS regulation. The Member State decided that test results obtained for one substance can be transferred to the other substance and that testing of both substances is usually not required. This decision is in accordance with the grouping of substances and read-across approach in Annex XI, 1.5 of the REACH Regulation.

Applicant's summary and conclusion

Executive summary:

An acute inhalation toxicity study with aspartic acid, N, N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester (containing approx. 10% n-butyl acetate as solvent) was performed by exposing Wistar rats nose only to the aerolized test item according OECD TG 403. In concentration groups of 0, 1436 and 4224 mg/m³ test item (analytical concentrations) in each case 5 males and 5 females were used. After exposure (4 hours) the animals were observed for two weeks. The test substance aerosol exhibited a particle-size indicating that it was of adequate respirability (mass median aerodynamic diameter (MMAD) ≈ 1.5 μm; geometric standard deviation (GSD) ≈ 1.8).

No mortality occured up to and including 4224 mg/m³. Signs (bradypnea, labored and unregular breathing pattern, bristled and ungroomed hair-coat, nostrils reddened, motility reduced, hindlimbs unable to support body weigth, hypothermia) were transient and resolved within the first postexposure week. Rats exposed to 1436 mg/m³ did not experience clinical signs except a mild hypothermia following cessation of exposure. Evidence suggests that the test substance is a mild respiratory tract irritant. The NO(A)EL (4 h) stated in this study to be 1436 mg/m³, and the LC50 was > 4224 mg/m³ for both sexes combined.

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