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Description of key information

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
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
Qualifier:
according to guideline
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Strain: BOR: WISW (SPF Cpb)
- Age at study initiation: approximately 8 weeks for males and 10 weeks for females
- Weight at study initiation: mean weight 182 g (for males) and 168 g (for females)
- Housing: in groups
- Diet and water: ad libitum
- Fasting period before study: approximately 16 hours
- Acclimation period: at least 4 days
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
The test substance was dissolved in peanut oil (DAB9) and intragastrally administered via a metal gastric tube.
Application volume: 10 ml/kg bw
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: day 0 several times, day 1 to 14 twice daily, except for the weekend (once daily).
- Frequency of weighing: immediately after application, on day 7 and day 14.
- Necropsy of survivors performed: yes
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
No mortality was observed after the application or during the 14-days post-application period.
Clinical signs:
No clinical signs were observed after the application or in the 14-days post-application period.
Body weight:
There were no effects on the body weight gain.
Gross pathology:
All animals were sacrificed at the end of the 14-days observation period. There were no gross pathological findings.
Other findings:
None

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.

Executive summary:

An oral toxicity study with a limit dose of 2000 mg test item/kg bw was performed with 5 male and 5 female rats according to EU method B.1. No mortality, no clinical signs and no gross pathological findings were observed after administration of the test substance (aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester) during the 14-days post-observation period.

Therefore the LC50 was estimated to be > 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

Acute toxicity: via inhalation route

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


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).
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.

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating conc.
Value:
4 224 mg/m³

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Strain: BOR: WISW (SPF Cpb)
- Age at study initiation: approximately 9 weeks for male rats and 16 weeks for female rats
- Weight at study initiation: mean weight 233 g (for males) and 222 g (for females)
- Housing: singly
- Diet and water: ad libitum
- Acclimation period: at least 5 days
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Approximately 24 h before application of the test substance the skin areas of the animals were carefully shorn.
- % coverage: approximately 10% of the bodies surface
- Type of wrap if used: covererd with gauze plus aluminium foil and fixed with a nonirritating bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: If there was remaining test substance after removal of the bandage, the skin was washed with tempered water.
Duration of exposure:
approximately 24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: day 0 several times, day 1 to 14 twice daily, except for the weekend (once daily)
- Frequency of weighing: immediately after application and on day 7 and day 14.
- Necropsy of survivors performed: yes
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
No mortality was observed after the application or during the 14-days post-application period.
Clinical signs:
2 of 5 males and 3 of 5 females exhibited a slight erythema at the application site. This was fully reversible within 1 day, except for one female, where full reversibility was achieved on day 6. No systemic symptoms were observed.
Body weight:
Body weight gain was retarded in female rats, no effects on body weight gain were observed for male rats.
Gross pathology:
All animals were sacrificed at the end of the 14-days observation period. There were no gross pathological findings.

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.

Executive summary:

A dermal toxicity study with a limit limit dose of 2000 mg test item/kg bw was performed with 5 male and 5 female rats according to EU method B.3. No mortality and no gross pathological findings were observed after application of the test substance (aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester) during the 14-days post-observation period. Some male and female rats exhibited a transient slight erythema after application of the test substance. Body weight gain was retarded in female rats, but the body weight gains of male rats were not affected.

Therefore the dermal LD50 was determined to be > 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

Additional information

The acute oral LD50 of the structural analogue aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester was > 2000 mg/kg bw in rats (Bomhard, Bayer AG, 1990); no mortality, no clinical signs and no gross pathological findings were observed.

The acute dermal LD50 was > 2000 mg/kg bw (Bomhard, Bayer AG, 1992a). No mortality and no gross pathological findings were observed. Some rats exhibited a slight erythema after application of the test substance. Body weight gain was retarded in female rats, but the body weight gains of male rats were not affected.

No mortality occurred in an acute aerosol inhalation toxicity study (OECD TG 403; Pauluhn, Bayer AG, 1998a) up to the highest concentration tested, which was 4224 mg/m³. Signs (bradypnea, labored and unregular breathing pattern, bristled and ungroomed hair-coat, nostrils reddened, motility reduced, hindlimbs unable to support body weight, 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 was 1436 mg/m³, and the LC50 was > 4224 mg/m³.

All studies investigating acute toxicity were 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.


Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – inhalation endpoint
Only one study available

Justification for selection of acute toxicity – dermal endpoint
Only one study available

Justification for classification or non-classification

No classification required according to EU-Directive 67/548/EEC, Annex I.

No classification required according to Regulation (EC) No 1272/2008, Annex VI.

The maximum concentration tested for the structural analogue in the acute inhalation study (4224 mg/m³ air) was tolerated without mortality. No effort was made to meet the limit concentration (5000 mg/m³) of the current OECD-Guideline because exposure of an additional group of animals does not appear that it would have changed the outcome and interpretation of the study at all. Therefore the non-classification of the acute inhalation toxicity is justified.