tetraethyl N,N'-(methylenedicyclohexane-4,1-diyl)bis-.sc.dl.sc.-aspartate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose 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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

, no ophthalmological examination

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

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

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:

other: Bor: WISW (Spf Cpb)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approximately 6 weeks for male rats and 7 weeks for female rats
- Weight at study initiation: mean weight 121 g (for males) and 117 g (for females)
- Housing: in groups
- Diet and water: ad libitum
- Acclimation period: at least 5 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

VEHICLE: PEG 400
APPLICATION VOLUME: 2 ml/kg bw

PREPARATION OF DOSING SOLUTIONS: daily, directly before application

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the applied doses was analytically verified by HPLC once during the study.

Duration of treatment / exposure:

29 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

for dose/recovery groups and control group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a pilot study on 3 male and female rats with repeated doses of 1000 mg/kg bw for 7 days no mortality, no clinical signs and no gross pathological lesions could be observed. An acute oral toxicity study on rats revealed no mortality and no symptoms at doses of 2000 mg/kg bw.

- Additional 5 males and females were used as recovery group (dosing of 0 and 1000 mg/kg bw) with a post-exposure recovery period of 2 weeks.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day, except on weekend (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly a detailled examination for each animal was performed

BODY WEIGHT: Yes
- Time schedule for examinations: body weight was determined before the first application and then once weekly until sacrifice of the animals.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes, performed in week 5. For recovery-groups hematology and clinical chemistry was done in week 7.
For determination of Glucose, blood was sampled from non-fasted non-narcotized animals (venae caudales). For all other determinations blood was sampled from non-fasted diethylether-narcotized animals (retro-orbital).
The following hematologic parameters were determined: Leukocyte differential count, Erythocyte count and -morphology, Hämoglobin, Hämatocrit, Leukocyte count, MCH, MCHC, MCV, Thrombocyte count, Thromboplastin time.
The following clinical chemistry parameter were determined: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, blood glucose, bilirubin, cholesterin, creatinine, total protein, urea, triglycerides, albumin, albumin/globulin ratio, determination of inorganic ions.

URINALYSIS: Yes, performed in week 4. For recovery-groups urinanalysis was done in week 6.
- Time schedule for collection of urine: 24 h
The following parameter were determined: blood, glucose, keton bodies, pH, bilirubin, urobilirubin, protein, sediment, density, volume.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organs were weighed: brain, heart, testes (pairwise), liver, spleen (fixed), kidneys (pairwise), adrenal glands (fixed, pairwise), ovaries (fixed, pairwise).

The following organs were fixed: abnormalities, adrenals, aorta, bone marrow (femur, sternum), brain, caecum, colon, rest of gastrointestinal tract, duodenum, epididymis, esophagus, exorbital lacrimal glands, eyes, eyelids, femur with knee-joint, head (rest), harderian glands, heart, hypophysis, ileum, jejunum, kidneys, larynx, liver, lymph nodes (mesenteric and mandibular), mammary glands, nervus ischiadicus, nervus opticus, ovaries/oviducts, pancreas, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle (femoralis), skin, spinal cord (3x) with bone marrow, spleen, sternum, stomach, tatoowed ears, testes, thymus, thyroid with parathyroids, tongue, tooth, trachea, uterus, urethra, ureters, urinary bladder, vagina, zymbal glands.


HISTOPATHOLOGY: Yes
The following organs were histopathologically investigated: heart, liver, spleen, adrenal glands and kidneys of all animals of dose group 0 and 1000 mg/kg bw.

Other examinations:

Additional histopathological investigation of the duodenum and jejunum of control and high dose males was performed in 2009 (reported in "1st Amendment to Report No. 21908 of December 08, 1992") after a dose finding study (4-week feeding study) for a two generation study revealed these organs to be targets.

Statistics:

rank-test of MANN and WHITNEY (1947) and WILCOXON (1945)

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY: No mortality and no clinical signs observed

BODY WEIGHT AND WEIGHT GAIN: No significant findings

FOOD AND WATER UPTAKE: No significant findings

OPHTHALMOSCOPIC EXAMINATION: not performed

HAEMATOLOGY: The hematological investigation at the end of the exposure duration revealed a non dose-dependent but statistically significant increased thrombocyte count in both sexes of the 1000 mg/kg group. At the end of the recovery period the thrombocyte count returned to the level of the control group. The MCHC is significantly increased in female rats of the 1000 mg/kg group, but nevertheless within normal range. Leucocyte count was reduced for male rats of the 1000 mg/kg recovery group.

CLINICAL CHEMISTRY: The activity of the alkaline phosphatase was slightly increased at the end of exposure for males and females of the 1000 mg/kg group. At the end of the recovery period the level returned to the level of the control group.

URINALYSIS: No significant findings

NEUROBEHAVIOUR: No data

ORGAN WEIGHTS: Slightly increased absolute liver weights in both sexes and slightly increased relative liver weights for the male rats were observed, both for the 1000 mg/kg group. At the end of the recovery period liver weights returned to the level of the control group. Other observed significant changes were singly and of no toxicological relevance.

GROSS PATHOLOGY: No significant findings

HISTOPATHOLOGY: No significant findings

HISTOPATHOLOGY of the duodenum and jejunum of control and high dose males performed in 2009 as additional investigation: No test substance-related findings.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: No adverse effects found up to the highest dose tested (1000 mg/kg).

Target system / organ toxicity

Critical effects observed:

not specified

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.

Further remarks on results of the study with Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester:

The results of the hematological and histological investigations gave no indication of substance-related damage to the blood or hematopoietic organs in the dose group up to and including 1000 mg/kg. Thus, there is no correlate for the significantly increased thrombocyte count in males and females of the dose group 1000 mg/kg at the end of the treatment period. No toxicological relevance is attributed to this finding, as the differences are very slight in males, the mean value in females is influenced by only one relatively high value, and almost all values are within the 2s range of historical controls.

The clinical laboratory investigations, necropsies and histological findings furnished no evidence of a change in the functional state or morphology of the liver.

The urea and creatinine concentrations in plasma as well as the results of the urinalysis, necropsies and histopathological investigations gave no indication of treatment-related damage to the kidneys.

The clinical laboratory investigations revealed a slightly (in the case of females significantly) increased activity of the alkaline phosphatase in plasma in males and females of the dose group 1000 mg/kg. As all values are within the normal range these results are most likely of no toxicological relevance.

Therefore the NOAEL was determined to be 1000 mg/kg bw.

Applicant's summary and conclusion

Executive summary:

An oral subacute dose study (OECD TG 407) with aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester was conducted on 5 male and 5 female rats with doses of 0 (vehicle control), 40, 200 and 1000 mg/kg bw per day. Additional 10 animals (5 per sex) were used for further observation in a 2 -weeks post exposure period following treatment with 0 and 1000 mg/kg.

No mortality, no clinical signs and no effects on food-/water-consumption or body weight gain were observed. Hematology and histopathology revealed no conclusive signs of toxicologic relevance on blood and hematopoetic organs.

Neither clinical chemistry nor gross/histopathology revealed signs indicating substance-related metabolic or organ impairment.

Thus, the NOAEL was determined to be 1000 mg/kg bw.