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Administrative data

Endpoint:
developmental toxicity
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:
2012
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF guidelines: Guideline on the Compiling of Test Results on Toxicity “Teratology Study”, 12-Nousan No. 8147 of November 24, 2000, amended June 26, 2001
GLP compliance:
yes (incl. QA statement)
Limit test:
no

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
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 12 - 19 weeks
- Weight at study initiation: males 397 - 586 g, females 225 - 276 g
- Housing: Starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings.
- Diet and water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approximately 55 %
- Air changes (per hr): > 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
polyethylene glycol
Details on exposure:
Administration volume: 5 mL/kg bw

VEHICLE
Polyethylene glycol 400
- Justification for use and choice of vehicle (if other than water): The vehicle was suitable as analytical investigations revealed that the test substance was stable in the vehicle for at least 8 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the homogeneity tests and content checks in samples with concentrations of 20, 60 (content checks, only), and 200 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value, except for the 20 mg/kg sample of August 23, 2011, which was therefore not used for administration.
Details on mating procedure:
The animals were mated by placing one female overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
Day 6 - 20 p. c.
Frequency of treatment:
once daily (between 06:00 and 12:00 CET)
Duration of test:
From experimental starting to end of in-life-Phase 22 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a previous pilot prenatal developmental toxicity study in rats with the test substance (T2081689).

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected once daily from day 0 to 21 p.c. (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the females were determined on day 0 p.c. and daily from days 6 to 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

FOOD AND WATER CONSUMPTION: Yes
- The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.
Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- The females were subjected to gross pathological evaluation at cesarean section on day 21 p.c. Necropsy was performed without knowledge of treatment groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Necropsies/cesarean sections were performed on day 21 p.c. without knowledge of treatment groups.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead and live fetuses: Yes
- Other: individual weight and appearance of the placentas
Fetal examinations:
- Sex of live fetuses
- Individual weights of live fetuses
- External examinations: Yes, findings in alive and dead fetuses are included
- Soft tissue and head examinations: Yes, evaluation of about half of alive fetuses per litter
- Skeletal and cartilage abnormalities: Yes, evaluation in about half of alive fetuses per litter
Statistics:
Differences between the control and test item treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01.
Statistical evaluation was performed on an Alpha 800 5/500 computer using the following methods:
- Analysis of Variance (ANOVA); in case of significance Dunnett's test for feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female.
- 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for fertility rate, gestation rate, number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group.
- Kruskal-Wallis test and in case of significant differences Dunn's test for number of preimplantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.
- CHI2 test (correction according to yates) for number of fetuses or litters with cartilaginous tissue observations.
Indices:
gestation rate

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Salivation after administration occurred in all dose groups, which is most likely related to the bad taste of the test item and considered as not adverse. One female of the 100 mg/kg group revealed piloerection for a single day, for which a treatment related effect is excluded due to a lacking dose dependency and because of the single occurrence.
Thus, appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to 1000 mg/kg.

No treatment related gross pathological findings occurred at dose levels up to 1000 mg/kg.

Fertility rate (percentage of inseminated females with implantations), the mean numbers of corpora lutea, preimplantation losses, and implantation sites in the dose groups did not differ to a meaningful extent from the control group values indicating a homogeneous distribution regarding these parameters.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by treatment at dose levels up to 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to 1000 mg/kg.

The overall incidences of fetuses or litters with malformations lay within the range of historical control data, revealed no statistical significance, and were thus unaffected by treatment at dose levels up to 1000 mg/kg. The types of malformations observed in this study are generally a representative sample of spontaneous malformations in the rat strain used (e.g. microphthalmia, dilation of lateral brain ventricles missing thyroid gland, ventricular septal defect of the heart, vertebral malformations with pelvis shift, malformation of ribs, supernumerary vertebrae) and comparable with spontaneous findings in the current control group and/or historical control groups. Malformations of the eyes (eye ball reduced in size) occurred in all dose groups and the control group in comparable incidences of affected fetuses and litters without a dose dependency and were thus unaffected by treatment at dose levels up to 1000 mg/kg. At the 1000 mg/kg level, one fetus revealed a hairline ventricular septal defect of the heart, for which a treatment related effect is not assumed due to its single occurrence, and since this finding lay well within the range of historical control data of the rat strain used. Furthermore, each one fetus of the 1000 mg/kg and 300 mg/kg groups showed a multiple malformation of cervical vertebrae with/without fusion of ribs, which is comparable with spontaneous findings in the rat strain used. Therefore, a treatment related effect is not assumed for these findings. All remaining findings were isolated findings, revealed no dose dependency and were thus also considered as incidental. Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to 1000 mg/kg.

The overall incidences of fetuses or litters with external and visceral deviations were unaffected at dose levels up to 1000 mg/kg and revealed the highest value on a fetal basis in the control group. The external and visceral deviations observed in this study were of a common type and comparable with spontaneous findings in the current control group and/or historical control groups and represented the normal range of scattering in the rat strain used. Histopathological evaluation of additional circumscribed hard whitish tissue in the nasopharynx was performed in a prenatal developmental toxicity study with the same rat strain (T0076746) and revealed calcium concrements without connection to the underlying tissue in the affected localizations. Calcium might have been dissolved from the fetal bones by the Wilson fixative and precipitated in the nasopharyngeal duct so that these findings were regarded as artifacts. Thus, a treatment related effect on external and visceral deviations was not evident at dose levels up to 1000 mg/kg.

At the 1000 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly progressed ossification of a few localizations (1st, 4th-5th distal phalanges of toes left, 2nd-3rd 3rd distal phalanges of toes bilateral), when calculation was done on a fetal basis and partially also on a litter basis, for which a treatment related effect is not assumed, since this is caused by the incidentally higher incidence of retarded ossification in the current control group. At the 300 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly progressed ossification of several localizations ((2nd, 4th-5th proximal phalanges of digits bilateral, 3rd proximal phalanges of digits left, 4thdistal phalanges of toes left, 4th proximal phalanges of toes left), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, since this is caused by the incidentally higher incidence of retarded ossification in the current control group. At the 100 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly progressed ossification of two localizations (1st distal phalanges of toes left, 7th caudal vertebral bodies, parietal bones bilateral), when calculation was done on a fetal basis and litter basis (1st distal phalanges of toes, only), for which a treatment related effect is not assumed, since this is caused by the incidentally higher incidence of retarded ossification in the current control group, and because a dose dependency was lacking. Evaluation of fetal cartilaginous tissue revealed no treatment related findings at dose levels up to 1000 mg/kg. Thus, a treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to 1000 mg/kg.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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 responsible for the notification of both substances under the NONS regulation. They 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.

Study key results in tabular form:

General Reproduction Data:

Dose (mg/kg b.w./day)

0

100

300

1000

inseminated females

25

25

25

25

inseminated females evaluated

25

25

25

25

females with implantations

23

22

22

22

in % of those inseminated

92.0

88.0

88.0

88.0

mean values per female with implantation sites

corpora lutea

14.4

15.0

15.8

14.5

preimplantation loss

0.9

0.9

2.0

1.1

implantations

13.5

14.2

13.8

13.4

A treatment related effect for the slightly increased preimplantation loss at the 300 mg/kg level is not assumed due to a lacking dose dependency, and because this value lay within the range of historical control data of the rat strain used.

Gestation Rate:

Dose

Females with

 

viable fetuses on day 21 p.c.

total resorption

mg/kg b.w./day

n

in % of females with implantations

n

0

23

100.0

0

100

22

100.0

0

300

22

100.0

0

1000

22

100.0

0

Mean Values of the Prameters of Intrauterine Development:

Dose (mg/kg b.w./day)

0

100

300

1000

number of females

with implantations (a)

23

22

22

22

with viable fetuses (b)

23

22

22

22

mean values per female

placental weight in gb

0.60

0.58

0.61

0.62

number of fetusesb

12.6

13.1

13.2

12.9

postimplantation lossa, b

1.0, 1.0

1.1, 1.1

0.6, 0.6

0.5, 0.5

males in %b

49.9

46.9

53.8

44.4

fetal weight in gb

4.92

4.94

4.90

4.90

Fetal Malformations:

Malformation

Dose (mg/kg b.w./day)

 

0

100

300

1000

eyeball reduced in size right

-

-

1

-

dilation of lateral brain ventricles bilateral

-

-

1

-

thyroid gland missing

-

1

-

-

hairline ventricular septal defect of the heart

-

-

-

1

multiple malformation of thoracic vertebrae and ribs

-

1

-

-

multiple malformation of cervical vertebrae with/without fusion of ribs

-

-

1

1

head of 1strib missing right

1

-

-

-

1stsacral vertebral arch has the shape of a lumbar vertebral arch right, pelvis shift to caudal right

1

-

-

-

one supernumerary lumbar vertebra

-

-

1

-

number of fetuses per group

289

288

290

283

number of fetuses with malformations

2

2

4

2

malformed fetuses per group (%)

0.7

0.7

1.4

0.7

number of litters per group

23

22

22

22

number of litters with malformations

2

2

4

2

malformed litters per group (%)

8.7

9.1

13.6

9.1

() number of litters affected

Fetal External and Visceral Deviations:

Deviation

Dose (mg/kg b.w./day)

 

0

100

300

1000

circumscribed hard whitish area at nasal cavities

3 (3)

2 (1)

2 (2)

-

slight dilation of lateral brain ventricle(s)

-

-

2 (2)

-

thyroid gland reduced in size

1

1

1

-

thymus extended cranially

10 (8)

9 (6)

9 (7)

7 (6)

thoracic cavity filled with brown mass

-

-

-

1

abdominal cavity filled with brown mass

9 (8)

7 (5)

5 (4)

4 (3)

brown spot(s) in the liver

12 (8)

6 (6)

12 (9)

4 (4)

slight dilation of renal pelvis

4 (4)

5 (5)

2 (1)

5 (5)

dilation of renal pelvis

-

1

-

-

slight dilation of ureter

-

3 (3)

2 (1)

1

dilation of ureter

-

-

-

1

testi(e)s lying on bladder

4 (4)

3 (3)

3 (2)

3 (2)

testi(e)s lying slightly more cranially

3 (3)

1

6 (6)

5 (5)

brown spot(s) at the throat under the skin

1

-

-

-

number of fetuses per group

289

288

290

283

number of fetuses with deviations

36

28

35

27

fetuses with deviat. per group (%)

12.5

9.7

12.1

9.5

number of litters per group

23

22

22

22

number of litters with deviations

15

16

19

13

litters with deviat. per group (%)

65.2

72.7

86.4

59.1

() number of litters affected

Applicant's summary and conclusion

Executive summary:

A developmental toxicity study according to OECD TG 414 was performed with the structural analogue Aspartic acid, N,N'-[methylenebis(2 -methyl-4,1 -cyclohexanediyl)]bis-,1,1',4,4'-tetraethyl ester. Twenty-five inseminated female Wistar rats were treated daily orally by gavage with doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw from day 6 to day 20 p.c. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to 1000 mg/kg bw.

No treatment related gross pathological findings occurred at dose levels up to 1000 mg/kg.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by treatment at dose levels up to 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to 1000 mg/kg.

Summarizing and evaluating all data investigated the NOAELs were determined to be 1000 mg/kg bw for maternal toxicity and 1000 mg/kg bw for developmental toxicity.

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