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Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
NOAEL based on subchronic and subacute oral study (OECD TG 408 and 407), where no effects on reproductive organs were observed.
Additional information

No definite study on reproductive toxicity/fertility (OECD TG 416 or 443) is available for the substance. However, histopathological examinations of reproductive tissues in repeated dose rodent-studies are of high value and high sensitivity for evaluation of reproductive toxicity, as confirmed by literature*. Histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility, whereas the absence of such effects give evidence that a substance does not influence fertility. In this respect repeated dose toxicity studies should be considered sensitive and sufficient information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered.

For Aspartic acid, N, N'-(2-methyl-1,5-pentanediyl) bis-, 1,1',4,4'-tetraethyl ester two repeated dose toxicity studies are available, i.e. an OECD TG 407 and 408 study, in which the histological examination of reproductive organs is covered (e.g. the 408 examined Epididymides, Ovaries/Oviducts, Prostate, Seminal vesicles (incl. coagulating glands), Testes, Uterus with Cervix, Vagina; cp. chapter Repeated dose toxicity). In these studies no substance related adverse effects on reproductive organs and tissues or other concerns in relation with reproductive toxicity were found up to the highest dose administered (1000 mg/kg bw), thus it can be concluded that the substance is no reproductive toxicant.

* 1) BAuA Forschungsbericht 984, 2003: Mangelsdorf, I.; Buschmann, J., Extrapolation from results of animal studies to humans for the endpoint male fertility. 1. Auflage. Bremerhaven: Wirtschaftsverlag NW Verlag für neue Wissenschaft GmbH 2003. (Schriftenreihe der Bundesanstalt für Arbeitsschutz und Arbeitsmedizin: Forschungsbericht, Fb984) ISBN: 3-89701-967-1, 156 Seiten, Projektnummer: F 1642, Papier, PDF-Datei; 2) Mangelsdorf, I. et al., Some aspects relating to the evaluation of the effects of chemicals on male fertility, Reg. Tox. Pharm. 37, 2003, 356-36914; 3) Ulbrich, B. & Palmer, A. K., Detection of effects on male reproduction – a literature survey, J Am. College of Toxicology 14, 1995, 293-327; 4) Dent, M. P., Strength and limitations of using repeated-dose toxicity studies to predict effects on fertility, Regulatory Toxicology and Pharmacology 48, 2007, 241-258; 5) Janer, G. et al., A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study, Reproductive Toxicology 24, 2007, 103-113; 6) Sanbuissho, A. et al., Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats, J Tox. Sci. 34, 2009, Special Issue SP1-SP22

 

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
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. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 11 - 17 weeks
- Weight at study initiation: males 434 - 587 g, females 208 - 258 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
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Administration volume: 2 mL/kg bw

VEHICLE
- 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 50, 150 (content checks, only), and 500 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.
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 38 days.
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 (T9081874).
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
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.
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.
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
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. multiple malformations, microphthalmia, situs inversus, retina folded, ventricular septal defect, vertebral malformations with pelvis shift, dysplastic limb bones) 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 and 300 mg/kg levels, one fetus each revealed a cleft palate (at the 300 mg/kg level in combination with a multiple malformation), which is a rare finding, but also known as a spontaneous finding in the rat strain used (for example 2 fetuses out of 1 litter in the unaffected low dose group of a prenatal developmental toxicity study, T7062991, conducted in the same laboratory in 2002). Furthermore, a concurrent study with a structural similar substance (Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester, CAS No. 136210-32-7, T5081691, performed in the same laboratory) showed no comparable finding so that a treatment related effect is not assumed. Furthermore, one fetus of the 1000 mg/kg group showed a finding of the vertebral column (3rd lumbar vertebral arch has the shape of a 4th lumbar vertebral arch bilateral, cartilaginous part of 6th lumbar vertebral arch fused with iliac bone, 1st sacral vertebrae missing, bilateral, pelvis shift to cranial, bilateral), which is comparable with spontaneous findings in the rat strain used. Therefore, a treatment related effect is not assumed for this finding. All remaining findings revealed no dose dependency and were thus 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 and litter 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 retarded ossification of several localizations (3rd distal phalanges of digits left, 3rd proximal phalanges of digits bilateral, 4th proximal phalanges of digits left, 2nd and 5th cervical vertebral bodies, 6th-8th caudal vertebral bodies, parietal bones bilateral), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, as these values were comparable with the control values of the concurrent study with a structural similar substance (Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester, CAS No. 136210-32-7, T5081691, performed in the same laboratory), and since statistical significance was lacking in all but one case, when calculation was done on a litter basis. At the 300 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of a single localization (8th caudal vertebral bodies), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, because statistical significance was lacking, when calculation was done on a litter basis, and due to its single occurrence. At the 100 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of two localization (9th caudal vertebral bodies, parietal bones bilateral), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, because statistical significance was lacking, when calculation was done on a litter basis, and since 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.
Abnormalities:
not specified
Developmental effects observed:
not specified

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

24

21

23

22

in % of those inseminated

96.0

84.0

92.0

88.0

mean values per female with implantation sites

corpora lutea

15.7

15.6

15.4

15.5

preimplantation loss

2.0

1.6

1.5

2.2

implantations

13.6

14.0

13.9

13.4

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

24

100.0

0

100

21

100.0

0

300

23

100.0

0

1000

22

100.0

0

Mean Values of the Parameters of Intrauterine Development:

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

0

100

300

1000

number of females

with implantations (a)

24

21

23

22

with viable fetuses (b)

24

21

23

22

mean values per female

placental weight in gb

0.57

0.59

0.57

0.61

number of fetusesb

13.1

13.4

13.4

12.8

postimplantation lossa, b

0.5, 0.5

0.6, 0.6

0.5, 0.5

0.6, 0.6

males in %b

55.9

50.7

50.4

52.4

fetal weight in gb

4.96

4.92

4.88

4.93

Fetal Malformations:

Malformation

Dose (mg/kg bw/day)

 

0

100

300

1000

umbilical cord shortened, generalized edema, domed head, skull bones partially missing, mandible shortened, ears missing, eye rudiment flat right, limbs reduced in size, abdominal hernia with protruding organs, absent genital tubercle, anal atresia, tail shortened and kinked

-

-

1

-

cleft palate

-

-

-

1

eye ball reduced in size with/without eye rudiment flat

2 (2)

2 (2)

1

1

situs inversus

-

1

-

-

retina folded

-

1

-

-

ventricular septal defect of the heart

-

1

-

-

malformation of 1stsacral vertebral arch with/without cartilage, pelvis shift

-

1

3 (2)

1

3rdlumbar vertebral arch has the shape of a 4thlumbar vertebral arch bilateral, cartilaginous part of 6thlumbar vertebral arch fused with iliac bone, 1stsacral vertebrae missing, bilateral, pelvis shift to cranial, bilateral

-

-

-

1

multiple skeletal malformation of the whole body, dysplasia of forelimbs and hind limbs, nearly completely missing ossification of head and sternebrae, cleft palate

-

-

1

-

number of fetuses per group

314

281

308

281

number of fetuses with malformations

2

6

5

4

malformed fetuses per group (%)

0.6

2.1

1.6

1.4

number of litters per group

24

21

23

22

number of litters with malformations

2

6

4

3

malformed litters per group (%)

8.3

28.6

17.4

13.6

() number of litters affected 

Fetal External and Visceral Deviations

Deviation

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

 

0

100

300

1000

eye rudiment flat

-

1

-

1

retina slightly folded

1

-

1

-

slight dilation of lateral brain ventricle(s)

2 (1)

2 (1)

-

1

slight dilation of 3rdbrain ventricle

-

2 (1)

-

1

thyroid gland reduced in size

2 (1)

4 (4)

3 (3)

4 (4)

thyroid gland enlarged

-

-

2 (2)

-

membranous part of trachea slightly folded, lying in tracheal lumen

1

1

2 (2)

-

thymus extended cranially

14 (10)

13 (9)

11 (7)

13 (8)

origin of left carotid artery displaced towards left subclavian artery on aortic arch

-

1

-

1

innominate artery and left carotid artery arise from aortic arch, left subclavian artery displaced and arises from descending aorta

1

-

-

-

thoracic cavity filled with brown mass

-

-

-

1

pericard filled with brown mass

1

-

-

-

brown mass in abdominal cavity

8 (6)

2 (2)

7 (6)

5 (5)

brown spot(s) in the liver

10 (7)

7 (6)

11 (9)

13 (8)

slight dilation of renal pelvis

10 (6)

7 (3)

6 (5)

7 (5)

dilation of renal pelvis

1

-

-

-

kidney flat and stretched

1

-

-

-

slight dilation of ureters

5 (4)

5 (2)

1

2 (2)

testi(e)s lying on bladder

5 (4)

4 (3)

6 (6)

2 (2)

testi(e)s lying slightly more cranially

6 (5)

1

7 (7)

4 (4)

right testis lying on the left side

-

1

-

-

circumscribed hard whitish area at choane, nasopharynx, larynx, nasal sinus, and/or palate

6 (4)

2 (2)

3 (3)

5 (3)

number of fetuses per group

314

281

308

281

number of fetuses with deviations

52

39

48

41

fetuses with deviat. per group (%)

16.6

13.9

15.6

14.6

number of litters per group

24

21

23

22

number of litters with deviations

23

17

23

19

litters with deviat. per group (%)

95.8

81.0

100.0

86.4

() number of litters affected

Executive summary:

A developmental toxicity study according to OECD TG 414 was performed with the substance. Twenty-five inseminated female Wistar rats received daily doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw by gavage 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 following NOAELs were determined: for maternal toxicity 1000 mg/kg bw, for developmental toxicity 1000 mg/kg bw.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

A developmental toxicity study according to OECD TG 414 was performed with the substance. Twenty-five inseminated female Wistar rats received daily doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw by gavage 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, andskeletal (including cartilage) deviations was not evident at dose levels up to 1000 mg/kg.

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


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available

Justification for classification or non-classification

No classification is required for reproductive toxicity according to Regulation (EC) No 1272/2008, Annex I.