Bis(4-(1,2-bis(ethoxycarbonyl)ethylamino)-3-methylcyclohexyl)methane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study with deviations: This study was originally planned as a one-generation study and later enhanced to a two-generation study. As deviation to the guidelines of a two-generation study histopathology of F1 parental rats was done only in liver and kidneys.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

(2001)

Deviations:

yes

Remarks:

, histopathology of F1 parental rats was done only in liver and kidneys.

Principles of method if other than guideline:

This study was originally planned as a one-generation study and later enhanced to a two-generation study. As deviation to the guidelines of a two-generation study histopathology of F1 parental rats was done only in liver and kidneys.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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:

1,4-diethyl 2-{[4-({4-[(1,4-diethoxy-1,4-dioxobutan-2-yl)amino]-3-methylcyclohexyl}methyl)-2-methylcyclohexyl]amino}butanedioate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: SPF-bred rats of the strain Wistar Crl: (Wi)WU BR
- Source: Charles River GmbH, Sulzfeld, Germany
- Age at study initiation: Animal age at delivery (F0): 4-5 weeks
- Weight at study initiation: (F0) Males: 110 (86-128) g; Females: 94 (79-113) g; the scatter of the animal weights (except one male) was less than 20% around the mean for each sex.
- Housing: singly, except when co-housed for matings or with litters in Makrolon cages Type IIIh. The animal room was accommodated within a special building domain, separated from other areas by a barrier system. The air pressure in the animal room was about 20 Pa above the normal pressure.
- Diet and water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2°C
- Humidity (%): 55 +/- 5%
- Air changes (per hr): >/= 10 passages per hour
- Photoperiod (hrs dark / hrs light): 12 hours rythm

IN-LIFE DATES: From: July 28, 2008 To: May 4, 2009 (281 days)

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
PREPARATION of Formulation(s). as needed, mostly once weekly

VEHICLE
Polyethylene glycol 400
- Justification for use and choice of vehicle (if other than water): The formulation was a clear liquid and and stability of it had been confirmed.

Details on mating procedure:

In general, the first adult F0 male was caged over night with the first F0 female from the same test group, whereas the third F1 female was mated with the first male of the corresponding dose group, and so on. Mating occurred at a maximum of 12 times during the three-week mating period until sperm cells in vaginal smears taken the next morning or a vaginal plug were observed. Inseminated females were not further co-housed with the below mentioned exception. Generally, if a male had to be necropsied prior to co-housing the previous or next male was co-housed with two females.
Rematings:
Remating with the same male
F0 and/or F1 females found sperm-positive after the first mating day but not shown to be pregnant (lack of weight gain within 14 days following insemination) were co-housed again over one week with the same male without checking insemination or food intake during possible further gestation. According to our experience occurrence of sperms after the first co-housing day without a following gestation can happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the estrus. Two pregnant F0 females of the control group were remated because a gestation could not be established by sperm positive vaginal smear, a vaginal plug or body weight gain.
Remating with another (fertile) male
F0 females, which presented no vaginal plug or sperm cells in the vaginal smears from the males appointed or did not show body weight gain indicating pregnancy, were co-housed additionally for two weeks with another male of the same group, which had been shown to be fertile. These additional co-housings were done to investigate the cause of lack of pregnancy of these females.
Three pregnant females [one of the 40 mg/kg group and two of the 200 mg/kg group] were remated with other males because a gestation could not be established by sperm positive vaginal smear, a vaginal plug or body weight gain.
One female of the control group and 3 females of the high dose group were mated with fertile males of the same dose group.
Remating occurred at a maximum of 11 times during the two-week remating period until sperm cells in vaginal smears taken the next morning or a vaginal plug were observed. Vaginal smears were also used for measurement of estrus cycling. Inseminated females were not further co-housed. Afterwards the females were kept individually until littering. During gestation clinical observations were made, body weights and food intake were measured as described for regular mating. During lactation food weight was taken on day 0 and 4 p.p. from one of three dams, body weights and clinical observations daily until necropsy from all dams. Body weights and clinical observations of pups were taken on day 0 and 4 p.p. The litters were nursed at least up to day 4 p.p. and then necropsied together with the dams.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For analytical verification a liquid chromatographic method was developed (HPLC on reversed phase (C18) with MSD-detection (MSD; SIM-Ions = 292.5 and 583.5). Content checks revealed that the formulations used were properly performed (checked at begin and near termination of the study and four times in between).

Duration of treatment / exposure:

Total duration of in-Life Phase: 281 days (day 1 = first day of administration). The test substance or vehicle was administered to the animals from the first day of the study (F0) or from Day 29 after birth (F1) until spontaneous death, moribund sacrifice or until scheduled necropsy

Frequency of treatment:

once daily

Details on study schedule:

The F0 animals were pretreated with the test substance for about 10 weeks up to the cohabitation period. Within the weeks 8-10 of the premating period examinations on estrus cycle were performed. Then the mating period of 3 weeks followed.
After a gestation period of about 22 days litters were born and the dams were allowed to rear them. If necessary, four days after birth the F1 litters were reduced (=culled) to eight pups. If possible, four male and four female pups remained per litter. Pups found in a moribund state on Day 4 were excluded from lactation. This was done to investigate possible malformations and to prevent cannibalism during the further rearing period. The remaining F1 pups were reared to an age of four weeks and then necropsied when not used as weanlings for further treatment. F0 females were killed and necropsied when about 28 days old F1 pups had been weaned. F0 males were killed after the mating period partly in the course of spermatological investigation.
25 male and 25 female F1 rats per group were selected for further treatment and to breed the F2 generation. This was done by randomly selecting one male and one female as far as possible from each litter. The weaned F1 offspring was treated further with the substance for at least 10 weeks premating period and then co-housed for mating. During the last three weeks of the premating period estrus cycle determinations were performed. The procedures during the mating, gestation and lactation period of F1 rats were the same as described for F0 rats. The F1 parental animals were killed as scheduled after their F2 litters had been weaned at about Day 28 p.p. as described for F0 rats.
Weaning of Pups: All pups were weaned on Day 28 p.p. The necropsy of weanlings and dams could be postponed up to three days later in case that Day 28 was a Saturday, Sunday or public holiday. However, selection of weanlings for the next generation was done exactly on Day 28 p.p.
Selection of Weaned F1 Pups for Further Treatment: One F1 male and one F1 female per litter were selected to appoint them to the study groups for further treatment. This was done by random selection (prepared on a HP 3000 computer system using a random-number generator) of one male and one female as far as possible from each litter. The selected F1 male out of the first litter of a group was appointed as the first F1 male of the dose group where it came from. The selected F1 female of the same litter was appointed as the first F1 female within its dose group. And so on. In cases where this procedure was not possible (severe clinical findings, missing pups or if pup weight differed extremely from the group mean) another pup or pups of other litters were chosen.
Selection Procedure for F1 co-Housings: As far as possible the first F1 male was co-housed group internally with a female taken from a litter with a different number. Using this procedure sibling matings among the F1 generation were excluded.

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on the results of a subacute gavage study in rats (21908, 1992; see chapter 7.5.1).
- As Pilot Study for this Two-Generation Study an Orientating Subacute Toxicity Study in Wistar Rats was conducted, with the test substance administrated via diet for 4 weeks (Bayer Health Care/Bayer Schering Pharma, AT 05474, study No. T4079062, Eiben, 2009-09-03). In this study duodenum and jejunum of male and female animals receiving 10000 or 15000 ppm test substance (corresponding to actual doses of 1176 and 1827 mg/kg bw in males and 1583 and 2319 mg/kg bw in females, respectively) exhibited a changed content (white mucous) at necropsy. Histopathology of the duodenum and jejunum revealed a certain change in villus epithelium (marked vacuolation) in all dose groups (beginning at 5000 ppm), indicating impaired resorption, which was presumably the reason for the increase in food and water intake without changes in body weight gain within this study. As such alteration was not seen in a previous study, where the test substance was administered by gavage (cf chapter 7.5.1 report no. 2190A, 2009) the effect on the intestine was considered to be most probably promoted by or related to administrationof the test substance in the diet. Therefore, the observations are not regarded to be directly related to test substance toxicity but promoted by the administration type and no NOAEL for administration of the test subtsance via diet could be established.

Examinations

Parental animals: Observations and examinations:

Observations on F0/F1 Parental Animals: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were done twice daily (once daily on weekends and public holidays). Clinical findings were recorded individually daily. General clinical examinations were made daily about 30-60 minutes after the last animal was administered (cage-side observation). All signs of illness or clinical reactions to treatment (especially during littering) were recorded daily. All further clinical symptoms were also recorded. This investigation included the observation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. Any further findings e.g. prolonged parturition were recorded as well.
A detailed clinical examination was done prior to the first administration of the test substance and then weekly as a rule, except females, which were investigated daily starting at mating period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded directly prior to the first administration and thereafter daily including the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The individual food consumption was measured (by weighing the quantity of food provided and back-weighing the amount, which remained unconsumed) as follows: - Males: Weekly from week 1 up to necropsy (except during mating period).
- Females: Weekly from week 1 up to mating (During gestation Day 0-7, 7-14 and 14-20 p.c., During lactation Day 0-4 and 4-7 p.p.)

WATER CONSUMPTION: No data

OTHER: Determination of Insemination
During the mating periods vaginal smears were taken in the morning after rats had been co-housed over night to determine time to insemination and gestation length. The vaginal smears were obtained using a flame-sterilized platinum loop and plated out on slides. Smears were stained for about 1 minute with MAY GRÜNWALD´S Eosine-methylene blue solution modified for microscopy.
The date on which a vaginal plug was noted or sperms were found microscopically was taken as gestation Day 0 for calculating the gestation length. Females which exhibited marked weight gains although insemination had not been established were not further co-housed. No duration of gestation could be determined for these animals.

Oestrous cyclicity (parental animals):

Estrus cycle length determination was done by evaluation of vaginal smears received daily over 19 consecutive days about 3 weeks prior to the mating period and during remating with a fertile male until the vaginal smear was sperm positive or a vaginal plug was found. The smears were examined microscopically for large serrated cells indicating that estrus had occurred. These data were used to determine the estrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigation was performed in all surviving F0 and F1 males of the 0 and 1000 mg/kg group on the day of necropsy.
- Determination of spermatozoa motility and viability: Just after testes and epididymides had been dissected sperm samples were collected from the right cauda epididymis and suspended in HAM`s F10 tissue culture medium (38°C). A sample of about 20 µl of the culture medium was placed on a 38°C warmed and siliconated slide and then covered with a warmed cover glass. Then sperm motility was recorded on 100 spermatozoa during minute 1 and minute 5 after preparation of the sample using semi-dark field microscope. The microscope stage was warmed up at 37°C. Spermatozoa showing a forward swimming movement were given a positive score.
Furthermore, the difference between motility recorded during the first minute and that measured during the fifth minute was calculated.
- Determination of spermatozoa morphology: Spermatozoa morphology was evaluated in a formalin citrate fixed and Eosin G stained sperm sample collected from the right cauda epididymis. Morphological changes of the head, upper and middle tail were evaluated on 200 spermatozoa using the microscope as mentioned above.
- Determination of spermatozoa in epididymis: Determination of spermatozoa density was performed in a suspension (0.9% NaCl) of minced cauda epididymis tissue by counting of spermatozoa in a hemocytometer and calculation of spermatozoa density per mg epididymis. The samples used for counting had been heated shortly. Each sample was counted in 5 tertiary squares of two secondary squares. All 10 values were averaged and taken for calculating spermatozoa density per mg epididymis.
- Determination of homogenization resistant spermatid heads in the testis: Determination of spermatid head density was performed in a suspension (0.9% NaCl plus Triton x-100) of homogenized testis tissue by counting of spermatid heads in a hemocytometer. Each sample was counted in 5 tertiary squares of two secondary squares. All 10 values were averaged and taken for calculating spermatid head density per mg testis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED:
Number of live pups, Pup weight, External alterations (=clinical and necropsy observation), Number of dead pups, Sex of each pup (M/F).
Developmental Milestones in F1 Weanlings were recorded: In all F1 weanlings selected for further treatment the age and body weight when balano-preputial separation or vaginal opening occurred were recorded. This was done by investigating daily the preputium (from Day 38 p.p. onwards) and vagina (from Day 28 p.p. onwards) up to the time point when the balano-preputial separation or vaginal opening occurred. Time of vaginal opening and balano-preputial separation of F1 weanlings was recorded together with actual body weight. Absolute and body weight related data were calculated per group.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
For males and non pregnant females: When no longer needed for matings, for females with pups: At weaning of at least 28 days old pups.

GROSS NECROPSY
Macroscopical investigation was done on all rats. Animals were subjected to detailed post-mortem examinations as follows. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Implantation sites were counted after staining the uterus with 10% aqueous ammonium sulfide. In cases where implausible discrepancies existed between the number of observed implantation sites and the number of pups delivered (more pups than implantation sites) the number of pups was used as number of implantation sites. In case of autolytic rats or rats killed pre-scheduled the number of pups was taken as the number of implantation sites.
Vaginal smears were performed and evaluated at scheduled necropsy as precaution but not reported.
Rats found dead or which had to be killed in moribund condition were necropsied as early as possible in the same way.
The following tissues of adult F0 and F1 rats were generally fixed in 10% formalin with the exception of ovaries, the left and two thirds of the right kidney and testes, which were fixed in Davidson’s solution: Abnormalities (if any), Adrenals, Brain, Epididymides unilateral, Esophagus, Kidneys, Liver, Ovaries and oviducts, Pituitary gland, Prostate, Physical identifier, Seminal vesicles and coagulating glands, Skin in mammary region, Spleen, Testes unilateral, Thyroid/Parathyroids, Trachea, Uterus (incl. cervix), Vagina, Head with scull cap.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Adrenals, Brain, Epididymides unilateral, Kidneys, Liver, Ovaries and oviducts, Pituitary gland, Prostate, Seminal vesicles and coagulating glands, Spleen, Testes unilateral, Thyroid/Parathyroids, Uterus (incl. cervix).
The following organs were evaluated histopathologically (F0) at least in control and high dose animals: Adrenals, Epididymides unilateral, Esophagus, Kidneys, Liver, Ovaries and oviducts, Pituitary gland, Prostate, Seminal vesicles and coagulating glands, Testes unilateral, Thyroid/Parathyroids, Trachea, Uterus (incl. cervix), Vagina. Kidneys were evaluated in all dose groups.
The following organs were evaluated histopathologically (F1) at least in control and high dose animals: Kidneys, Liver. Kidneys were evaluated in all dose groups.

Postmortem examinations (offspring):

SACRIFICE
All F1 weanlings not selected for further treatment were killed when they were at least 4 weeks old and examined macroscopically.
All F2 weanlings were killed after a 4 week lactation period and examined macroscopically.

GROSS NECROPSY
All pups culled for standardization of litter size, found dead or which had to be killed moribund during lactation were necropsied and investigated macroscopically with particular attention to the organs of reproduction except for cases of autolysis or cannibalism.
This included also visible skeletal abnormalities as far as possible.
A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth (live birth) or not.
Grossly abnormal tissues if any were fixed in all pups/weanlings. In F1 and F2 weanlings brain, spleen, thymus and uterus of one male and one female out of the firstly necropsied 5 litters per group were fixed in 10% formalin.

ORGAN WEIGTHS
The brain, spleen, thymus and uterus of one male and one female per F1/F2 litter were trimmed and weighed as soon as possible after dissection. The ratio of organ weights to body weights was calculated. Therefore, all these weanlings were weighed at day of necropsy.

Statistics:

Statistical evaluation was performed on an Alpha 800 5/500 computer (TASC-system) using the following methods:
a) Analysis of Variance (ANOVA) and in case of significant results Dunnett’s test as post hoc test for:
• Body weights and body weight gains of the male and female animals
• Food consumption of the male and female animals
• Number of implantation sites per female
• Number of viable pups per female
• Organ weights at necropsy
• Number of estruses
• Time to insemination
• Life birth, viability and lactation rate
• Determination of estrous length
b) 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for:
• Number of viable pups per group based on the number of implantations
• Insemination, fertility, gestation and rearing rate
c) Kruskall-Wallis test and in case of significant differences Dunnett's test for:
• Number of prenatal loss per litter
The sperm and spermatid count data were not evaluated statistically, because no meaningful differences occurred between the high dose and control groups.
Generally, differences between the control group and groups treated with the test substance groups were considered as statistically significant when p ≤ 0.05. Significant differences from the control are indicated with * for p ≤ 0.05 and ** for p ≤ 0.01.

Reproductive indices:

The following reproductive indices were calculated: Insemination index (%), Fertility index (%), Gestation index (%), Rearing index (%).

Offspring viability indices:

The following viability indices were calculated from lactation records of litters in the study (index calculation from litter means): Live birth index (%), Viability index (%), Lactation index (%).

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no changes in survival and clinical appearance up to 1000 mg/kg in parental rats.
In both generation rats died sporadically in each dose group (dose groups 0-40-200-1000 mg/kg: F0 males 0-0-1-2 and females 1-0-1-2; F1 males 0-0-0-1 and females 0-1-0-0). No test substance-specific death causes including signs of administration error were seen in these rats. According to our experience during long term administration normally some rats die because of administration errors, if animals defend themselves or when rats try to regurgitate administration formulation.
Therefore, survival of parental rats was not influenced by the treatment with the test substance up to 1000 mg/kg in F0 and F1 rats.
No toxicologically relevant changes in clinical appearance and behavior of parental rats were observed during daily in-cage or detailed clinical observations up to 1000 mg/kg (F0 and F1 in both sexes).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At the end of the premating periods no adverse effect on body weights and body weight gain was noted up to 200 mg/kg in F0 males and up to 1000 mg/kg in F0 females and F1 rats. At 1000 mg/kg F0 males exhibited statistically significant lower body weights and body weight gain than corresponding control rats.
The food consumption was comparable with that of controls up to 1000 mg/kg in both sexes and generations.
No toxicologically relevant body weight depression and changes in food consumption were noted in pregnant or lactating F0 or F1 females up to 1000 mg/kg.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Results from the evaluation of vaginal smears performed at the end of the premating periods indicate that there were no toxicologically relevant effects on the estrus cycle up to 1000 mg/kg neither in F0 nor in F1 rats.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No biologically relevant effects on sperm parameters (epididymal sperm counts, sperm motility and morphology and testicular spermatids counts) were detected neither in F0 nor in F1 rats at 1000 mg/kg.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Concerning the indices for the insemination, fertility and gestation as well as the mating behavior (co-housings until sperm positive findings) no relevant difference between treated and control rats occurred. The gestation length was not changed by the treatment up to 1000 mg/kg in either generation. At 1000 mg/kg a relatively low rearing index was calculated for F0 rats, which was outside the reference range of historical controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 rats: Organ weight measurements revealed increased (pAbsolute liver weights were not dose-dependently changed in F0 rats, whereas relative liver weights were statistically significantly increased in F0 rats at 1000 mg/kg (8%). The statistical significance of absolute liver weights in 40 and 1000 mg/kg F0 females, which were above the control value, is considered to be incidental as a dose correlation is missing.
The mean relative brain weight of 1000 mg/kg F0 males was statistically significantly higher than at 0 mg/kg due to the lower body weight in this group.
Statistically significantly reduced means for the weights of adrenals at 40 and 200 mg/kg in F0 males do not reflect a toxic effect, because a dose-dependence is missing. The absolute (by 16%) and relative (by 17%) adrenal weights of F0 females were increased (pF1 rats: There was a dose-dependent and partly statistically significant increase in absolute and/or relative kidney weights beginning at 40 mg/kg in F1 males and females up to 10%.
Absolute and relative liver weights were not dose-dependently changed up to 200 mg/kg but increased in 1000 mg/kg F1 males (13%).
The relative adrenal weights were statistically significantly increased in F1 females at 1000 mg/kg (9%). Decreased absolute and relative adrenal weights of males at 40 and 200 mg/kg, marked as statistically significant, do not reflect a toxic effect, because a dose-dependence is missing.
Other statistical significancies among absolute or relative weights are considered incidental, because a clear dose correlation is missing (left epididymides of F1 males at 40 mg/kg) or the statistical significance is based on relatively wide spreading of absolute individual values without correlation to relative weights (pituitary of F1 females).

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy no macroscopical findings due to the treatment were observed up to 1000 mg/kg in F0 and F1 rats.

HISTOPATHOLOGY (PARENTAL ANIMALS)
F0 generation: In males of the F0 generation basophilic tubules in the kidneys were slightly increased by severity and focal tubular dilation/casts were slightly more frequent at 1000 mg/kg, which is considered as an adverse effect.
In F0 females a slight tubular change of the inner renal cortex was observed mainly at 1000 mg/kg. The morphology of this finding did not indicate any adverse impact and might, therefore, result from functional changes.
The remaining organs investigated were morphologically inconspicuous. Reproduction organs of the few F0 breeding pairs lacking offspring did not demonstrate any test substance-related changes.
F1 generation: The kidneys and liver of treated F1 rats showed no treatment-related alterations.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: General toxicity: Mild kidney effects (basophilic tubules, focal tubular dilation/hyaline casts) in F0 male rats combined with elevated absolute and/or relative kidney weights in F0 males and females and F1 rats at next higher dose group (1000 mg/kg)

Remarks on result:

other: Generation: P (F0 and F1) (migrated information)

2

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Reproduction toxicity: no changes in reproductive performance up to the highest dose tested (1000 mg/kg)

Remarks on result:

other: Generation: P (F0 and F1) (migrated information)

3

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Developmental toxicity: no indications for developmental toxicity up to the highest dose tested (1000 mg/kg)

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The live birth and viability indices, the mean litter size at birth, the percentage of males born, the number of born pups and the number of implantation sites including prenatal loss were not changed toxicologically relevantly up to 1000 mg/kg in both generations.
The lactation index of F1 pups was slightly but statistically significantly (p
CLINICAL SIGNS (OFFSPRING)
Clinical observations on pups revealed no remarkable increase in clinical symptoms up to 1000 mg/kg in F1 and F2 pups. Some incidences of F1 and F2 pups being thin, relatively small and/or lacking milk spots were comparable with historical controls.

BODY WEIGHT (OFFSPRING)
Offspring body or litter weights were not toxicologically relevantly reduced up to 1000 mg/kg in F1 generation and up to 40 mg/kg in F2 generation. At 200 and 1000 mg/kg body weights and weight gain of F2 pups were statistically significantly lower from day 14 p.p. onwards, if compared with concurrent controls.
However, because the mean weights of 200 mg/kg pups were mostly not remarkably different from the means calculated for 0 mg/kg F1 pups at the same age, a test substance-related effect on the reproduction and pup development is unlikely. This interpretation was supported by the fact that the appearance of pups was inconspicuous up to 1000 mg/kg and F2 litter weights were roughly comparable with those of F1 and/or F2 controls up to 200 mg/kg.
At 1000 mg/kg F2 pup/litter weights were reduced test substance-relatedly.

SEXUAL MATURATION (OFFSPRING)
The mean age of vaginal opening was unchanged in F1 females up to 1000 mg/kg and that of balano-preputial separation was unchanged in F1 males up to 1000 mg/kg.

ORGAN WEIGHTS (OFFSPRING)
No statistically significant changes in absolute or relative organ weights were observed in male and female F1 or F2 weanlings up to 200 mg/kg.
There were slightly but partly statistically significantly increased relative brain weights in F1 and F2 female weanlings at 1000 mg/kg. These deviations are considered most likely to differences in body weights.
Slightly but statistically significantly (p
GROSS PATHOLOGY (OFFSPRING)
No remarkable changes in incidences of macroscopical findings were found at pup or weanling necropsies up to 1000 mg/kg.

HISTOPATHOLOGY (OFFSPRING)
Not performed, cause no findings were apparent at gross pathology.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

A two-generation reproduction study (OECD 416) was performed with Aspartic acid, N,N'-[methylenebis(2 -methyl-4,1 -cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester administered to 25 male and 25 females F0 and F1 rats at doses of 0 (vehicle control; polyethylene glycol), 40, 200 and 1000 mg/kg bw via gavage. Parental F0 animals received the test substance for a period of about 10 weeks and a subsequent mating period of up to 3 weeks. F1 offspring were nursed up to an age of 4 weeks, Some of them were selected for further treatment and for breeding a F2 generation. Generally, the test substance was administered to each parental animal up to its necropsy.

The study was originally planned as a one-generation study and later enhanced to a two-generation study, therefore as deviation to the guideline histopathology of F1 parental rats was done only in liver and kidneys.

There was no change in survival and clinical appearance up to 1000 mg/kg in parental rats. At 1000 mg/kg body weights and body weight gain were decreased in F0 males during the premating phase. The food intake was not influenced up to 1000 mg/kg in F0 and F1 rats.

Kidney histopathology revealed an increase in severity of basophilic tubules and in frequency of focal tubular dilation/casts at 1000 mg/kg in F0 males considered as adverse. Slight non-adverse tubular change of inner renal cortex was noted slightly more frequent in 1000 mg/kg F0 females. These findings correlated with elevated absolute and/or relative kidney weights, which were seen in 1000 mg/kg F0 males and females as well as in 1000 mg/kg F1 rats, where, however, kidney histopathology was inconspicuous. Taken together, there were signs of kidney damage in 1000 mg/kg F0 males and indications of changes in kidney function in 1000 mg/kg F0 females and F1 rats based on increase of kidney weights and/or tubular change. Necropsy and histopathology of the remaining organs revealed no treatment-related changes.

Investigations of estrus cycling and sperm analyses revealed no substance-related effects.The parameters of the reproductive performance such as insemination, fertility and gestation indices as well as gestation length and the number of litters born were not influenced by the test substance up to 1000 mg/kg.

The litter data such as mean number of implantations, prenatal loss, live birth index, sex distribution, numbers of pups born and litter sizes at birth were not affected by the test substance at levels of up to 1000 mg/kg. The rearing and lactation indices were reduced in F0 generation at 1000 mg/kg. As these changes were minor and not seen in F1 females, a reprotoxic effect is not assumed. The viability index was not test substance-relatedly reduced up to 1000 mg/kg.There were no clinical and necropsy findings in F1 and F2 pups up to 1000 mg/kg. In F1 pups body and litter weights were unaffected up to 1000 mg/kg. In F2 pups body and litter weights were test substance-related depressed at 1000 mg/kg. Concerning the marginal lower pup weights of 200 mg/kg F2 pups a test substance-related effect is unlikely. The occurrence of sexual maturation in F1 post-weanlings revealed no test substance-related effect up to 1000 mg/kg.

Overall in this study there was no indication of effects on reproductive performance up to 1000 mg/kg. Test substance-related effects on offspring were limited to a decrease in F2 pup weight gain starting at lactation day 14 without any other obvious effect on F2 pups at 1000 mg/kg.

Thus, under the conditions described, the NOAEL is established at 200 mg/kg bw for kidney histopathology in F0 males and indications of changes in kidney function in F0 females and F1 males/ females.