N,N''-hexane-1,6-diylbis[N'-benzylurea]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental 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: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-hexane-1,6-diylbis[N'-benzylurea]

EC Number:

254-274-3

EC Name:

N,N''-hexane-1,6-diylbis[N'-benzylurea]

Cas Number:

39072-70-3

Molecular formula:

C22H30N4O2

IUPAC Name:

N',N'''-hexane-1,6-diylbis(1-benzylurea)

Details on test material:

- Test Item: N,N“-Hexane-1,6-diylbis[N`-benzylurea]
- BASF Test Item No.: 11/0726-1
- Batch Number: 259-635
- Purity: 98.0 g/100 g determined by 1H NMR spectroscopy using the internal standard method
- Physical state, appearance: White solid
- Homogeneity: The test substance appeared to be homogeneous.
- Storage conditions: At room temperature, no direct sunlight, protect against humidity

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: body weight of all animals within ± 20% of the sex mean.
- Housing:
Pre-mating:
Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating:
Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating:
Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation:
Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal no. 55. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (27 August 2012), according to a validated method (Project 499988, BASF Project 05Y0726/11X510). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 44 (Group 1) 53, 56 (Group 2), 71 and 72 (Group 4) were not dosed during littering.

Frequency of treatment:

once daily

Details on study schedule:

N/A

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the 14-Day dose range finding study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
At least twice daily for mortality. Daily, detailed clinical observations were made in all animals, at least immediately (0-15 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Since no clinical observations were noted in the dose range finding study, observations were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Litter observations:

Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made for all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight, the evening before the scheduled necropsy (with a maximum of 24 hours), but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Females which delivered on Lactation Days 5-7.
The female which failed to deliver (no. 73) on Post-coitum Day 26 (female with evidence of mating)
Female with litter loss (no. 55) within 24 hours.
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females. These numbers were not reported for non-pregnant and non-mated females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve Duodenum, Seminal vesicles, Epididymides[1], Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland)[1], (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes[1], Jejunumm, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

[1]: Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
A peer review on the histopathology data was performed by a second pathologist.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs[*] of all animals of Group 1 and 4 and of male no. 15 and 33 (failed to sire) and female no. 55 (litter loss) and no. 73 (no offspring).
[*] Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate[2], Liver, Seminal vesicles including coagulating glands[2], Ovaries, Thyroid including parathyroid[2],
[2] weighed when fixed for at least 24 hours.
All remaining males:
Epididymides and Testes

Postmortem examinations (offspring):

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation

Offspring viability indices:

For each group, the following calculations were performed: Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One female (no. 55) was found littering on 26 September 2012 (post-coitum Day 22), but no pups were found on 27 September 2012. This animal was therefore sent for unscheduled necropsy due to “total litter loss”. At necropsy, one pup was found left in the uterus.

No toxicologically relevant clinical signs were noted.

Pale faeces noted at 1000 mg/kg bw/day in both sexes from week 1 of treatment onwards, was attributed to the white colour of the test substance, and considered not to be of toxicological relevance.

Salivation seen after dosing for most males at 1000 mg/kg bw/day during the last week of treatment was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.
Salivation was also incidentally noted for one female at 300 mg/kg bw/day. Together with other incidental findings including rales, alopecia, green discolouration of the urine and scabs, these were considered signs of no toxicological relevance. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted.

The statistically significant higher body weight of females at 1000 mg/kg bw/day on Day 8 of the premating period (including statistically significant higher body weight gain) and on most occasions during post-coitum/lactation remained within the range considered normal for rats of this age and strain, and showed no clear dose-related trend over the dose groups. No toxicological relevance was therefore ascribed to these changes.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic staging profiles were normal for all Group 1 and 4 males, and for all males suspected to be infertile.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted.

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.

The statistically significant higher liver weight and liver to body weight ratio of females at 300 mg/kg bw/day occurred in the absence of a dose-related trend and was therefore considered to be of no toxicological relevance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy did not reveal any toxicologically relevant alterations.

The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend, and occurred in the absence of treatment-related histopathological changes. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age.

No histopathological changes were noted in the reproductive organs of two Group 2 animals (male no. 15 and female no. 55) and two Group 4 animals (male no. 33 and female no. 73) that could account for failing to deliver healthy offspring.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

parental, reproduction and developmental

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Generation: P and F1 (migrated information)

Results: F1 generation

Details on results (F1)

MORTALITY
A total of 10 pups of the control group were found dead/missing postnatally, resulting in a statistically significant higher viability index at 100, 300 and 1000 mg/kg bw/day. Among the test substance treated groups, 4 pups at 100 mg/kg bw/day, 3 pups at 300 mg/kg bw/day and no pups at 1000 mg/kg bw/day were found dead or missing during lactation. One female at 100 mg/kg bw/day (no. 55) was found littering on 26 September 2012, but no pups were found on 27 September 2012. At necropsy, one pup was found left in the uterus. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

SEX RATIO
The statistically significant higher sex ratio at 100 mg/kg did not show a dose-related trend and was slight in nature. No toxicological relevance was attributed to this change.

CLINICAL SIGNS
Incidental clinical symptoms of pups consisted of cannibalism, scabs on various body parts, a wound on the abdomen or back, a nodule in the abdominal subcutis, blue discolouration of the whole body, a blue spot on the neck and hypothermia. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHTS
No toxicologically relevant changes in pup body weight were noted.

Body weights of male and female pups were statistically significantly higher than controls on Day 1 and 4 of lactation. Body weight gain over Days 1-4 of lactation was however similar across the groups, and mean body weights remained essentially within the range considered normal for pups of this age. Mean litter size was similar between litters at 1000 mg/kg bw/day and control litters, and hence this difference could not be ascribed to a clear difference in litter size. Given the slight nature of this change, these higher body weights were considered to be of no toxicological relevance.

MACROSCOPY
Incidental macroscopic findings of pups that were found dead included scabbing of the abdomen and/or throat region, absence of milk in the stomach and a nodule in the abdominal subcutis. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Developmental data

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and earlypostnatalpup development (mortality, clinical signs, body weight and macroscopy) were observed.

 

Gestation

Gestation index and duration of gestation were similar across the groups.

 

Parturition/maternal care

One female at 100 mg/kg bw/day (no. 55) was found littering on 26 September 2012, but no pups were found on 27 September 2012. This animal was therefore sent for necropsy due to “total litter loss”. At necropsy, one pup was found left in the uterus, which suggests that parturition was prolonged for this female. The female also has a relatively low number of corpora lutea (5) and implantation sites (2). Given that this was an incidental occurrence, and no signs of difficult or prolonged parturition were noted among the other pregnant females, this was considered not to be of toxicological relevance.Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.No deficiencies in maternal care were observed.

 

Earlypostnatalpup development

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.