Hexane, 1,6-diisocyanato, homopolymer, N-butyl-1-butanamine-blocked

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:

1-[6-(3-{6-[3,5-bis({6-[(dibutylcarbamoyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(dibutylcarbamoyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-3,3-dibutylurea; 1-{6-[3,5-bis({6-[(dibutylcarbamoyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,3-dibutylurea; 3,3-dibutyl-1-[6-(3-{6-[(dibutylcarbamoyl)amino]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]urea

EC Number:

924-903-2

Cas Number:

1190401-47-8

Molecular formula:

Unspecified

IUPAC Name:

1-[6-(3-{6-[3,5-bis({6-[(dibutylcarbamoyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(dibutylcarbamoyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-3,3-dibutylurea; 1-{6-[3,5-bis({6-[(dibutylcarbamoyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,3-dibutylurea; 3,3-dibutyl-1-[6-(3-{6-[(dibutylcarbamoyl)amino]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]urea

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Hexane, 1,6-diisocyanato, homopolymer, N-butyl-1-butanamine-blocked
- Test-substance No.: 11/0689-1
- Lot/batch No.: IB 60/0053
- Purity: approx. 100 %
- Homogeneity: The test item was homogeneous by visual inspection
- Storage stability: stable
- Storage conditions: At room temperature in the dark
- Physical state/ colour: Solid / colourless to yellowish

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. The test substance was warmed before weighing (maximum temperature=66.9°C, maximum duration=6 hours) and the formulations were also warmed to facilitate mixing (maximum temperature=69°C, maximum duration=57 minutes). 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. To obtain at least 9 mated females in group 3, two proven (non-selected) group 3 males (nos. 26 and 29) were paired with female nos. 64 and 70 on 31 August 2012.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis.

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% 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 42-56 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).

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. 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
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 5-6 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. 54) on Post-coitum Day 27 (female with evidence of mating)
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. 14 (failed to sire) and female no. 54 (failed to deliver healthy pups).
[*] 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
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 5-6 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. No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included alopecia, scabs on the neck and piloerection. A single female at 300 mg/kg bw/day had a wound and swelling along with dull appearance and opacity of the left eye. 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. At the incidence observed, these were not considered treatment related or toxicologically relevant.

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

Absolute body weights were significantly lower on Day 1 of the mating period and Day 4 of the lactation period for females at 300 and 100 mg/kg bw/day, respectively. In the absence of a treatment related distribution, this was not considered to be toxicologically relevant.

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

Absolute food consumption was significantly lower for females at 100 mg/kg bw/day on Days 11-14 of the post coitum period and Days 1-4 of the lactation period. Food consumption was also significantly lower for females at 300 mg/kg bw/day on Days 17-20 of the post coitum period. As the difference from controls was only slight and occurred in the absence of a treatment related distribution, it was not considered toxicologically relevant.

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. There were 10, 9, 10 and 10 pregnant females in the control and 100, 300 and 1000 mg/kg bw/day groups, respectively.

The 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.

Relative liver weights were significantly lower for males at 1000 mg/kg bw/day. This was not toxicologically relevant as an increase in liver to body weight ratios would be expected if treatment related toxicity was evident. Similarly, the significantly lower terminal body weight seen for females at 100 mg/kg bw/day was not considered toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were attributable to treatment.

All macroscopic findings noted were incidental in nature. These included pelvic dilation of the kidneys, tan, red-brown or reddish discoloration of the thymus and mandibular lymph node, reduced size of the left prostate, several tan foci on the right clitoral gland, thickened limiting ridge of the stomach, enlarged spleen, mesenteric lymph node and/or liver, cloudy left eye, yellowish, hard nodule on the uterine adipose tissue, scab on the back of the neck and alopecia on the throat, flank, or foreleg. These findings remained within the range of findings encountered among rats of this age and strain, and none were attributable to treatment. None of these were considered to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS:
There were no treatment-related microscopic findings. All recorded microscopic findings were within the normal range of background alterations encountered in Wistar (Han) rats of this age and strain.

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility.

Furthermore, the spermatogenic staging profiles were normal for all Group 1 and 4 males, and for all males suspected to be infertile.

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

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There were four pups of the control group and nine and four pups in the 100 and 300 mg/kg bw/day groups, respectively, that were found dead or went missing during the first days of lactation. No pups died or went missing at 1000 mg/kg bw/day. Missing 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.

CLINICAL SIGNS (OFFSPRING)
At 100 mg/kg bw/day, almost all pups from one litter (no. 55) were noted with red spots on the body, blue spots on the paws, and calm during the first few days of lactation. These mostly disappeared by Day 4. Blue spots on the hind paws or hind leg was also noted for a few pups in individual litters from the 300 and 1000 mg/kg bw/day groups (litters 68 and 77, respectively). As these signs did not impact viability of the pups, had a limited occurrence, and were transient in nature, they were not considered to be indicative of treatment related toxicity.

Incidental clinical symptoms of pups consisted of blue spot on the neck, head or back, blue tail apex, missing tail apex, and a wound and scab on the abdomen, pale appearance and cold to the touch. 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 WEIGHT (OFFSPRING)
Pup body weights were significantly lower on Days 1 and 4 at 100 mg/kg bw/day and body weights were significantly lower for male pups at 1000 mg/kg bw/day on Day 4. In the absence of a dose related distribution this was not considered toxicologically relevant.

GROSS PATHOLOGY (OFFSPRING)
No milk in the stomach was noted for 2 control litters (nos. 41 and 50) and 1 litter from 100 mg/kg bw/day (no. 53). This was not considered to be treatment related or toxicologically relevant since it occurred mainly for control litters and these pups survived until the scheduled necropsy, indicating they were all sufficiently fed during the first days of lactation. This finding for the animals surviving to the scheduled necropsy is likely secondary to the time when the actual necropsies were performed, and is not attributable to treatment.

Absence of milk in the stomach was also noted for pups that were found dead; this was incidental. Missing tail apex was the only macroscopic finding seen for surviving pups. The nature and incidence of all of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.

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

The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg bw/day.

 

The significant increase in the duration of gestation seen for females at 100 mg/kg bw/day was attributable to a relatively short gestation period seen for control animals.

 

Parturition/maternal care

No signs of difficult or prolonged parturition were noted among the pregnant females.

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.No deficiencies in maternal care were observed.

 

Earlypostnatalpup development

The 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.