Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

EC Number:

253-281-9

EC Name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

Cas Number:

36938-15-5

Molecular formula:

C14H30N4O4

IUPAC Name:

2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol

Constituent 2

Reference substance name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Molecular formula:

C14H30N4O4

IUPAC Name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Test material form:

solid: bulk

Details on test material:

- Batch no.: EGGE 2806-1
- Currenta sample no.: 2187

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (15 to 25 °C)
- Stability under test conditions: analytically confirmed (Formulations prepared for use in Weeks 1, 3, and 6 of the study were analyzed to determine achieved concentration.)
- Homogeneity and stability of the test substance in the solvent/vehicle: analytically confirmed (at 0.5 and 200 mg/mL for 7 days at room temperature (Bayer Study T102142-0) and 10 and 100 mg/mL at room temperature after 2 and 8 days (Covance Study Number: 8332715))


FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

Test Guideline is designed for use with the rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 9 to 10 wks; Females 8 to 9 wks
- Weight at study initiation: (P) Males: 338.7-445.5 g; Females: 186.5-244.7 g
- Housing: During the pre pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group housing after the pairing phase.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for at least 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 27 April 2016 To: 29 June 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Kolliphor HS15/ethanol/purified water (40/10/50) (v/v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):a commonly used vehicle for solid test items to reach stable suspension
- Concentration in vehicle: 10 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 10 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared for use in Weeks 1, 3 and 6 of the study were analysed to determine achieved concentration. Three samples were removed from the test item formulations. Two of the samples were analysed with the third to be analysed only if the samples results were outside the target range (90 to 110% of nominal concentration). Two samples were taken from control formulations. One of these samples was analysed

Duration of treatment / exposure:

Males were dosed for 42 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post pairing phase]). Females were dosed for up to 64 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until Lactation Day (LD) 13, inclusive).

Frequency of treatment:

once daily for 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 7 day range-finding study in rats was performed at the selected dose levels and did not elicit any overt toxicity (Covance Study 8332717). For details see chapter 7.5.1

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination once daily from Day 1 of the dosing period until necropsy. These observations were also made on four occasions during the predose period (but this data is not reported). An individual record of the clinical condition of each animal was maintained.

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The amount of food consumed was determined twice weekly prior to pairing (both sexes) and post-paring for males. Daily food consumptions were recorded for females from GD 0 to 20 and LD 1 to 13. Consumption was calculated as g/animal/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
- Postdose Observations: Animals were observed upon return to the home cage and approximately 1 hour postdose. Observations were not recorded from GD 20 to LD 4 (inclusive) for females.

Oestrous cyclicity (parental animals):

Daily vaginal lavage samples were taken from all females during acclimatization (predose), from 1 week after arrival until the day prior to dosing; the stage of estrus was recorded; only females showing a regular 4 to 5 day cycle were included on study. Daily vaginal lavage samples were taken from females from the start of dosing, until the confirmation of mating.

Sperm parameters (parental animals):

Parameters examined in [highest dose group and the control group] male parental animals:
testis weight, epididymis weight

Litter observations:

LITTER SIZE AND SEX DETERMINATION
Litter sizes and offspring sex were recorded on PND 1, 4, 7, and 13. Daily records of mortality and changes in litter sizes were maintained.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter ([5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

- Clinical Observations: Each animal was given a detailed physical examination on days of body weight recording.
- Body Weights: Individual pup body weights were recorded on PND 1, 4, 7, and 13.
- Ano-Genital Distance: Ano-genital distance was recorded for all pups on PND 4.
- Nipple/Areolae Count: The numbers of nipples/ areolae were counted for all male pups on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, where possible

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 43 of the study
- Maternal animals: All surviving animals on LD 14

GROSS NECROPSY
After termination, macroscopic examinations were conducted, and all lesions were recorded.
The uterus of each female was immersed in a 10% ammonium sulfide solution, and implantation sites were counted.

Clinical PATHOLOGY
Blood samples for thyroid hormone analysis (T4, TSH) were withdrawn at necropsy; the sampling was performed at a similar time on each occasion. Adult animals had samples withdrawn from the abdominal aorta on Day 43 (males) or LD 14 (females).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues from each adult animal were preserved in 10% neutral-buffered formalin, unless otherwise indicated indicated in the Table below.

Organ/Tissue Organ/Tissue
animal identification ovary E
Cowper’s gland (bulbo-urethral gland) W pituitary
epididymisa W; E prostate
Glans penis W seminal vesicle (with coagulating glands)
gross lesions testis (including Tunica albuginea)a W;E
kidneys W thyroid and parathyroid Wb, c
levator ani plus bulbocavernosus muscle complex W uterus (including cervix) E
liver W vagina E
mammary gland

W = Organs weighed (all groups).
E = Tissues processed and examined microscopically (Groups 1 and 4 only).
a = Tissue taken in Modified Davidsons fixative and processed to at least block stage.
b = tissues weighed together.
c = tissues weighed approximately 24 hours post-fixation.

Postmortem examinations (offspring):

SACRIFICE
- Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13
- These animals were subjected to postmortem examinations (macroscopic examination and clinical pathology) as follows:

GROSS NECROPSY
- After termination, macroscopic examinations were conducted, and all lesions were recorded.

CLINICAL PATHOLOGY
Blood samples for thyroid hormone analysis (T4, TSH) were withdrawn at necropsy. Pups culled on PND 4 had samples withdrawn via decapitation to provide one pooled sample for each litter, where possible.
Blood samples collected from pups at terminal kill on PND 13 were withdrawn by cardiac puncture to provide two pooled samples for each litter (one sample from two males and one sample from two females, where possible).

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid and parathyroid were removed from one pup/sex/litter on PND 13 and fixed in 10% neutral buffered formalin. The tissue was weighed 24 hours post-fixation.

Statistics:

Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data for each sex was analyzed separately, unless stated otherwise. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant when p≤0.05. By default, significant results were reported as * p≤0.05, + p≤0.01, and/or # p≤0.001.
Body weight, body weight gains, food consumption (gestation and lactation phases), absolute organ weights, organ:terminal body weight ratios, and terminal body weights were analyzed using analysis of variance (ANOVA).
Male and female mating, fecundity, and fertility indices were analyzed using a one sided lower tail Fisher’s exact test..
Mean number of estrus cycles and mean cycle length were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.
Percent pregnant, percent delivering, and gestation index were analyzed using a one sided lower tail Fisher’s exact test.
Percent of females with stillborn pups was analyzed using a one-sided upper tail Fisher’s exact test.
The number of pups delivered, live-born pups, implantation sites and live-birth index, Day 4 viability index, weaning index, duration of gestation, and percent post-implantation loss were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.
Pup weights (male, female, and combined) were analyzed using analysis of covariance (ANCOVA), with litter size as the covariate.
Live Pups/Litter with Live Pups were analyzed using the Kruskal-Wallis and Wilcoxon rank sum test.

Reproductive indices:

Female mating index = Mated females/Females cohabitated (excluding females sacrificed during cohabitation) X 100

Male mating index = Number of males mating with at least 1 female/Number of males cohabitated with at least 1 female X 100

Female fecundity index = Pregnant females/Mated females (excluding females with an undetermined pregnancy status) X 100

Male fecundity index = Number of males impregnating at least 1 female/Number of males mating with at least 1 female X 100

Female fertility index = Pregnant females/Females cohabitated (excluding females sacrificed during cohabitation or with an undetermined pregnancy statu s) X 100

Male fertility index = Number of males impregnating at least 1 female/Number of males cohabitated with at least 1 female X 100

% pre-implantation loss = Number of corpora lutea – number of implantations/Number of corpora lutea X 100

% post-implantation loss = Number of implantations – number of live embryos/Number of implantations X 100

Offspring viability indices:

Livebirth Index
DAY 4 Viability Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical observations were noted.
Clinical observations generally consisted of salivation, sores and staining of the skin and/or fur; these were noted throughout dose groups, including controls. As such, these observations were considered low incidence findings and unrelated to EGGE 2806-1 toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No unscheduled deaths considered related to toxicity of the test item occurred.
One control male (Animal 1M) was sent to necropsy during the pairing phase (Day 27 of dosing) following clinical observations of rapid respiration, raised hair, yellow staining of the abdominal region, and brown staining of the mouth. One control female (Animal 105F) was found dead on GD 8. At necropsy, both animals had esophageal ruptures, with clear fluid in the thoracic cavity. These finding were consistent with mis dosing and were unrelated to test item toxicity.
One female administered 300 mg/kg/day (Animal 130F) was sent to necropsy on LD 0 due to total loss of the litter. Macroscopic examinations confirmed that a litter was present due to fetal/placental tissue found in the stomach.
One control female (Animal 103F) was sent to necropsy on GD 23 following clinical observations consistent with prolonged parturition (pale extremities, subdued/sluggish, red discharge from the urogenital region, and raised hair). Necropsy showed fetal content in the stomach.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effect of the test item was noted on body weight change.
Slightly lower body weight gains were evident during the first week of dosing for males at all dose levels compared with controls, and a clear dose-relationship was evident (p<0.05, p<0.01, or p<0.001 for 100, 300, or 1000 mg/kg/day, respectively). Subsequently, the weight gain of these animals was similar to controls. Overall, this resulted in a 22% lower body weight gain during the 42 day dosing phase for males administered 1000 mg/kg/day, compared with controls; however, statistical significance was not achieved. Therefore, this minor difference in body weight gain for 1000 mg/kg/day was considered to be a transient response to the start of dosing and was unrelated to test item toxicity.
No effects on body weight change were evident throughout the pre-pairing, gestation, or lactation phase for females administered the test item, when compared with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings attributed to administration of the test item occurred as microscopic findings in the limited tissue list examined were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and stage of the reproductive cycle.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings attributed to administration of the test item occurred as microscopic findings in the limited tissue list examined were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and stage of the reproductive cycle.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed regular estrus cycles, approximately 4 days in length.
No effect of the test item administration was noted on the number or length of estrus cycles during the pre-pairing phase.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effect on mating, fecundity, or fertility was noted.
The majority of animals mated within 4 days of pairing, and all females from all dose groups were pregnant after mating.
One male administered 1000 mg/kg/day failed to mate, but this was considered a low incidence finding, is occasionally observed in studies of this type, and, in isolation, was unrelated to the test item administration.
There were 8, 10, 9 or 10 females administered 0, 100, 300 or 1000 mg/kg/day test item that gave birth to a live litter and maintained the litter until LD 13.
One control female was found dead on GD 8. One control female was sent to necropsy due to prolonged parturition, and one female administered 300 mg/kg/day had a total litter loss soon after parturition and was sent to necropsy.
Gestation lengths were essentially similar for control and test item-treated females, and no EGGE 2806-1-related effects on the number of still births, live births, or litter size were noted.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical observations were noted for pups from test item-treated females, when compared with controls.
Incidental findings (blue coloration, pup decedents, skin sores or thin fur) were observed throughout groups, including controls, and therefore, were considered unrelated to test item toxicity.

Mortality / viability:

no mortality observed

Description (incidence and severity):

no test item-related effect on pup survival occurred.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test item administration was noted on pup body weights.
Slightly lower mean body weights were noted for pups from 1000 mg/kg/day litters, compared with control litters. The differences were no more than 10% up to PND 4, with differences smaller after PND 7. These differences were most likely attributable to the slightly larger litter sizes in the 1000 mg/kg/day dose groups because it appeared to regress after the PND 4 cull to standardize litter sizes; as such, differences were considered not to have represented an effect of test item administration.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects on T4 or TSH were noted at the end of the study in male or females pups.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted for thyroid weights of PND 13 pups, when compared with those of concurrent controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic finding suggestive of an effect due to administration of the test item occurred in PND 13 pups .

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No differences were noted in the ano-genital distance from litters from test item 1 treated groups, when compared with controls.
No nipples/areolae were present for any male pup from any dose group, including controls.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

The objective of the study was to screen for potential adverse effects of the test item, EGGE 2806-1, on reproductive performance in the rat, including offspring development. Four groups of 10 male and 10 female Crl:WI(Han) strain rats were administered 0 (control article [vehicle]), 100, 300, or 1000 mg/kg bw/day EGGE 2806-1 by gavage, at a constant dose volume of 10 mL/kg. The control article(vehicle) was Kolliphor HS15/ethanol/water (40/10/50; v/v/v). The test item was formulated as a suspension. Formulations prepared for use in Weeks 1, 3, and 6, including the vehicle control were analysed for accuracy. Before the start of dosing, all females were screened for regular estrous cycles; only females showing regular estrous cycles were included in the study.

In accordance with the OECD test guidelines 421, males were dosed once daily for 42 consecutive days (two weeks prior to mating, during the mating period and, approximately, two weeks post-mating) and sent to necropsy on Day 43. Females were dosed for up to 64 days (two weeks prior to mating, during the mating period and until Day 13 post-partum) and sent to necropsy on Lactation Day (LD) 14. Following 2 weeks of dosing, animals were paired for mating on a one male:one female basis within each dose group. Once mating was confirmed, females were individually housed and males were returned to the home cage.

Assessment of toxicity in adults was based on clinical observations, body weights, food consumption, estrous cycling, mating, fertility and pregnancy indices, offspring parameters and anatomic (microscopic) pathology evaluations for adults. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Organ weights were performed for all animals, and microscopic examinations were conducted for control and high dose animals. Pup clinical observations, litter size, pup sex and pup body weight were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was also recorded for male pups on PND 13. One pup/sex/litter was selected from each dose group for thyroid weight and retention. Thyroid hormone assessments (T4, TSH) were performed in adults and offspring. Accuracy of formulations was demonstrated by analyses.

No unscheduled deaths, clinical observations, and changes in body weight gain considered related to the toxicity of EGGE 2806-1 occurred in parental animals. No test item-related effects were noted on food consumption, estrous cycles, mating, fecundity, or fertility indices and parturition, litter data collected. No macroscopic findings, organ weight and/or organ weight ratio changes, or microscopic changes considered related to administration of EGGE 2806-1 were noted. No test item related effects were noted on the parameters investigated in the offspring. No toxicologically significant effect on thyroid hormones were noted in parental animals and offspring of both sex.

In conclusion, once daily oral gavage administration of 100, 300, or 1000 mg/kg bw EGGE 2806-1 to male and female rats for at least 42 days (in accordance with the OECD 421 test guidelines), did not result in any treatment-related effects in parental animals. Furthermore, no test item-related effects were noted in the offspring at any dose level examined. With regard to reproduction/developmental toxicity, the no observed effect level (NOEL), therefore, is considered to be 1000 mg/kg bw/day.