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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jun 2020 - 31 May 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
The study design was chosen based on the requirements specified in the final ECHA decision notification for the substance (CCH-D-2114495620-46-01/F, 05 Feb 2020).
- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: Doses were chosen based on previous toxicity data of a 90-day toxicity study according to OECD 408, and a systemic/reproductive toxicity screening study according to OECD 422, where doses up to 1000 mg/kg bw/day (limit dose) were tolerated without serious signs of toxicity.
- Inclusion/exclusion of extension of Cohort 1B: exclusion
- Termination time for F2: not applicable
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: exclusion
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: exclusion
- Route of administration: oral by gavage

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N''-(isobutylidene)diurea
EC Number:
228-055-8
EC Name:
N,N''-(isobutylidene)diurea
Cas Number:
6104-30-9
Molecular formula:
C6H14N4O2
IUPAC Name:
N,N''-(2-methylpropane-1,1-diyl)diurea
Details on test material:
technical grade (purity ca. 92%)

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Limited, Kent, UK
- Females nulliparous and non-pregnant: not reported
- Age at study initiation: (P) 8 - 9 weeks; (F1) 3 weeks
- Weight at study initiation: (P) Males: 298 - 405 g; Females: 203 - 266 g
- Housing: Males were housed in groups of 2 or 3 and females were housed in groups of 4 or 5 animals per cage in solid-floor cages with appropriate bedding and environmental enrichment devices provided.
- Diet: pelleted rodent diet, VRF1 (SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England; ad libitum
- Water: mains tab water (in bottles); ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % w/v, medium viscosity
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated daily, at a minimum volume of 500 mL to minimize aeration of the formulations. For each preparation, a quantity of pre-ground test item (adjusted to account for purity) was weighed into a mortar; the weighed test item was also ground using a pestle, where required, to break down any larger granules to a fine powder. The test item was wetted in the mortar with a small quantity of vehicle and initially made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a container and made up to final quantity, ensuring that the mortar was thoroughly rinsed out with vehicle and adding these rinsings to the suspension. The formulations were then mixed with a laboratory homogeniser and placed in an ultrasonic bath for up to 35 min. The formulations used for dose administration were dispensed into amber glass bottles and were continuously stirred at room temperature until the end of dosing; they were used within 6 h of the completion of the formulation preparation procedure. The dosing volume was 10 mL/kg and individually adjusted based on most recent body weight recordings.

VEHICLE
- Concentration in vehicle: 10, 30 and 300 mg/mL for the low-, mid-, and high-dose groups, respectively
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm in vaginal smear (and presence / absence of copulation plugs) referred to as Day 0
- After successful mating, each pregnant female was caged individually with their litter in solid-floor cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first day of dosing for the P generation, the achieved concentration of the 30 and 100 mg/mL formulations administered to the 300 and 1000 mg/kg bw/day animals was below the acceptance criteria. The individual concentrations were up to 11% to 19% lower and mean concentrations up to 16% lower than nominal for both concentrations, thereby falling outside of the acceptance criteria (± 15% and ± 10% for individual and mean concentrations, respectively). The concentration of the 10 mg/mL formulation on the same day was within the acceptance criteria, with individual concentrations ranging from -1 % to +4% and a mean concentration at +1% of nominal, respectively. Despite the low concentrations at 30 and 100 mg/mL, test item formulations at all concentrations were shown to be homogeneous, as confirmed by a relative standard deviation (% RSD) of 3.8 % or lower (acceptance criteria: no greater than 5 %).
Subsequent investigations within the following week demonstrated that the test item formulations had attained achieved concentrations within the acceptance criteria at all concentrations (within 6%, 4% and 11% for individual concentrations and 5%, 2% or 7% for mean concentrations, at 10, 30 or 100 mg/mL, respectively). A constant preparation volume of 500 mL was found to be beneficial in achieving the concentrations required, although a 250 mL preparation volume also provided similar results at 30 or 100 mg/mL (from the investigations conducted as detailed in Section 3.4.5). The 500 mL preparation volume was also thought to reduce the degree of formulation aeration, and so was used on all dosing days apart from two days at the start of the dosing period.
Thereafter, analysis of the test item formulations also confirmed achieved concentrations within the acceptance criteria had been attained (mean concentrations within 3%, 9% and 5% of nominal at 10, 30 or 100 mg/mL over the remaining three analysis occasions – corresponding to the gestation period for P generation females and formulations used at the start and towards the end of the F1 generation).
Overall, it was considered that the animals had received formulations of the correct concentration throughout the study, and that the minimal reduction in the concentration of the first 30 and 100 mg/mL formulations administered did not impact on the overall dose levels that the animals received.
No test item was detected in vehicle used to dose the controls on the four preparation occasions that were analysed (coinciding with the start of the P generation, gestation for the P generation and formulations used at the start and towards the end of the F1 generation).
Duration of treatment / exposure:
(P) Males: at least 2 weeks before mating (up to a maximum of 20 days), during mating, and at least 6 weeks after mating
(P) Females: at least 2 weeks before mating (up to a maximum of 20 days), during mating, throughout gestation and up to Day 20 of lactation (females mid-parturition not dosed on that day)
(F1) Cohort 1A: Day 21 - Day 90 of age (10 weeks)
(F1) Cohort 1B: Day 21 - Day 97 of age (11 weeks)
Frequency of treatment:
Once daily, 7 days / week
Details on study schedule:
- Age at mating of the mated animals in the study: 11 - 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
96 P males and 96P females (24/sex/dose)
40 F1 males and 40 F1 females, split into 20 males and 20 females each for cohorts 1A and 1B
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in consultation with the Sponsor on the basis of previous toxicity data. In an OECD 408 (90-day toxicity) and an OECD 422 (systemic and reproductive toxicity screening study) with oral (gavage) administration of the test item to rats, doses of up to 1000 mg/kg bw/day were tolerated without serious signs of toxicity. For the females, effects were limited to reduced food consumption and body weight gain at 1000 mg/kg bw/day. In males, the only effects seen were rat-specific kidney lesions related to α2μ-nephropathy. No effects were noted in females at 300 mg/kg bw/day. In a truncated OECD 416 study (no second generation), slight effects on food consumption and body weight development were noted on in males and females at 600 and 1200 mg/kg bw/day. On the basis of these data, the high dose level for this study was selected as 1000 mg/kg bw/day, with dose levels of 100 and 300 mg/kg bw/day selected to assess a potential dose response.
- Rationale for animal assignment: Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on arrival using the electronic data capture system, Provantis™.
- Fasting period before blood sampling for clinical biochemistry: no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included checking for mortality, morbidity, changes in behaviour and appearance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing and then at weekly intervals until necropsy (males); females weighed additionally on Days 0, 7, 14 and 20 of gestation, and on Days 0, 1, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, recorded per cage at weekly intervals during pre-pairing and post-pairing periods (males), and at weekly intervals during pre-pairing plus on Days 0 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 20 of gestation, and on Days 1 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 21 of lactation.

WATER CONSUMPTION: No data

OTHER: haematology, coagulation, blood chemistry, thyroid hormone evaluation (T4/TSH)
For details on the evaluated parameters, please refer to Attachment 1 under section "Attached background material".

Urine samples were not collected from the P generation animals, on the basis that there were no adverse effects on urinalysis during a previous 90 day toxicity study in the rat (BASF Report Number: 50C0113/00S036, 06 February 2012). For the F1 generation Cohort 1A animals only, urine samples were collected from 10 males and 10 females in each group towards the end of the dosing period (Week 8 of the F1 generation for males and females). Samples were collected overnight, whilst the animals were housed individually in metabolism cages under food deprivation.
Oestrous cyclicity (parental animals):
From 14 days before the start of the pairing period and until the start of the pairing period, vaginal smears were taken from all surviving P generation females daily by lavage. A vaginal smear was also recorded on the day of necropsy; this procedure was not conducted for premature decedents where the animals’ clinical condition did not permit the procedure to be conducted.
Sperm parameters (parental animals):
Not performed (only in F1, cohort 1A)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- A maximum litter size of 4/sex/litter as nearly as possible was yielded and excess pups were killed and discarded. Litters of 8 pups or lower were not altered. Culled pups were assigned to blood sample collection for thyroid function subject to macroscopic necropsy.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: body weights, ano-genital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, food intake, sexual development (age of vaginal opening / balano-preputial separation), oestrous cyclicity (cohort 1A only), urinalysis (cohort 1A only)

GROSS EXAMINATION OF DEAD PUPS:
yes, subjected to full necropsy procedures, where possible

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes, cohort 1A (10 animals per sex); axillary and mesenteric lymph nodes weighed and prepared for microscopic evaluation, bone marrow smears examined, splenic lymphocyte sub-population analysis (CD4+/CD8+ lymphocytes, B-lymphocytes and natural killer cells) conducted
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, six weeks after completion of the mating phase (after successful littering)
- Maternal animals: All surviving animals on Day 21 of lactation, at weaning of their litters. Apparently non-pregnant females that mated during the pairing period were euthanised from Day 24 of gestation.

GROSS NECROPSY
- Gross necropsy consisted of dead body weight recordings and examination of major organs and tissues. If macroscopic abnormalities occurred, they were recorded and the respective organs retained.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Attachment 2, Tables 4 and 5 were weighed and prepared for microscopic examination, respectively.
Postmortem examinations (offspring):
SACRIFICE
- Necropsy was conducted on all F1-Cohort 1A pups, including those killed / found dead during lactation, all culled pups and all unselected pups on Day 21 of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: organ weights of brain, spleen and thymus; tissue retention of brain, site of mammary, spleen, thymus and all gross lesions for microscopic evaluation.

GROSS NECROPSY
- Gross necropsy consisted of examinations of the major organs. Organs or tissues showing any macroscopic abnormalities were removed and preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were weighed: please refer to Attachment 2, Tables 6, 8 and 10 for pups, Cohort 1A and Cohort 1B, respectively, under section: "Attached background material."
- The following tissues were prepared for microscopic examination: please refer to Attachment 2, Tables 7, 9 and 11 for pups, Cohort 1A and Cohort 1B, respectively, under section: "Attached background material."
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Depending on the nature of the data set that was to be analysed, appropriate tests were applied (for details, see section: "Any other information on materials and methods, incl. tables). Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied. Probability values of less than 5% were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Reproductive indices:
Copulation indices, fertility indices, gestation index and post-implantation loss were calculated. For details, please refer to section "Any other information on materials and methods incl. tables".
Offspring viability indices:
live birth index, viability indices, lactation index, percentage of male pups and anogenital distance index were calculated. For details, please refer to section "Any other information on materials and methods incl. tables".

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no abnormal clinical signs in surviving animals that were attributed to the test substance administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1000 mg/kg bw/day: There were 2 male and 2 female decedents, whose decline in clinical condition was attributed to accidental trauma from the dosing procedure, as as multiple adhesions were observed in the thoracic cavity macroscopically, which correlated with inflammation/fibrosis and/or eosinophilic contents (presumably test item) microscopically. In addition to the signs of dosing trauma, both decedent males had diffuse tubular basophilia (slight to moderate) and multifocal tubular mineralisation (moderate) in the kidneys and decreased cellularity of the bone marrow in the femur (slight). The importance of these findings and its relationship to the test item was considered equivocal, as these were isolated incidences.
300 mg/kg bw/day: One female was euthanised due to difficulties in parturition (dystocia). As also control females were affected, this effect was not considered treatment-related.
100 mg/kg bw/day: There was 1 female decedent which developed a wet lesion and dark appearance of a palpable mass in its ventral posterior region, which had grown rapidly during late gestation, along with a separate mass in the inguinal region. Histopathological examination defined it as an adenocarcinoma, but not considered treatment-related as it was an isolated incidence and such tumours are often seen in SD rats. Due to the euthanasia of the dam on Day 8 of lactation, the F1 pups from this litter were also euthanised on Day 8 of age.
Control group: Two females were euthanised due to dystocia, and one additional female was euthanised on Day 3 of lactation following total litter loss. The 18 pups of this female were euthanised due to poor clinical condition.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For the males given 300 or 1000 mg/kg bw/day test substance, a slight but statistically significant reduction in body weight gain over Weeks 5 to 6 (p≤0.01) contributed to a slight, non-dose-related decrease in group mean body weight gain over the dosing period compared with controls (Weeks 0 to 10: 9% or 6% lower than controls, respectively). Given the minimal difference between the groups, and that body weight gains after Week 6 were generally comparable with controls, this apparent intergroup difference was considered to reflect normal biological variation and not to be associated with administration of the test substance.
For details on body weight and body weight change, please refer to Attachment 3, Figure 1 and Tables 1 - 2 under section "Attached background material."
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food intake was unaffected by the test substance administration, except for Days 10 - 14 of lactation, where an increase of 12% was found in treated groups, compared to controls (p≤0.05 or p≤0.01). In the absence of a clear effect during the remaining study period, this was considered to reflect normal biological variation and not to be associated with the test substance.
For details on food intake, please refer to Attachment 3, Table 3 under section "Attached background material."
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on haematology or coagulation considered to be treatment-related, with any minor intergroup differences from controls considered to reflect normal biological variation as individual values remained within the historical control reference ranges.
For details on haematology, please refer to Attachment 3, Table 12 under section "Attached background material."
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: There was a statistically significant increase in group mean thyroid stimulating hormone (TSH) in males compared with controls (0.6 fold; 56% increase, p≤0.05). Since only one of the individual TSH concentrations in this group was above the range seen in the concurrent controls and there was no corresponding change in total thyroxine concentration (T4) and no test item-related organ weight or pathology changes in the thyroids or pituitary, this apparent difference from controls was considered not to be adverse.
For details on clinical chemistry and thyroid hormone analysis, please refer to Attachment 3, Tables 13 and 15, respectively, under section "Attached background material."
Endocrine findings:
not specified
Description (incidence and severity):
The following endocrine parameters were evaluated in the study:
- Oestrous cyclicity and stage of oestrous
- Sperm analysis (motility, morphology, concentration)
- Pregnancy parameters (count of primordial follicles and naked oocytes, primary follicles, number of corpora lutea; copulation index, fertility index, gestation index, post-implantation loss)
- Litter size (pup body weights, viability index, live birth index, lactation index, percentage of male pups)
- Anogenital distance, nipple count in males
- Thyroid hormone measurements (TSH, T4)
- Weight of adrenal glands, brain, epididymides, cauda epididymis, liver, ovaries, pituitary, prostate, seminal vesicles (incl. coagulating gland), testes, thyroids, and uterus
- Histopathology of adrenal glands, epididymides, liver, ovaries, pituitary, prostate, seminal vesicles (incl. coagulating gland), mammary gland, testes, thyroids, uterus (incl. uterine cervix and ovdiucts), vagina, vas deferens

For details on these results, please refer to the respective sections.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no microscopic findings that were considered to be related to the test substance. The spectrum of changes was generally consistent with those normally encountered in rats of this age and strain kept under laboratory conditions.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
One female killed in extremis (100 mg/kg bw/day) had developed an adenocarcinoma, but it was considered incidential and not test item-related.
Other effects:
not specified
Description (incidence and severity):
2/24 females given 1000 mg/kg bw/day had ductal dilatation (moderate) and inflammation/fibroplasia (slight) observed in the mammary gland. For one female, these findings correlated with a cream, irregular, firm mass with cream, amorphous contents, noted macroscopically in the mammary gland situated in the non protocol thoracic skin/subcutis.
An additional request was made for the examination of a third female given 300 mg/kg bw/day, which had a macroscopic abnormality at the site of the mammary gland noted as a cream, irregular, firm mass with cream granular contents. Microscopically, this correlated with marked dilatation of the ducts and minimal inflammation.
The described above findings were considered to be equivocal.

There were two animals (one male and one female) given 300 mg/kg bw/day that mated during the mating period, but the female was not pregnant. Therefore, the reproductive organs from these animals were examined microscopically. It was concluded that the findings of decreased corpora lutea and follicular cysts observed in the female would account for the failure of pregnancy. As this was an isolated incidence, these findings were considered not related to the test item.

For the other 6 non-pregnant females with signs of reduced fertility there were no macroscopic or microscopic findings to indicate infertility. In the pairing males, however, atrophy of the epididymides and testes were seen in four of the males. Since these findings were seen in the affected males from both the controls and dose groups, it was concluded that they were not related to test substance administration and that there was no indication of a test item-related effect on fertility.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
For details on oestrous cyclicity, please refer to Attachment 3, Table 4 under section "Attached background material."
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of the test substance on fertility or mating performance at any dose level; time to mating and fertility indices were unaffected, as were post-implantation loss, mean number of pups born alive, post-natal survival, and percentage of male pups. Non-pregnant females were found in control and treatment groups (3, 1 and 3 in groups 1, 3, and 4, respectively), but the incidence of apparent infertilities was comparable between these groups, and thus, no relationship to the test substance was assumed.
For details on mating and fertility parameters, please refer to Attachment 3, Tables 5 - 6 under section "Attached background material."
There were no effects on the length of gestation or on parturition that were considered to be associated with the test substance. Although the group mean gestation lengths were comparable across the groups, at the 300 mg/kg bw/day dose, the mean length was slightly longer than controls and attained statistical significance (p≤0.05). This was, however, considered not to be associated with test substance administration, as similar differences were not seen at 100 or 1000 mg/kg bw/day and the difference from the control was minimal (mean gestation length: 22.13 days for controls and 22.43 days at 300 mg/kg bw/day). With the exception of the decedents that showed signs of dystocia and the pregnant female that was euthanised during gestation following a decline in clinical condition, all of the pregnant females gave birth to live litters.
There was no effect of the test item on post-implantation loss, the mean number of pups born alive, or on the post-natal survival of the pups to Day 21 of age. The percentage of male pups per litter was also comparable across the groups. For details on pregnancy and litter data, please refer to Attachment 3, Table 7 under section "Attached background material."

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive and developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormal clinical signs during the F1 generation (both pre-and post-weaning) that were attributed to treatment.
1000 mg/kg bw/day: one male had two clonic convulsions for up to two minutes on Day 61 of the F1 generation. As this was an isolated occurrence for this animal, and convulsions were not seen in any other study animal, this occurrence was considered not to be associated with the test item. All other clinical signs were considered to be isolated occurrences and within the normal limits for laboratory animals.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no mortality in the pups that were attributed to maternal dministration of the test substance at any dose level, as demonstrated by pup survival indices in the treated groups that were comparable with or higher than the controls.
There were 6 premature decedents during the filial (F1) generation; however, none of these was directly attributed to the test substance administration.
1000 mg/kg bw/day: 2 males were found dead on Days -2 and 2, and 2 females were euthanized in extremis due to decline in clinical condition on Day 3. 3 of these decedents were from the same litter and exceptionally small. Clinical signs the day before mortality were observed and could be associated with macroscopic and/or microscopic changes from post mortem examinations. The decline of these animals was considered to be a likely result of inadvertent trauma (from housing these animals with larger animals) and nutritional disturbance, and not directly related to the test item. Furthermore, similar instances of low body weight or impaired survival were not observed in the other selected animals from the high dose group, and therefore the findings seen in pups from this litter were considered not to be related to the test substance. The second male found dead, (that died after dosing on Day -2 relative to the start of this generation, equivalent to Day 22 of age) in this dose group did not show any adverse clinical signs or macroscopic abnormalities at necropsy. There were, however, challenges with the dosing procedures at this stage of the study due to the granularity of the test item formulation, and so an indirect association with the dosing procedure cannot be discounted. As this was an isolated occurrence, this decedent was considered unlikely to be directly-related to test substance administration.
Another female in the high-dose group was euthanised on Day 74 in extremis. The animal’s abdomen was noted to be abnormally firm, dark and distended; the animal also had pale eyes and extremities. Macroscopic examination identified a pale pituitary gland, adrenals, liver and lungs, a large spleen and an abnormally large and dark liver with roughened surface and a depressed cream area. In the absence of similar findings in the remaining F1 generation animals, this occurrence was considered not to be associated with the test item.
300 mg/kg bw/day: one female was euthanised on Day 12 of the F1 generation due to clinical signs of decreased activity, laboured breathing and hunched posture. These signs were attributed to dosing trauma and not related to test substance administration, as the macroscopic examination identified fluid in the thoracic cavity with adhesions to various organs within the thoracic cavity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no adverse effect of maternal administration of the test substance on group mean pup body weights or body weight gain at any dose level. For all dosed groups (both sexes) on Day 1 of age (day after birth), the male and female pups were slightly heavier than the controls, typically in a dose related manner (3% to 7% higher). This was most prominent in female pups, where the difference from Controls was statistically lower (p≤0.05). Considering that this apparent difference was exacerbated by relatively low pup body weights in a small proportion of control group litters and that pup body weights were comparable across the groups by the next measurement on Day 4 of age, this finding was considered not to be adverse.
Body weights and body weight gains were unaffected by the administration of the test item during the F1 generation.
For details on body weight and body weight change, please refer to Attachment 3, Figure 1 and Table 1 under section "Attached background material."
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect on food intake during the F1 generation that was considered to be related to treatment. For the Cohort 1B males only, food intake was generally marginally higher than the controls throughout the F1 generation for all test item groups, and resulted in a statistically significant increase in food intake for the overall dosing period at 300 or 1000 mg/kg bw/day (Weeks 0 to 12: p≤0.05). Given the minimal nature of the increase (generally +2 g per day) and that a similar response was not seen in the Cohort 1A males, this apparent difference was considered to reflect normal biological variation and not to be associated with the test item.
For details on food intake, please refer to Attachment 3, Table 3 under section "Attached background material."
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Group mean total white blood cell counts were slightly higher than the controls for the males at all dose levels and for the females at 300 or 1000 mg/kg bw/day at the end of the F1 generation; in the males, there was no clear dose –related difference between the test item groups (up to 32% higher, p≤0.05). This change was primarily attributed to increases in lymphocytes and large unstained cells in the males and females, with an increase in eosinophils also seen in males (p≤0.05 or p≤0.01). There was a large degree of individual variation in the data, however, the total white blood cell concentrations for one, two and three males at 100, 300 or 1000 mg/kg bw/day were slightly higher than the upper limit of the normal range. The concentrations in the females were generally within the historical Control range. Although slight increases in thymus weights were seen in males, there were no correlating microscopic changes, and in consideration that the increases were relatively slight, these findings were considered to be non-adverse.
For details on haematology, please refer to Attachment 3, Table 12 under section "Attached background material."
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no changes in total thyroxine (T4) or thyroid stimulating hormone (TSH) concentrations on Day 21 of age that were considered to be related to treatment. The slight and non-statistically significant increase in group mean TSH concentration for the male F1 Cohort 1A pups at 1000 mg/kg bw/day (19%) was considered to be within the limits of normal variation, as shown by both the highest and lowest TSH concentrations being observed in this group.
No effects of test substance administration on the mean total thyroxine (T4) or thyroid stimulating hormone (TSH) concentrations at any dose level were observed.
There were no adverse test item-related blood chemistry changes at the end of the F1 generation following the administration of the test substance. In males, group mean globulin concentrations were slightly higher than controls in all treated groups, with corresponding increases in calcium (p≤0.05 or p≤0.01). This apparent dose-related increase was considered to be exacerbated by a particularly low globulin concentration for one control group male. In consideration of this, and that individual values in the treated groups remained within or only marginally above the upper limit of the normal range of this historical control data, these apparent increases were considered not to be adverse. Furthermore, group mean potassium concentrations in the males given 300 or 1000 mg/kg bw/day only were slightly higher than controls, in a non-dose related manner (p≤0.01). In the case of the 300 mg/kg bw/day group, this was largely attributed to a particularly high concentration for one male only. In addition, group mean chloride concentrations were statistically lower than controls in males from all dose groups. Since most concentrations for the treated males remained within the historical control range, this apparent difference from controls was considered to reflect normal biological variation. Lastly, urea concentrations were higher than controls for the females given 100 mg/kg bw/day, but not at 300 or 1000 mg/kg bw/day (p≤0.01). In the absence of a dose- response, and in consideration that individual values remained within the normal range of the historical control range, this was apparent difference was considered not to be test item-related and instead to reflect normal biological variation.
For details on clinical chemistry and thyroid hormone analysis, please refer to Attachment 3, Tables 13 and 15, respectively, under section "Attached background material."
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no changes in urinary volume or urine composition in the F1 generation animals following test substance administration that were considered to be adverse. In males, group mean urine volumes were slightly higher than the controls at all dose levels, however, the apparent increases were largely due to a high degree of individual variation and there was no notable change in urinary composition.
For details on urinalysis, please refer to Attachment 3, Table 14 under section "Attached background material."
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: In Cohort 1B males only, the average day for completion of balano-preputial separation was marginally later than the controls (almost two day delay). This was largely due to a slight delay for only 2 males, whereas the day of completion for the remaining high dose males was comparable with the variation seen in the controls. In consideration of this, and that an effect was not seen in the Cohort 1A animals, this apparent intergroup difference for Cohort 1B was considered not to be associated with the test substance.
For the females, there was no difference in the day of vaginal opening between the control and test item groups in either cohort. For the Cohort 1A females, the day of detection of the first oestrus after vaginal opening was also comparable across the groups.
For details on sexual maturation, please refer to Attachment 3, Tables 10 - 11 under section "Attached background material."
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance of male and female pups was unaffected by treatment with the test substance.
For details on anogenital distance, please refer to Attachment 3, Table 8 under section "Attached background material."
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no nipples/areolae in the male pups from any group on Day 12 of age.
For details on nipple counts, please refer to Attachment 3, Table 9 under section "Attached background material."
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Absolute and body weight-related group mean spleen weights were slightly higher than controls for the F1 Cohort 1A male pups (up to 22%; p≤0.05) only. There was a large degree of variation in the individual values, with this apparent difference exacerbated by relatively high spleen weights for the male pups from the litters of two females. In the absence of subsequent findings in the spleen when the F1 animals were dosed directly after weaning, this apparent difference was considered to be non-adverse. All other intergroup differences were considered not to be related to maternal administration of the test substance and to represent normal biological variation.
Thymus: For males at all dose levels, the group mean absolute and body weight-related thymus weights were higher than the controls, in a non-dose related manner (Cohort 1A: up to 22% higher; p≤0.05 or p≤0.01). There was, however, a high degree of individual variation in the data, and all of the body weight-related values remained within the upper limit of the normal historical control range. In consideration of this, and that there were no corresponding microscopic changes, this change was considered to be non-adverse.
Seminal vesicles: Lower group mean absolute and body weight-related seminal vesicle weights compared with controls were seen for males at all dose levels, in a dose-related manner (Cohort 1B: up to 5%, 14% or 19% lower at 100, 300 or 1000 mg/kg bw/day; p≤0.05 or p≤0.01). In the absence of a similar organ weight change or microscopic changes in the seminal vesicles in the Cohort 1A males, and that the individuals in the Cohort 1B groups were generally comparable with the variation seen in the controls, this finding was considered not to be related to the test substance.
Brain: At 1000 mg/kg bw/day, group mean absolute brain weights for the males were slightly lower than the controls (Cohort 1A: 3%; p≤0.05). This was primarily attributed to the relatively low brain weight for one male only. Since there were no corresponding pathology changes in the brain for any of the examined animals, the relevance of this finding is unclear.
Liver: Also at 1000 mg/kg bw/day, the group mean liver weights for the males were slightly higher than the controls (Cohort 1A: absolute and body weight-related: up to 10% higher; p≤0.05 to p≤0.01). This small degree of increase was considered to represent an adaptive response to the test substance and not to be adverse.
There were no organ weight changes for the females that were attributed to the administration of the test substance, with any minor intergroup differences considered to reflect normal biological variation, as values generally remained within the normal limits of the historical control range.
For details on organ weights, please refer to Attachment 3, Table 17 under section "Attached background material."
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Kidneys: There was a slightly increased incidence of pelvic dilatation in the kidney in males given 300 and 1000 mg/kg bw/day, which correlated with minimal to slight pelvic dilatation at the microscopic examination (Cohorts 1A and/or 1B). In addition, a mottled discolouration was noted in the kidneys of two males at 300 mg/kg bw/day and three males at 1000 mg/kg bw/day from Cohort 1B, which correlated microscopically with the accumulation of hyaline droplets and therefore was considered to be test item-related.
Other observed abnormalities were generally consistent with those normally encountered in rats of this age and strain kept under laboratory conditions.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 - Cohort 1A:
Kidneys: Test item-related changes were limited to the kidneys, where an increased incidence of accumulation of hyaline droplets (minimal to slight) was seen in the tubules of kidneys in 7/20 males given 300 and in 10/20 males at 1000 mg/kg bw/day. Heidenhain’s Azan staining was used to improve visualisation and quantification of the hyaline droplets, indicating that the increased number of hyaline droplets was positive for α2µ-globulin. This finding was accompanied by an increased incidence of pelvic dilatation (minimal to slight) in the kidneys of 6/20 males given 1000 mg/kg bw/day. However, α2µ-globulin nephropathy is considered to be male rat specific and therefore of no human relevance.
Other findings: 1/20 males given 1000 mg/kg bw/day had bilateral tubular atrophy of the testes (slight), which was accompanied with changes in the epididymides (decreased sperm and germ cells/cell debris in the lumen). As this was a single animal, the findings were considered equivocal and could be included as part of the background.
Other findings: Lobular atrophy of the mammary gland (slight to moderate) was observed in 3/20 control males compared to 7/20 high dose males, which, due to the large variation in the mammary gland sections, was considered not test item-related.
F1 - Cohort 1B:
Kidneys: For the Cohort 1B males, only kidneys from males with macroscopic abnormalities noted at necropsy were examined microscopically. For these animals, accumulation of hyaline droplets (minimal) was found in the tubules of kidneys in 1/20 males from each group given 300 or 1000 mg/kg bw/day. In addition, 1/20 males given 1000 mg/kg bw/day had bilateral pelvic dilatation (moderate). At 300 mg/kg bw/day, these changes were considered to be non-adverse due to the lower incidences of droplet accumulation, but adverse at 1000 mg/kg bw/day; α2µ-globulin nephropathy is, however, considered to be male rat specific and therefore of no human relevance.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Oestrous cyclicity:
There were no effects on the oestrous cycles for the F1 generation females that were attributed to the test item. For all treated groups, there was a slight increase in the mean length of each oestrous cycle compared with the controls, with a subsequent slight decrease in the mean number of cycles per female within the 14 day monitoring period (p≤0.05). This was, however, considered not to indicate a test item-related effect, as the oestrous cycles for the individual animals demonstrated normal cyclicity in most animals. Furthermore, it was expected that no more than three cycles would typically be seen within a 14 day period for rats based on the expected normal cycle of four to five days and therefore the cyclicity in the test item groups was within the normal variation expected (mean number of cycles in test item groups: 2.8 or 2.9). For details on oestrous cyclicity, please refer to Attachment 3, Table 4 under section "Attached background material."
Sperm evaluation and morphology:
There was no test-item related effect on sperm concentration or velocity at any dose level. At 1000 mg/kg bw/day, the group mean percentage of morphologically abnormal sperm was slightly higher than the controls (p≤0.05). This was attributed to a relatively high number of headless sperm for one male only; additionally, the sperm concentration for this male was also relatively low. As the sperm morphology for the remaining males was comparable with the variation seen in the concurrent controls, this occurrence was not attributed to the test substance and instead considered to represent normal biological variation. For details on sperm analysis, please refer to Attachment 3, Table 18 under section "Attached background material."
Ovarian follicle and corpora lutea quantification:
At 1000 mg/kg bw/day, the group mean total number of primary follicles was slightly lower than the controls. This difference was attributed to a large degree of individual variation, where the total primary follicle counts ranged from 6 to 28 in the controls and 1 to 27 at 1000 mg/kg bw/day. Therefore, this apparent intergroup difference was considered not to be associated with the test substance. The mean total number of primordial (naked oocytes) and small follicles and the number of corpora lutea were comparable between the controls and 1000 mg/kg bw/day F1 generation animals.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, treatment-related
Description (incidence and severity):
Lymph node weights:
For the males at 1000 mg/kg bw/day, the group mean axillary lymph node weights (representative of a distal lymph node) were slightly higher than the controls, but not statistically significant. This was predominantly due to weights that were higher than those seen in the individual controls for four high dose group males; although one of these males was also shown to have high white blood cell counts during the haematology analysis, similarly high concentrations were not seen in the remaining males. The weights for the mesenteric lymph nodes (representative of a proximal node to the site of exposure) were, in contrast, comparable across the groups and therefore the relevance of the axillary lymph node result is unclear.
There were no other lymph node weight differences from the controls that were considered to be related to the test substance administration; the degree of variation between the individual weights was generally comparable across the groups.
Splenic lymphocyte sub-population analysis:
There were no clear effects of test substance administration on the splenic lymphocyte sub populations in the males or females at any dose level; the group mean percentages of T-cells, B-cells and natural killer cells in the total lymphocytes were comparable across the groups, along with the percentage of CD4+ and CD8+ in the T-cells.
Bone marrow smears:
There were no changes in the bone marrow cells that were considered to be related to test substance administration. The proportion of myeloid to erythroid cells was relatively comparable between the control and test item groups for both males and females.
For details on immunotoxicity assessments, please refer to Attachment 3, Table 16 under section "Attached background material."

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive and developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effect observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
Systemic toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effect observed up to and including the highest dose tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The present study was conducted in compliance with GLP conditions and OECD test guideline 443 and investigated the effects of the test substance N,N' (isobutylidene)diurea on reproduction and development in a transgenerational study in male and female rats. Under the present experimental conditions, no adverse effects relevant to humans were observed at any dose level. Thus, the No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day. The NOAEL for systemic toxicity was considered to be 300 mg/kg bw/day for males and 1000 mg/kg bw/day for females.