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Effects on fertility

Description of key information


In a reproductive/developmental toxicity screening test, and in a modern one generation study, the test substance N,N' (isobutylidene)diurea had no effects on the reproductive function of rats. In addition, no effects on reproductive parameters (organs, sperm and estrous cycle) were seen in a 90-day study.


Additionally, an extended one-generation reproductive toxicity study was recently conducted (UKK0004) in rats. In this study, no adverse effects on developmental and reproductive endpoints were observed up to and including the highest dose tested.


 


Reproductive/developmental screening test (95R0113/009039; 2003; RL1; GLP; according to OECD 422)


NOAEL P0 (reproductive toxicity): 1000 mg/kg bw/day in males and females


NOAEL P0 (systemic toxicity): 1000 mg/kg bw/day in males; 300 mg/kg bw/day in females


LOAEL P0 (systemic toxicity): 1000 mg/kg bw/day in females, based on reduced body weight and body weight gain during pregnancy and lactation


NOAEL F1 (reproductive toxicity): 1000 mg/kg bw/day in males and females


NOAEL F1 (systemic toxicity): 1000 mg/kg bw/day in males and females


 


Extended One-Generation Reproductive Toxicity Test (77R0113/009069; 2003; RL1; GLP; according to OECD 416 without the second generation)


NOAEL P0 (reproductive toxicity): 1200 mg/kg bw/day in males and females


NOAEL P0 (systemic toxicity): 1200 mg/kg bw/day in males


LOAEL P0 (systemic toxicity): 600 mg/kg bw/day in females, based on decreased body weight gain in females during pregnancy


 


90-Day Repeated-Dose Toxicity Study (50C0113/00S036; 2017; RL1; GLP; according to OECD 408)


NOAEL P0 (reproductive toxicity): 1000 mg/kg bw/day in males and females


 


Extended One-Generation Reproductive Toxicity Test (UKK0004; 2021 (study pending); RL1; GLP; according to OECD 443)


NOAEL P0 (reproductive and developmental toxicity): 1000 mg/kg bw/day in males and females


NOAEL P0 (systemic toxicity): 1000 mg/kg bw/day in males and females


NOAEL F1 (reproductive and developmental toxicity): 1000 mg/kg bw/day in males and females


NOAEL F1 (systemic toxicity): 300 mg/kg bw/day in males and 1000 mg/kg bw/day in females


LOAEL F1 (systemic toxicity): 1000 mg/kg bw/day in males, based on alpha-2-microglobulin nephropathy in the kidneys


**Final histopathology results are still pending (draft report expected 24 May 2022, final report expected 01 Jul 2022). In case future results would impact the NOAELs described above, this section will be immediately updated.**



Link to relevant study records

Referenceopen allclose all

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 - 30 Apr 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
Missing tissues are currently under histopathological evaluation; result sections will be updated once results become available (24 May 2022; latest 01 July 2022)
GLP compliance:
yes
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
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
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
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
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
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".
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.
The following tissues are currently under histopathological evaluation with results pending: GI tract, eyes + optic nerve, peripheral nerve, skeletal muscle, spinal cord, and urinary bladder.
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."
- The following tissues (cohort 1A) are currently under histopathological evaluation with results pending: GI tract, eyes + optic nerve, lungs, mammary gland, peripheral nerve, skeletal muscle, spinal cord, thyroid + parathyroid, trachea, urinary bladder, and vas deferens.
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".
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.
The results for the tissues still under evaluation will be provided as soon as possible.
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.
The results for the tissues still under evaluation will be provided as soon as possible.
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: 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."
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.
Key result
Critical effects observed:
no
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.
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.
The results for the tissues currently still under evaluation will be provided as soon as they become available.
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.
Behaviour (functional findings):
not examined
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."
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:
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:
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:
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.
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
Key result
Reproductive effects observed:
no
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.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: males: 8 weeks; females: 10 weeks
- Weight at study initiation: males: 276 g mean bw; females: 228 g mean bw
- Housing: housed individually in suspended wire-mesh cages, females shifted 4 days before lactation to polycarbonate cages in a barrier rodent unit.
- Diet: A04 C pelleted diet ad libitum, distributed weekly
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2
- Humidity (%): 50 +- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 69H0028, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL and then homogenized using a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours).
Details on mating procedure:
- M/F ratio per cage: 1 male/1 female of the same dose group
- Length of cohabitation: during the night
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- Each female was placed with the same male until mating occured or 14 days had elapsed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations: concentration and homogeneity.
Three samples of each control and test substance dosage form (top, middle and bottom of the flasks) prepared for use during the first week and the last week of treatment were taken. They were stored at -20 °C pending dispatch, to the Sponsor, for analysis of concentration and homogeneity
Duration of treatment / exposure:
Exposure period: males: pre-mating, mating (14days) and post-mating, for a total of 34 days, females: pre-mating, mating, pregnancy and lactation until day 4 post partum
Premating exposure period (males): 15 days
Premating exposure period (females): 15 days
Duration of test: males: until sacrifice in the post-mating period, females: until day 4 post partum
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
no futher data
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were specified by the Sponsor, following the results of a prev iously conducted prenatal developmental toxicity study in Wistar rats (BASF Project No. 30R0727/90116). The oral route was selected since it is the route of exposure, which is requested by regulatory authorities for this type of product.
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at least once

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
male animals: first day of treatment, then once a week until sacrifice
female animals: first day of treatment, then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE :
The quantity of food consumed by each animal was recorded once a week, from the first day of each of the pre-mating, gestation and lactation periods. During the mating period, the food consumption was noted for neither males nor females.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
[testis weight, epididymis weight, morphological examination of the testes: tailed and round spermatids, spermatocytes, spermatogonia, different stages of spermatogonic cycle]
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after the completion of the treatment period: after the end of the mating period: on study day 35 (total treatment period was 34 days)]
- Maternal animals: All surviving animals [after the completion of the treatment period: the females and their pups were killed on day 5 post partum. Female animals showing no evidence of mating were killed 24-26 days after the last day of mating]

GROSS NECROPSY
- A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. In the parent females, the corpora lutea and implantation sites were recorded. In apparently non-pregnant or un-mated females, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
body weight and organ weights (adrenals, brain, heart, kidneys, liver, spleen, thymus, additionally in males: testes, epididymides; females:ovaries), Histopathology: macroscopic lesions, adrenals, brain, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids and parathyroids, trachea, urinary bladder, uterus
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations: externally for gross abnormalities, pup body weight, sex ratio, clinical signs

Statistics:
Dunnett, Fisher`s exact, Dunn, Mann-Whitney, Wilcoxon tests.
Clinical signs:
no effects observed
Description (incidence and severity):
In the male and female treated groups, there were no clinical signs, which were considered to represent adverse effect of the test substance in any animal.
Mortality:
no mortality observed
Description (incidence):
no deaths in any group, which were attributed to treatment with the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight change of the treated males was similar to that of the control animals.
The body weight change of the females given 100 or 300 mg/kg/day was similar to that of the control group during the pre-mating, pregnancy and lactation periods. In the 1000 mg/kg/day treated group, the body weight change was similar during the pre-mating period, but was slightly lower during pregnancy (day 0 to day 20 of pregnancy: - 10%, not statistically significant) and lactation (day 1 to day 4 post-partum: -38%, not statistically significant). Mean body weights on day 0 of pregnancy and day 4 post-partum statistically differed from that of the control group. These differences, correlating with a reduction of food consumption, (during the pregnancy period) was considered to be related to treatment with the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of all the treated males was similar to that of the control males, during the pre-mating period.
The food consumption of all the treated females was similar to that of the control females, during the pre-mating and post-mating periods except for females of the 1000 mg/kg/day group in which a slight reduction in food consumption was recorded during the pregnancy (day 0 to day 20 of pregnancy : -6%, not statistically significant). This difference correlating with an effect on body weight gain was considered to be related to treatment with the test substance.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There appears to be a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the treated male rats, given 300 and 1000 mg/kg/day. As no tubular degeneration/necrosis was observed in the kidneys of the males of any group, the presence of acidophilic globules was considered to be of minor toxicological importance and most probably due to the accumulation of the sex-linked alpha 2 micro-globulin in the cortical tubular cells of the male rats. This is a sex- and species-specific effect, as no such accumulation occurs in female rats or in humans. Thus, this finding has no relevance for humans in terms of risk assessment.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The pre-coital interval was similar in the control and the treated groups. All paired animals (except one control pair) mated within 1 to 4 days of cohabitation, i .e. within the duration of a single estrous cycle. No particular microscopic finding was noted in the treated ovaries as their morphological characteristics were normal (particularly the number of ovarian follicles and corpora lutea).
Hyalinosis of the blood vessels, hemosiderosis and sometimes capillary hemorrhage, were seen with similar incidence and/or severity in the uterus of the treated and control females.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No treament-related abnormalities were found in the treated animals. The tailed and round spermatids were unaffected and the different stages of spermatogenic cycle were undisturbed by treatment with the test substance. In almost all treated as well as control males, a few degenerated necrotic cells sloughed in the lumen (minimal or slight), multinucleated giant cells (minimal), vacuoles of the seminiferous tubules (minimal or slight) were noted with similar incidence and/or severity in all control and treated groups. These findings with such severity are commonly recorded as spontaneous changes in the rat and were considered to be of no toxicological importance. The seminiferous tubules were lined with Sertoli cells only (minimal or slight) in 1/10 males given 300 mg/kg/day and in 2/10 males given 1000 mg/kg/day. For a third male from the same group, tubules lined with Sertoli cells only were considered to be tubuli recti as they were situated beneath the capsule. For 1/10 males given 300 mg/kg/day and another given 1000 mg/kg/day, minimal reduction in the number of spermatids was observed in very few seminiferous tubules. Minimal vacuolization of Sertolicells was observed in 1/10 males given 1000 mg /kg/day. Although not found in the control males, these microscopic abnormalities recorded with minimal severity in few or very few seminiferous tubules of a few males were considered to be without relationship to the treatment and most probably fortuitous.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The mating index was 100% for the pairs of the control, 100 and 300 mg/kg/day groups. The mating index was lower in the 1000 mg/kg/day group: 7/10 pairs (70%) mated within the two weeks of cohabitation. As no treatment-related morphological changes were noted in the genital organs of the high-dose male and female animals, this finding is considered to be accidental. The fertility index (pregnant/mated) was similar in the control and the treated groups, ranging from 90 to 100% without indication of dose- or treatment -relationship.
Key result
Dose descriptor:
other: NOAEL, systemic toxicity male
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Overall effects
Key result
Dose descriptor:
other: NOAEL, systemic toxicity female
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects
Clinical signs:
no effects observed
Description (incidence and severity):
There were no notable clinical signs in the pups of the control or treated groups after birth.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of pups which died during the four days of observation after birth was low (0 to 5 %) and similar in all the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The weights of pups were similar in the control and the treated groups on day 1 and day 4 post-partum (being slightly higher in the high -dose group, as expected for lower-sized litters).
Sexual maturation:
no effects observed
Description (incidence and severity):
No deviations from the control group were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross external abnormalities in the pups of the control and the treated groups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effets on the F1 generation observed up to the highest dose tested
Reproductive effects observed:
no
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
the study focused on fertility/reproduction only and there was no 2nd generation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
the study focused on fertility/reproduction only and there was no 2nd generation and no DIT and no DNT cohorts
Principles of method if other than guideline:
The study follows OECD 416 guideline, focussing on fertility/reproduction only. There was no 2nd generation (modified one generation study).
GLP compliance:
yes
Justification for study design:
Based on the available data, there are neither triggers for a 2nd generation nor a DIT or DNT cohort. Thus the available data is sufficient to cover the data requirements and no new OECD 443 study is necessary.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle, France
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 171-210 g (mean: 192 g); Females: 142-179 g (mean: 161 g)
- Housing: The animals were housed individually in suspended wire-mesh cages (43 .0 x 21 .5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage. The cages were placed in numerical order on the racks. On a monthly basis, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a 6-day acclimation period to the conditions of the study preceded the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-2 °C
- Humidity (%): 50+-20 %
- Air changes (per hr): about 12 cycles/hour filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 101K0185, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 60 and 120 mg/mL
and then homogenized using an Ultraturrax laboratory mixer pending 5 minutes and a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours at room temperature).
Details on mating procedure:
- M/F ratio per cage: 1 male/1 female of the same dose group
- Length of cohabitation: during the night
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- Each female was placed with the same male until mating occured or 14 days had elapsed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and homogeneity were determined on samples of each control and test item dosage form prepared for use in week 1, 7 and 13.
Three samples of each dosage form (from top, middle and bottom of the container) were taken on each occasion. They were stored at -200 °C pending dispatch (two consignments). The samples were sent on dry ice, to the Sponsor. These analyses were carried out under the responsibility of the Sponsor.
Duration of treatment / exposure:
Exposure period: males: throughout the pre-mating period (10 weeks), mating period (2 weeks), and until sacrifice;
females: throughout the pre-mating period (10 weeks), mating period (2 weeks), and pregnancy until day 14 post-coitum inclusive.
Frequency of treatment:
daily/ 7 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Dose / conc.:
1 200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were specified by the Sponsor and based on the available toxicological data obtained in a combined repeat dose toxicity/developmental toxicity screening test with the test item : 600 mg/kg/day as the expected No Adverse Effect Level, 1200 mg/kg/day as themaximum feasible dose-level.
The oral route was selected since it is a possible route of exposure in human and it is requested by the regulatory authorities for this type of test item.
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded once a week until sacrifice. The body weight of each female was recorded once a week during the pre-mating and mating periods, then on days 0, 7 and 15 post-coitum.


Oestrous cyclicity (parental animals):
For technical and scientific reasons, the estrous cycle was not taken into account for the interpretation of the data.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
not examined
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after most hysterectomies of the females had been performed]
- Maternal animals: All surviving animals [on day 15 post coitum, females which did not mate: at least one week after the end of the mating period]

GROSS NECROPSY
- Gross necropsy consisted of a macroscopic post-mortem examination of the principal thoracic and abdominal organs (with particular attention paid to the reproductive organs) was performed on all animals, including those that died during the study or were killed prematurely. For all females, the number of corpora lutea and implantation sites was recorded whenever possible. Whenever necessary, photographs were taken to document findings and kept with the study archives.

HISTOPATHOLOGY / ORGAN WEIGHTS
No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment.
The body weight of all animals killed at terminal sacrifice was recorded and the following organs were weighed (wet) as soon as possible after dissection:
all males: testes, epididymides, prostate, seminal vesicles together with coagulating glands, pituitary gland and adrenals, testes and epididymides were weighed separately, all females: uterus, ovaries, pituitary gland and adrenals.
Postmortem examinations (offspring):
The pups were discarded without further investigation.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Reproductive indices:
Mating data, Male fertility data, Female fertility data (Female fertility and gestation indices, Hysterectomy parameters)
Offspring viability indices:
not examined
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
The few clinical signs recorded in three males given 600 mg/kg/day (piloerection or chromodacryorrhea and chromorhinorrhea) and one male given 1200 mg/kg/day (ptyalism) were not considered to be of toxicological importance since the incidence of these findings was low and not dose-related.
Females:
The few clinical signs (ptyalism) recorded in one female given 600 mg/kg/day and three females given 1200 mg/kg/day did not represent an adverse effect. Other observations (area of hair loss on the forelimb or hindlimb) recorded in some males and females are among findings commonly observed in rats of this strain and age.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males:
No mortality was recorded in the control or the 1200 mg/kg/day groups.
One male given 600 mg/kg/day was found dead on day 44. At macroscopic post-mortem examination, esophagus perforation was noted together with serous contents in the thoracic cavity and whitish deposit on the pleura. This death was considered of accidental nature and without any relationship to treatment with the test item.
Females:
There was no death, in any control or treated group over the treatment period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
There was a minimal non significant decrease in body weight gain in males given 600 (-6%) and 1200 mg/kg/day (-5%) over the treatment period (days 1 to 92). Because these minor changes in body weight gain were not statistically different, not dose-related and did not correlate with any change in food consumption, they were considered to be marginal and of no toxicological significance.
Females:
During the pre-mating period (days 1 to 71), the body weight gain at 600 mg/kg/day was similar to that of the controls while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption, were considered to be related to treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
The food consumption of the treated males was similar to that of the control group.
Females:
During pre-mating (days 1 to 71), the food consumption was similar in the control and the treated groups.
During pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0 .01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels respectively.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
The minimal fluctuations recorded in the seminology parameters in the treated groups were not dose-related, not notably different from the controls, and in the range of CIT historical control data. Consequently, they were not considered of toxicological significance.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The female mating index (mated/paired) and pre-coital interval were similar in all groups. Most paired animals mated within 1 to 4 days of cohabitation except for a few pairs in each group, for which the duration was slightly longer. This finding, commonly recorded at this low incidence was not attributed to treatment with the test item. The slight fluctuations in male fertility parameters which were not dose-related are commonly recorded in rats of this strain and age, consequently, they were not considered to be related to the treatment with the test item.
The treatment with the test item did not disturb fertility and gestation indices of the females at any dose-level. The number of corpora lutea was minimally lower in the treated groups when compared to the controls. Since the difference was small, not statistically significant and within the range of CIT historical control data, these findings were considered to have occurred by chance. Hence at 600 and 1200 mg/kg/day the number of implantation sites and concepti was also lower than the control, gaining statistical significance only at-the top dose-level.
Since these values were also within CIT historical control data, they were considered to be a consequence of the fortuitous lower number of corpora lutea rather than a test item related effect. The slight fluctuations in the pre- and post-implantation losses were not considered to be related to the treatment with the test item since they were not dose-related and/or not significantly different from either the concurrent control or the CIT historical control data.
Key result
Dose descriptor:
other: LOAEL, systemic toxicity (female)
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Overall effects
Key result
Dose descriptor:
other: NOAEL, systemic toxicity (male)
Effect level:
1 200 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Overall effects
Key result
Dose descriptor:
NOAEL
Remarks:
fertility, gonadal function and reproduction
Effect level:
1 200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed on number of corpora lutea, number and distribution of dead and live concepti, number and distribution of early and late resorptions and number and distributionof implantation sites
Remarks on result:
other: the study focused on fertility/reproduction only and there was no 2nd generation
Reproductive effects observed:
no

Additional data:

ORGAN WEIGHTS: Females: significantly lower absolute and relative ovary and uterus weights (ovaries: low, high-dose/absolute: -12, -13%; low,  high-dose/relative: -7, -4%  as compared to controls; uterus: low,  high-dose/absolute: -19, -21%; low, high-dose/relative: -14, -12% as compared to controls). These differences were considered not to be treatment related, as most of the values were within the range of the control group, and changes were only due to the contribution of a few individual values of two animals. Males: significantly, but not dose-related higher absolute and relative adrenal weights (low, high-dose/absolute: +24, +18%; low, high-dose/relative: +29, +23% as compared to controls).  Because of the lack of a dose-response and because only seen in males, these effects were considered as of no biological significance.  MATING DATA: The female mating index (mated/paired) and pre-coital interval were similar in all groups (control, low, high-dose: female mating index 96 -  96 - 96%, mean pre-coital interval 2.7 - 2.8 - 3.2 days).

MALE FERTILITY DATA:

The male mating index and the male fertility index were similar in all groups (male mating index: control, low, high-dose: 100 - 96 - 96 %, male fertility index: 100 - 91.3 - 91.3 %).

FEMALE FERTILITY DATA:

The following was obtained for the control, low, and high-dose groups, respectively:

Mated females: 24, 24; 24;

Pregnant females: 24, 21, 22;

Female fertility index (%): 100, 87.5, 91.7;

Females with live concepti: 24, 21, 22;

Gestation index (%): 100, 100, 100.

NUMBER OF CORPORA LUTEA, NUMBER OF IMPLANTATIONS (control, low, high-dose groups): corpora lutea: 17.6, 16.4, 16.0; Implantation sites: 16.6, 14.9, 14.5* (p<0.05); Pre-implantation loss (%): 5.7, 9.6, 9.1; Concepti: 15.2, 14.3, 13.0* (p<0.05); Post-implantation loss (%): 8.3, 4.2, 11.0.

Conclusions:
There were no substance-related effects on the male and female reproductive performance. In the light of these results, the lower mating index in a previous study is considered to be incidental and not related to the test substance.
Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 408 guideline
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 2015-03-24
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models GmbH, Sulzfeld, Germany.
- Age at study initiation: 42 ± 1 days
- Weight at study initiation (mean): Males:164.4 g; Females: 128.6 g
- Fasting period before study: No
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm²)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Yes, 8 days.

DETAILS OF FOOD AND WATER QUALITY:
- Food analyses: The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1 × 10^5/g food.
- Drinking water analyses: The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings, the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod: 12 h light/ 12 h dark
Route of administration:
oral: gavage
Vehicle:
other: Drinking water containing 0.5% Carboxymethylcellulose
Details on exposure:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 0.5% Carboxymethylcellulose was filled up to the desired volume and subsequently homogenized with an Ultraturrax. During administration, the test substance preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced once a week, at least. The administration volume was 10 mL/kg body weight.
Details on mating procedure:
Not performed
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. The stability of the test substance in drinking water containing 0.5% Carboxymethylcellulose at room temperature for a period of 7 days was proven before the start of the study (study code 15L00246). Homogeneity was verified in 3 samples in the highest and lowest concentrations (were used as a concentration control at the same time) before the start of the administration period; additional concentration control analyses were verified in the mid concentrations. Moreover, concentration control analyses were verified in 1 sample of each concentration towards the end of the administration period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Details on study schedule:
see 7.5.1
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
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:
see 7.5.1
Positive control:
none
Parental animals: Observations and examinations:
see 7.5.1
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters were determined in the right
testis or right epididymis of all male F0 parental animals and cohort 1A males sacrificed on
schedule:
Sperm motility examinations were carried out in a randomized sequence.
Parameters:
- Sperm motility [%]
- Sperm morphology [%]
- Sperm head count (cauda epididymis) [Mio/g cauda epididymis]
- Sperm head count (testis) [Mio/g testis]

Litter observations:
none
Postmortem examinations (parental animals):
see 7.5.1
Postmortem examinations (offspring):
none
Statistics:
see 7.5.1
Reproductive indices:
none
Offspring viability indices:
none
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation shortly after treatment was observed in 7 male and 8 female animals of test group 3 (1000 mg/kg bw/d) on several days of the study. From the temporary, short appearance immediately after dosing it was concluded that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. No test substance-related effects were obtained in test groups 1 and 2 (100 and 300 mg/kg bw/d).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related impairment in body weight parameters were observed for male and female animals in test groups 1-3 (100, 300 and 1000 mg/kg bw/d). Mean body weight of male animals in test group 3 (1000 mg/kg bw/d) was slightly but not significantly lower towards the end of the administration period, with a maximum of -4.2% on study day 91. The same was true for female animals of test group 2 (300 mg/kg bw/d), showing a maximum deviation of -6.5% on study day 77. These findings were assessed to be incidental and not related to treatment. Mean body weight change values were significantly decreased in female animals of test groups 2 (300 mg/kg bw/d) study day 77. The change was assessed to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of male animals in test group 3 (1000 mg/kg bw/d) was slightly lower, with a maximum reduction of -6.0% at the end of the administration period. Due to the marginal occurrence, these changes were assessed as being incidental and not related to treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
After the three months administration period in males of test group 2 (300 mg/kg bw/d) mean corpuscular volume (MCV) was lower compared to controls, but it was not dose dependently changed.
In males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) absolute monocyte counts were higher compared to controls, but the values were within the historical control range (absolute monocytes 0.07-0.15 Giga/L).
Therefore, both alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
After the three months administration period in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d) urea levels were increased. The mean of test group 3 was within the historical control range, that one of test group 1 above this range (urea 5.31-7.10 mmol/L). However, the urea level was not dose-dependently changed. In females of test group 3 (1000 mg/kg bw/d) sodium levels were lower compared to controls, but the values were within the historical control range (sodium 139.8-145.4 mmol/L). Therefore, both alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
In females of test group 3 (1000 mg/kg bw/d) urine volume was decreased and urine specific gravity was increased. These findings without any other changes of kidney parameters reflect the physiological adaptation of the kidneys towards lesser fluid income and, therefore, were regarded as adaptive and not adverse.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. The following examinations were performed during FOB and were assessed individually:
- Home cage observations
- Open field observations
- Sensorimotor tests/reflexes
- Quantitative parameters
Overall, no test substance related effects were observed.

Motor activity measurement:
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). At interval No. 7 a decreased value was measured for male animals of test group 1 (100 mg/kg bw/d). The individual deviation was assessed not to be related to treatment as no dose-response relationship occurred.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the kidneys of male animals there was a dose- and treatment-related increase in incidence and severity in eosinophilic droplets visualized by CAB staining, which was accompanied by an increased incidence of basophilic tubules (mostly of minimal severity) and, in test group 3, by tubular casts. Incidences and grading are shown in the table 3 in section "Any other information on results incl. tables".
In females, 3 animals of test group 3 (1000 mg/kg bw/d) showed a tubular cast. This was, however, unilateral and CAB staining for protein was negative in tubules. Therefore, these findings were regarded as incidental. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test substance-related effects on estrous cycle length and the number of cycles were obtained.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment-related effects were observed.
Reproductive performance:
not examined
Dose descriptor:
NOAEL
Remarks:
fertility parameters
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed on male and female reproductive organs
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
other: No F1 generation
Generation:
F1
Sex:
male/female
Basis for effect level:
other: 90-day study, no F1 generation
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The toxicity to reproduction was assessed in an extended one-generation reproductive toxicity test conducted with male and female rats according to OECD TG 443 (2018), under GLP conditions (RL1). The study is considered of reliable quality and validity, fulfills the criteria of a key study and thus, is suitable for assessment of the present endpoint.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity studies via the oral route


Rat


 


N,N' (isobutylidene)diurea was tested in several modern studies for its effects on the reproductive function:


 


The first study (BASF AG, 2003) was performed in accordance with the OECD guideline 422. 10 male and 10 female Sprague-Dawley rats per group were treated by gavage with 0, 100, 300 or 1000 mg IBDU/kg bw/day. Males were treated during pre-mating (15 days), mating (max. 14 days) and post-mating for a total of 34 days, and females during pre-mating (15 days), mating (max. 14 days), and pregnancy and lactation until day 4 post partum. The mating index in this study was 100% for the pairs of the control, 100 and 300 mg/kg bw/day groups, but was lower in the 1000 mg/kg bw/day group (70%). As no treatment-related morphological changes were noted in the reproductive organs, this finding was considered to be incidental. No effects of the substance were seen with regard to the time to insemination, fertility, duration of gestation, gestation index, number of implantation sites, and on postimplantation and neonatal losses. The number of corpora lutea was similar in the 0, 100 and 300 mg/kg bw/day groups. In the group treated with 1000 mg/kg bw/day the number was slightly lower (18 versus 23 per female). As the counts in all groups (including the high dose) were higher than the laboratory`s historical controls, and because there were no microscopic changes in the ovaries, this finding was considered as a chance event. A few microscopic abnormalities were recorded with minimal severity in seminiferous tubules of a small number of treated males. These incidences were considered to be without relationship to the treatment and most probably fortuitous, although not found in the control males. There were no stillborn pups or runts. The number of pups which died during the four days of observation after birth was low (0 - 5%) and similar across all groups. Moreover, there were no notable clinical signs in the pups, and the pup body weights were comparable across all groups. Lastly, the treatment with IBDU had no effect on the sex ratio of the pups up to the highest dose tested


 


The following “No Observed Adverse Effect Levels” (NOAELs) were deduced from this study:


NOAEL, General Toxicity (male): 1000 mg/kg bw/day (highest tested dose)


NOAEL, General Toxicity (female): 300 mg/kg bw/day (reduced body weight gain in females during pregnancy and lactation at 1000 mg/kg bw/day).


NOAEL, Reproduction (male, female): 1000 mg/kg bw/day (highest tested dose).


NOAEL, F1 (male, female): 1000 mg/kg bw/day (highest tested dose)


 


The second study (BASF AG, 2003) was performed as a one-generation study partially following OECD test guideline 416 and beeing similar to an OECD 443 lacking the 2nd generation and the DIT and DNT cohorts. This was because the study focused on fertiliy and reproduction endpoints and therefore lacked the 2nd generation, and only had a limited assessment of endpoints regarding the parental (1st) generation. The study was undertaken to further evaluate the toxicological relevance of the reduced mating index that was found at 1000 mg/kg bw/day in the earlier study (BASF AG, 2003). 25 male and 25 female Sprague-Dawley rats per group were treated by gavage with 0, 600 or 1200 mg IBDU/kg bw/day. Females were treated throughout the pre-mating period (10 weeks), during mating (max. 2 weeks), and during pregnancy until day 14 post-coitum, inclusive. Males were treated throughout the pre-mating period (10 weeks), during mating (2 weeks), and until sacrifice (i.e. when most of the hysterectomies in females were completed).There were no substance- related deaths, nor were any substance-related clinical signs observed. Females showed lower body weight gain during pregnancy, and during pre-mating. No significant effects were found in males. The food consumption was also slightly lower in females of the high-dose group during pregnancy, and during pre-mating and pregnancy in the low-dose, but not statistically significant. There were no pathological findings at necropsy. Slight changes in organ weights (females: ovary and uterus, males: adrenals) as compared to the controls were not considered to be substance-related, because of the lack of a dose-response in males, and as most of the values were within the range of the control group, and changes were only due to the contribution of a few individual values (females). Seminology findings in the treated groups were not different from controls. Furthermore, the female mating index (96% at 0, 600 and 1200 mg/kg bw/day) and pre-coital interval were similar in all groups, as were the male mating index (100%, 96% and 96% at 0, 600 and 1200 mg/kg bw/day respectively) and the male fertility endpoints. The treatment had also no effect on female fertility endpoints and gestation indices. The number of corpora lutea was minimally lower in the treated groups when compared to the controls, but since the difference was small, not statistically significant, and within the range of the laboratory`s historical control values, this finding was considered as a chance event. The number of implantation sites and concepti was consequently also lower than in the controls, gaining statistical significance only at the top dose. However, also the values for implantation sites and concepti were within the laboratory’s historical controls. The slight fluctuations in the pre- and post-implantation losses were not considered treatment-related, since they were not dose-related and/or not different from either the concurrent or historical controls. Thus, there were no effects observed on the offspring based on these limited examinations.


 


The following “No Observed Adverse Effect Levels” (NOAELs) were deduced from this study:


NOAEL, General Toxicity (male): 1200 mg/kg bw/day (highest tested dose)


LOAEL, General Toxicity (female): 600 mg/kg bw/day (reduced body weight gain during pregnancy)


NOAEL, Reproduction (male, female): 1200 mg/kg bw/day (highest tested dose).


 



Additionally, a 90-Day Repeated-Dose Toxicitiy study (BASF SE, 2017) is available, where the toxicity of IBDU was assessed according to OECD guideline 408 (1998). The test substance was administered by oral gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300 and 1000 mg IBDU/kg bw/day over a period of 3 months. Drinking water containing 0.5% Carboxymethylcellulose served as vehicle. No treatment-related, adverse effects were observed on sperm parameters, estrous cycle length and the number of cycles, as well as reproductive organs up to the limit dose of 1000 mg/kg bw/day. For further details please refer to the repeated dose toxcicity section.


 


Finally, the most recent study available on reproductive toxicity is an Extended One-Generation Reproductive Toxicity Test (CompoExpert, 2021) according to OECD guideline 443 (2018) - basic test design (Cohorts 1A and 1B without extension). The test substance was administered by oral gavage to groups of 24 male and 24 female Sprague-Dawley rats at dose levels 100, 300 and 1000 mg/kg bw/day, plus one concurrent vehicle control (0.5% Carboxymethyl cellulose). For the P-generation, the treatment period started two weeks before mating, was continued during mating and until at least 6 weeks after mating for males, or throughout gestation and up to Day 20 of lactation for females. The F1-generation was treated from Day 21 to Day 90 of age (100 weeks) for Cohort 1A animals, and from Day 21 - Day 97 of age (11 weeks) for Cohort 1B animals.


In the parental generation, no mortality attributable to the test substance occurred. The treatment did not affect food intake or estrous cycling, gestation or parturition. Furthermore there was no effect 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, but the incidence of apparent infertilities was comparable between these groups, and thus, no relationship to the test substance was assumed. There were no effects on body weight gain. At 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. Furthermore, there were non-adverse changes in organ weights: in males at all dose levels and females at 1000 mg/kg bw/day, the absolute group mean brain weight was slightly, but statistically significantly, lower than controls (up to 4% or 3% lower for males and females, respectively; p≤0.01 for males and p≤0.05 for females); in the males, the decrease was not dose-related. Although the aetiology of this finding is unclear as there were no corresponding microscopic changes, the individual values were generally within the lower limit of the normal historical range and/or within the values for the concurrent controls. Also in males, there was a non-dose related increase in group mean body weight-related spleen weights at all dose levels compared with controls (up to 12% higher; p≤0.01). There was a large degree of individual variation in the data and no corresponding pathology findings. At 1000 mg/kg bw/day, there was a slight increase in group mean body weight-related liver weight for the males and females compared with controls (up to 4% or 5%, respectively; p≤0.05 for the adjusted weights in females only). Due to the minimal nature of the increase and the absence of a corresponding microscopic change, this was considered to represent an adaptive change and not to be adverse. There were equivocal findings in the mammary glands of two females receiving the high dose and one female receiving the middle dose, presenting with ductal dilataion and/or inflammation/firbroplasia, which correlated with macroscopic findings of an irregular, firm mass with cream contents in two cases.


In the filial generation (F1), no mortality or abnormal clinical signs attributed to maternal administration of the test substance occurred. Pups in treatment groups were heavier than controls the day after birth, but due to a relatively low pup body weight in a small proportion of control group litters and comparability of body weight by Day 4, this was not considered to be adverse. Food intake was slightly higher in cohort 1B males in the mid- and high-dose groups. Even though this effect reached statistical significance, 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 substance administration. Anogenital distance and nipple counts were unaffected by the treatment with the test substance. Sexual maturation was not affected in either sex, even though balanopreputial separation at the high-dose group was completed marginally later when compared to controls. This was, however, attributable to the delay in two individuals only. Since the effect was only noted in cohort 1B males, but not in cohort 1A, it was not considered to be related to the treatment. 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. Minimal changes to the immune system were observed in terms of haematology data, i.e. a non-adverse increase in total white blood cell concentrations in males at all dose levels and females of the mid- and high doses, and increases in thymus weights at all dose levels, increases in spleen weights in pups and increases in distal lymph node weights at 1000 mg/kg bw/day. These findings were considered non-adverse and not indicative of systemic immunotoxicity due to the minmal nature and absence of corresponding pathological changes. Sperm parameters remained unaffected by treatment with the test substance; the effects seen could be attributed to normal biological variation or a singular atypical case. Similarly, due to a high individual variation in the number of primary follicles, the slightly lower group mean total number at 1000 mg/kg bw/day was concluded not to be associated with the treatment. There was a slight and non-statistically significant increase in mean TSH concentration for male pups of the 1000 mg/kg bw/day dose group (19%), which was considered to fall within the limits of normal variation. Macroscopic findings were confided to pelvic dliatation in the kidney for males given 300 and 1000 mg/kg bw/day, which was correlated with mottled discolouration in cohort 1B animals and microscopically with hyaline droplets accumulation. This effect was indeed considered test item-related, but not relevant to humans. Apart from the aforementioned changes in spleen and thymus weights, brain and liver weights were also slightly affected at the high dose animals, but relevance to the test item administration remains unclear for the decrease in brain weights. Liver weight changes were considered adaptive, and non-adverse. Lastly, there were no adverse changes in blood chemistry parameters during either generation, with the only test item-related finding seen in the F1 generation males being a slight increase in globulin concentration, with corresponding increase in calcium.


 


The following “No Observed Adverse Effect Levels” (NOAELs) were deduced from this study:


NOAEL P0 (reproductive and developmental toxicity): 1000 mg/kg bw/day in males and females


NOAEL P0 (systemic toxicity): 1000 mg/kg bw/day in males and females


NOAEL F1 (reproductive and developmental toxicity): 1000 mg/kg bw/day in males and females


NOAEL F1 (systemic toxicity): 300 mg/kg bw/day in males and 1000 mg/kg bw/day in females


LOAEL F1 (systemic toxicity): 1000 mg/kg bw/day in males, based on alpha-2-microglobulin nephropathy in the kidneys


 


In addition to these studies, the following information on toxicity to reproduction/developmental toxicity is available from QSAR models:


 


The estrogen-receptor binding potential of CAS # 6104-30-9 was assessed in the QSAR Toolbox 3.3.0.152 and the VEGA QSAR Models Relative Binding Affinity model (IRFMN) 1.0.1. Both models come to the conclusion that CAS # 6104-30-9 is inactive/ a non-binder towards the ER receptor.


 


The potential of CAS # 6104-30-9 to induce Developmental/Reproductive Toxicity was assessed in the VEGA QSAR Models Developmental/Reproductive Toxicity library (PG) 1.0.0. The model predicts that CAS# 6104-30- 9 is a NON-Toxicant with regards to Developmental and Reproductive activity.


 


In addition, the potential of CAS # 6104-30-9 to induce Developmental Toxicity was assessed in the VEGA QSAR Models Developmental Toxicity model (CAESAR) 2.1.7. This models also predicts that CAS # 6104-30-9 is a NON-Toxicant with regards to Developmental toxicity activity.


 


Reproductive toxicity studies via the inhalation route


No studies are available addressing reproductive toxicity via inhalation exposure to the test substance.


 


Reproductive toxicity studies via the dermal route


No studies are available addressing reproductive toxicity via dermal exposure to the test substance.


 


Conclusion on reproductive toxicity


Even though there were several studies available that provide information on the reproductive toxicity potential of the test substance, only the most recent study (UKK0004, Compo Expert) complied with current guidelines. In this study, the test substance was administered to male and female rats orally via gavage at the three dose levels 100, 300, and 1000 mg/kg bw/day, plus a concurrent vehicle control group. No effects with respect to reproductive toxicity and attributable to the test substance adminstration were observed. Therefore, the NOAEL for reproductive toxicity of the test substance was 1000 mg/kg bw/day in males and females.



Effects on developmental toxicity

Description of key information

Data from a screening test (BASF AG, 2003), two developmental toxicity studies (BASF AG, 1993, BASF SE, 2017) and an extended one-generation reproductive toxicity test (CompoExpert, 2022) indicate that the test substance has no effects on the development of the offspring and that no teratogenic effects were observed.


 


Developmental/reproductive screening test (BASF, 2003)


NOAEL (maternal general toxicity): 300 mg/kg bw/day


LOAEL (maternal general toxicity): 1000 mg/kg bw/day


NOAEL (teratogenicity, developmental toxicity): 1000 mg/kg bw/day (highest dose tested)


 


Developmental toxicity study (BASF AG, 1993)


NOAEL (maternal general toxicity): 1000 mg/kg bw/day (highest dose tested)


NOAEL (fetotoxicity, teratogenicity): 1000 mg/kg bw/day (highest dose tested)


 


Developmental toxicity study (BASF SE, 2017)


NOAEL (maternal general toxicity): 100 mg/kg bw/day (nominal)


LOAEL (maternal general toxicity): 300 mg/kg bw/day (nominal)


NOAEL (developmental toxicity): 100 mg/kg bw/day (nominal); considered secondary due to maternal toxicity


LOAEL (developmental toxicity): 300 mg/kg bw/day (nominal); considered secondary due to maternal toxicity


 


Extended One Generation Reproductive Toxicity test (CompoExpert, 2022)


NOAEL (P0; general toxicity): 1000 mg/kg bw/day (highest dose tested)


NOAEL (P0; developmental/reproductive toxicity): 1000 mg/kg bw/day (highest dose tested)


NOAEL (F1; general toxicity; males): 300 mg/kg bw/day


NOAEL (F1; general toxicity; females: 1000 mg/kg bw/day (highest dose tested)


NOAEL (F1; developmental toxicity): 1000 mg/kg bw/day (highest dose tested)


Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 2015-03-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
rabbit
Strain:
New Zealand White
Remarks:
(Crl:KBL(NZW))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-17 weeks
- Weight at study initiation: 3347 - 4302 g
- Fasting period before study: None
- Housing: Singly in Type 4X03B700CP cages (floor space 4264 mm2, internal height 450 mm). For enrichment, wooden gnawing blocks were added (Typ KNH E-041, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet: Pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Portable tap water in water bottles; ad libitum
- Acclimation period: Yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs light / hrs dark): 12/12

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.



Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The analyses of homogeneous distribution of the test substance in the vehicle as well as correctness of the prepared concentrations are performed in a separate GLP study which was not completed by the end of January 2017. The present study report is being finalized without the results of these analyses because of urgent authority submission requirements of the toxicology results. This has no impact on the outcome of the present toxicology study. The results of these analyses will be covered in a GLP-compliant Amendment to the Report for submission to sponsor/authority as soon as they become available.

The concentrations in the endpoint study record and the endpoint summary are thus currently the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: artificial insemination (day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1)
Duration of treatment / exposure:
Gestation days (GD) 6 through 28
Frequency of treatment:
Daily
Duration of test:
Terminal sacrifice on GD 29
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily (GD 0-29)
- Cage side observations checked: morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (GD 6-28)

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Daily during GD 0-29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: heart, kidneys
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Statistical analyses were performed according to the following:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females preg-nant at terminal sacrifice, number of litters with fetal findings.
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
Historical control data:
Yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female, each, of the control (No. 21 - 0 mg/kg bw/d, GD 22), mid-dose (No. 57 - 300 mg/kg bw/d, GD 29) and high-dose (No. 91 - 1000 mg/kg bw/d, GD 28) groups had blood in bedding.
In total, reduced defecation was observed in four control, three low-dose, two mid-dose and nine high-dose females (0, 100, 300 and 1000 mg/kg bw/d). No defecation was observed in two females of test group 3. Incidence and distribution of these findings do not indicate a relationship to the test substance.
There were no clinical findings in the other does in the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (No. 3 - 0 mg/kg bw/d), one low-dose female (No. 37 - 100 mg/kg bw/d) and two high-dose females (Nos. 83 and 84 - 1000 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, these were considered to be spontaneous incidents.
Low-dose female No. 28 (100 mg/kg bw/d) was found dead on GD 21. The gross pathological examination of this animal revealed findings indicative of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight:
No statistically significant difference was observed for the mean body weights (BW) of the high-dose females (1000 mg/kg bw/d) when compared to the concurrent control group. However, from GD 19 onwards the high-dose body weights were distinctly lower than the body weights of the other groups including the control. Also, the mean body weight gain (BWC) of the high-dose rabbits was statistically significantly below control on GD 25-28. During the treatment period (GD 6-28) the does of the high-dose group gained 69% less body weight than the control does.
Though not statistically significant, the body weight gain during treatment (GD 6-28) was also reduced at the mid-dose level by 33%, which indicates beginning systemic toxicity at 300 mg/kg bw/d.
The mean BW and the average BWC of the low-dose group (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Corrected (net) body weight gain:
The corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 3 (1000 mg/kg bw/d). Furthermore, the carcass weight of the high-dose does was also reduced, but without attaining statistical significance (about 5% below controls). Though not statistically significant, the corrected body weight gain during treatment (GD 6-28) was also reduced in the mid-dose does. These effects are assessed as test substance-related signs of maternal toxicity.
Mean carcass weights and the corrected body weight gain were not significantly different in test groups 0 and 1 (0, 100 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in test group 3 (1000 mg/kg bw/d) was reduced during major parts of the treatment period, attaining statistical significance on GD 14-17 and GD 27-29. During the treatment period (GD 6-28) the high-dose does consumed 16% less food than the concurrent control does.
Though not statistically significant, the food consumption during treatment (GD 6-28) was also reduced at the mid-dose level by 8%.
The food consumption of the low-dose rabbits (100 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Uterus weights:
The mean gravid uterus weights of the rabbits of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Weight of the placentae:
The mean placental weights in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were lower than in the concurrent control group, however, the difference was small and did not gain statistical significance. The mean placental weights in test group 1 (100 mg/kg bw/d) were similar to the control value.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Petechiae in lungs of one high-dose doe (No. 84 - 1000 mg/kg bw/d).
Stomach filled with very dry, hard feed in one high-dose doe (No. 78 - 1000 mg/kg bw/d).
Watery feces in two mid-dose does (Nos. 52 and 67 - 300 mg/kg bw/d) and eight high-dose does (Nos. 77, 78, 79, 83, 84, 85, 90, 97 - 1000 mg/kg bw/d).
Findings indicative of misgavaging, i.e. thoracic cavity filled with blood and test substance, in low-dose doe No. 28 (100 mg/kg bw/d - died on GD 21).
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One control female (No. 3 - 0 mg/kg bw/d), one low-dose female (No. 37 - 100 mg/kg bw/d) and two high-dose females (Nos. 83 and 84 - 1000 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, these were considered to be spontaneous incidents.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for the pre- and the post-implantation losses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In the mid-dose group (300 mg/kg bw/d) the mean number of early resorptions was slightly, but statistically significantly, increased. This is assessed as incidental, as the total number of resorptions (10.1 mean%) was well within the historical control range (HCD Re-sorptions: mean% 7.2 [2.4 - 12.6]) and there is no dose-response.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found at cesarean section of mid-dose doe No. 52. Alike abortions this is considered as an expression of maternal toxicity in rabbits, in particular as this animal suffered from markedly reduced food consumption and body weight loss near term.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Female rabbits were placed into the study in four cohorts. The conception rate was 84% in the mid-dose group (300 mg/kg bw/d), 88% in the low- and high-dose groups (100 and 1000 mg/kg bw/d) and 92% in the control group (0 mg/kg bw/d). There were no test substance-related biologically relevant differences between the different test groups in conception rate.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Details on maternal toxic effects:
Test group 1 (100 mg/kg bw/d):
- No test substance-related adverse effects observed.

Test group 2 (300 mg/kg bw/d):
- Reduced mean food consumption, 8% below control on GD 6-28
- Reduced mean body weight gain, 33% below control on GD 6-28
- Corrected body weight gain below control (-267.2 g vs. -209.4 g in control)

Test group 3 (1000 mg/kg bw/d):
- Reduced mean food consumption, 16% below control on GD 6-28
- Reduced mean body weight gain, 69% below control on GD 6-28
- Corrected body weight gain distinctly below control (-410.6 g vs. -209.4 g in control), lower carcass weight (about 5% below controls).
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Weight of fetuses:
The mean fetal weights of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were statistically significantly reduced (about 11% and 13% below control - both sexes combined). The mean fetal weights of test group 1 (100 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
no effects observed
Description (incidence and severity):
External malformations did not occur in any fetuses in this study and no external variations were recorded.
Two unclassified external observations, i.e. necrobiotic placentae and discolored placentae, were recorded in one litter of the mid-dose group (300 mg/kg bw/d) and in two individual litters of the high-dose group (1000 mg/kg bw/d).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in test groups 1 and 2 (100 or 300 mg/kg bw/d). No statistically significant differences between the test substance-treated groups and the control were noted and no dose-response relationship was observed.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton. Generally, all skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at comparable or higher frequency.
One skeletal variation (incomplete ossification of talus; cartilage present) was noted at an incidence above the concurrent and historical control in the high-dose group (1000 mg/kg bw/d). The other increased incidences of skeletal variations were either not related to dose or they were inside the historical control range. Therefore, these minor changes are not considered as treatment-related adverse events.
As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this New Zealand White rabbit strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. They are most likely related to the lower pup body weights which were considered to be a consequence of a distinctly lower body weight gain of the dams in the high-dose group, particularly in the last week of pregnancy. Notably, the marginal increase of these changes did not affect the overall rate of variations.

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups 0-3. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of soft tissue malformations occurred in all test groups including the control (0, 100, 300 or 1000 mg/kg bw/d). Nine cases of small spleen were observed in one litter of the high-dose group - dam No. 94. No findings in organs of related ontogenetic origin is seen in the other offspring of this test group. Thus the clustering of this observation in just one litter suggests a non-treatment related origin of the spleen findings. Other malformations, such as absent subclavian, absent gallbladder and misshapen thoracic vertebra were scattered observations in individual fetuses of test groups 0-2. No statistically significant differences between the groups were noted. They were not dose-related and some of them can be found in the historical control data. An association of these findings to the treatment is not assumed.

Soft tissue variations occurred in all test groups including the control. The examinations of the organs revealed dilated cerebral ventricle, malpositioned carotid branch, malpositioned carotid origin and a small gallbladder in individual fetuses of test groups 1 and 2 (100 and 300 mg/kg bw/d). An absent lung lobe (lobus inferior medialis) was noted in all test groups and showed a slightly but statistically significantly increased incidence in the high-dose group (2.8% affected fetuses/ litter vs. 0.6% in control); also slightly above the historical control range. (0.0 - 2.7%). As this is a commom variation in this rabbit strain which is unlikely to adversely affect health of the developing offspring, and the incidence was just above the historical range, no toxicological relevance is attributed to this finding. Although the total incidence of soft tissue variations were statistically significantly increased in test groups 2 and 3, these incidences are well covered by the historical control range (HCD: 2.2% [0.4 - 5.0]). Thus these increases are also not considered to be of toxicological relevance.

Two unclassified soft tissue observations were recorded: a (reddish) discolored thymus in one mid-dose and three high-dose fetuses in three different litters (300 and 1000 mg/kg bw/d), and a blood coagulum around urinary bladder in one control fetus (0 mg/kg bw/d). The affected fetuses/litter incidence of discolored thymus was statistically significantly increased in test group 3. However, this is considered to be a mere notice which is not supposed to have any adverse effect on the developing offspring. In terms of the overall rates of soft tissue unclassified observations no statistically significant differences between the groups were noted.
Details on embryotoxic / teratogenic effects:
Test group 1 (100 mg/kg bw/day):
- No test substance-related adverse effects observed.

Test group 2 (300 mg/kg bw/day):
- Reduced fetal weight (about 11% below control)

Test group 3 (1000 mg/kg bw/day):
- Reduced fetal weight (about 13% below control)
- Increased incidence of one fetal variation indicating developmental delay

Additional information on fetal external, soft tissue and skeletal observations:
External malformations did not occur in any fetuses in this study. There were noted soft tissue and skeletal malformations in all test groups (0, 100, 300 or 1000 mg/kg bw/d) which are described in detail in the respective sections obove.
Four fetuses were multiple-malformed. Female low-dose fetus No. 27-07 (100 mg/kg bw/d) had a malpositioned, small kidney and an absent subclavian. For male mid-dose fetus No. 55-08 (300 mg/kg bw/d) a thoracic hemivertebra, a misshapen thoracic vertebra and a fused rib were recorded, while the findings in male fetus No. 9 of the same litter (No. 55-09 - 300 mg/kg bw/d) consisted of a thoracic hemivertebra, a misshapen thoracic vertebra and a branched rib. Furthermore, female high-dose fetus No. 94-07 (1000 mg/kg bw/d) had a small spleen and multiple malformations of the great vessels, i.e. narrowed pulmonary trunk, dilat-ed aorta, dilated carotid (right side) and absent carotid (left side).
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Table 1: Mean maternal body weight change during gestation

 

 

0 mg/kg bw/d

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Days 0 to 6

Mean

S.D.

N

190.4 D

78.16

23

170.9

105.47

21

187.0

68.53

21

163.7

66.91

22

Days 6 to 28

Mean

S.D.

N

222.3 D

129.20

22

272.5

189.21

20

172.8

165.01

21

66.9*

202.66

20

Days 0 to 29

Mean

S.D.

N

438.6 D

162.11

22

477.3

207.14

20

366.4

205.74

21

263.0*

225.45

20

Statistics: D=Dunnett-test (two-sided); *: p<=0.05, **: p<=0.01

Table 2: Individual fetal soft tissue malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

24-01 M

absent subclavian

1 (100 mg/kg bw/d)

27-07 F

 

34-05 M, 34-09 M

absent subclavian, malpositioned kidney, small kidney

absent gallbladder

2 (300 mg/kg bw/d)

66-06 F

72-11 M

absent subclavian

absent gallbladder

3 (1000 mg/kg bw/d)

94-07 F

 

94-03 M, 94-04 M, 94-05 M, 94-06 M, 94-08 F, 94-09 F, 94-10 F, 94-11 F

multiple malformations of the great vessels, small spleen

small spleen

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 3: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

1 (0.6)

 

3 (1.6)

 

2 (1.0)

 

9 (4.7)

Litter incidence

 

N (%)

 

1 (4.5)

 

2 (10)

 

2 (9.5)

 

1 (5.0)

Affected fetuses/litter

 

Mean%

 

0.6

 

1.4

 

1.0

 

4.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 4: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

1 (0.6)

 

6 (3.2)

 

7 (3.6)

 

8 (4.2)

Litter incidence

 

N (%)

 

1 (4.5)

 

3 (15)

 

5 (24)

 

6 (30)*Fi

Affected fetuses/litter

 

Mean%

 

0.6

 

2.8

 

3.5*Wi

 

3.7*Wi

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent
*Fi = p ≤ 0.05 (Fisher’s exact test [one-sided])        ** Fi= p ≤ 0.01 (Fisher’s exact test [one-sided])
*Wi = p ≤ 0.05 (Wilcoxon-test [one-sided])              ** Wi= p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 5: Individual fetal skeletal malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

40-02 M

misshapen thoracic vertebra

2 (300 mg/kg bw/d)

55-08 M

misshapen thoracic vertebra, thoracic hemivertebra, fused rib

 

55-09 M

misshapen thoracic vertebra, thoracic hemivertebra, branched rib

3 (1000 mg/kg bw/d)

none

 

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Table 6: Total sekeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

0.0

 

1 (0.5)

 

2 (1.0)

 

0.0

Litter incidence

 

N (%)

 

0.0

 

1 (5.0)

 

1 (4.8)

 

0.0

Affected fetuses/litter

 

Mean%

 

0.0

 

0.6

 

1.0

 

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 7: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

167 (93)

169 (89)

170 (88)

178 (93)

Litter incidence

N (%)

22 (100)

20 (100)

21 (100)

20 (100)

Affected fetuses/litter

Mean%

92.6

89.5

89.5

92.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 8: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean% (range)

Incomplete ossification of cervical centrum; unchanged cartilage

4.5

6.0

7.5

11.9*

17.5

(1.9 - 33.4)

Supernumerary thoracic vertebra

9.5

18.7

24.8**

14.2

20.7

(12.6 - 29.9)

Misshapen sacral vertebra

1.9

4.7*

3.2

3.3

6.4

(2.6 - 12.1)

Incomplete ossification of talus; cartilage present

4.8

3.9

8.5

10.8*

1.1

(0.0 - 2.2)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = percent

*= p ≤0.05 (Wilcoxon-test [one-sided]); ** = p0.01 (Wilcoxon-test [one-sided])

Table 9: Total fetal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

1 (0.6)

4 (2.1)

4 (2.1)

9 (4.7)

Litter incidence

N (%)

1 (4.5)

3 (15)

3 (14)

1 (5.0)

Affected fetuses/litter

Mean%

0.6

2.0

1.9

4.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 10: Total fetal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

167 (93)

196 (89)

171 (88)

178 (93)

Litter incidence

N (%)

22 (100)

20 (100)

21 (100)

20 (100)

Affected fetuses/litter

Mean%

92.6

89.5

88.4

92.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

 

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of N,N’-(isobutylidene)diurea to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at dose levels of 300 mg/kg bw/d and above, such as reduced food consumption and body weight gain.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 100 mg/kg bw/d. However, reduced fetal body weights (at 300 and 1000 mg/kg) as well as a temporary delay of prenatal development of offspring (at 1000 mg/kg only) were associated with congenerous effects in their mothers and thus not considered as an independent effect.
There is no evidence for selective developmental toxicity of N,N’-(isobutylidene)diurea. The test substance is not teratogenic in rabbits at the tested dose levels.
Executive summary:

N,N’-(isobutylidene)diurea was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 100, 300 and 1000 mg/kg bw/day.

No test substance related adverse clinical observations were noted in any test group.

The mean food consumption of the high-dose dams (1000 mg/kg bw/d) was reduced during major parts of the treatment period, during the treatment period (GD 6-28) the high-dose does consumed 16% less food than the concurrent control does. Though not statistically significant, the food consumption during treatment (GD 6-28) was also reduced at the mid-dose level by 8%. This effect is regarded to be treatment-related.

The high-dose of the test substance distinctly affected the gross and corrected (net) body weight gain of the does in the respective test group. These does gained about 69% less gross weight or lost twice as much net weight than the concurrent control during the treat-ment period (GD 6-28). In the mid-dose group the gross weight gain was 33% below control and the corrected (net) body weight change was also reduced compared to control, which indicates beginning systemic toxicity at 300 mg/kg bw/d. These effects are assessed as test substance-related signs of maternal toxicity.

The mean fetal weights of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were slightly but statistically significantly reduced (about 11% and 13% below control - both sexes combined). These slight reductions are considered to be subsequent to an overall lower body weight gain of the does in the mid- and high-dose groups.

Fetal examinations revealed that there is no effect of the compound on the respective mor-phological structures up to the highest dose tested (1000 mg/kg bw/d). Incidences of incom-plete ossifications of skeletal elements at the tips of the lower limbs represent temporary de-lays in development which have no permanent effect on morphology and function of the af-fected structures.

Thus, the test item is not teratogenic in rabbits.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: males: 8 weeks; females: 10 weeks
- Weight at study initiation: males: 276 g mean bw; females: 228 g mean bw
- Housing: housed individually in suspended wire-mesh cages, females shifted 4 days before lactation to polycarbonate cages in a barrier rodent unit.
- Diet: A04 C pelleted diet ad libitum, distributed weekly
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2
- Humidity (%): 50 +- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 69H0028, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL and then homogenized using a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours) .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations: Concentration and homogeneity.
Three samples of each control and test substance dosage form (top, middle and bottom of the flasks) prepared for use during the first week and the last week of treatment were taken. They were stored at -20 °C pending dispatch, to the Sponsor, for analysis of concentration and homogeneity.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/1 female of the same dose group
- Length of cohabitation: during the night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- Each female was placed with the same male until mating occured or 14 days had elapsed.
Duration of treatment / exposure:
males: pre-mating, mating (14 days) and post-mating, for a total of 34 days; females: pre-mating, mating, pregnancy and lactation until day 4 post partum.
Frequency of treatment:
daily, 7 days/week
No. of animals per sex per dose:
10 male and 10 female animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were specified by the Sponsor, following the results of a previously conducted prenatal developmental toxicity study in Wistar rats (BASF Project No. 30R0727/90116). The oral route was selected since it is the route of exposure, which is requested by regulatory authorities for this type of product.
- Rationale for animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: female animals: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post coitum and days 1 and 4 post partum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: the females and their pups were killed on day 5 post partum. Female animals showing no evidence of mating were killed 24-26 days after the last day of mating.
- Organs examined: Examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. In the parent females, the corpora lutea and implantation sites were recorded. In apparently non-pregnant or un-mated females, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. Body weight and organ weights (adrenals, brain, heart, kidneys, liver, spleen, thymus, additionally in males: testes, epididymides; females:ovaries), Histopathology: macroscopic lesions, adrenals, brain, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids and parathyroids, trachea, urinary bladder, uterus
Ovaries and uterine content:
The ovaries and uterine were examined. As the females were allowed to give birth to their only the following examinations were performed:
- In the parent females, the corpora lutea and implantation sites were recorded.
- In apparently non-pregnant or un-mated females, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique.
Fetal examinations:
Pup examinations:
- External examinations: Yes: [all per litter]
Statistics:
Dunnett, Fisher`s exact, Dunn, Mann-Whitney, Wilcoxon tests.
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
1000 mg/kg/day of the test substance revealed slight signs of maternal (but not paternal) toxicity, confined to:
- a slightly lower food consumption in the females during gestation (-6%, compared to controls, day 0-day 20 of pregnancy)
- a slightly lower body weight gain in the females during gestation (-10%, compared to controls, day 0-day 20 of pregnancy) and lactation (-38%, compared to controls , day 1-day 4 post-partum).
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

There were no indications for a substance-induced developmental toxicity up to and including the highest tested dose of 1,000 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, Germany
- Age at study initiation: 53-56 days (65-74 days at day 0 post coitum)
- Weight at study initiation: 225.4 g (day 0 post coitum)
- Fasting period before study: no data
- Housing: during the study period, the rats were housed singly in type DK III stainless steel wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each day the test substance suspensions were freshly prepared shortly before the test substance was administered. For the preparation of the suspensions, an appropriate amount of the test substance was weighed and subsequently suspended in a 0 .5% aqueous carboxymethyl cellulose solution using a high speed sonicator (Ultra Turrax, JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

DIET PREPARATION
The food used was ground Kliba 343 feed rat/mouse/ hamster supplied by KLINGENTALMÜHLE AG, CH-4303 Kaiseraugst, Switzerland which was assayed for chemical as well as for microbiological contaminants.

VEHICLE
- Justification for use and choice of vehicle: 0.5% aqueous CMC solution, no justification for choice of vehicle given
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance suspensions up to 3 hours were carried out during the study . Furthermore, samples of the test substance suspensions were sent to the analytical laboratories of BASF Aktiengesellschaft twice during the study period for verification of the concentrations. The samples which were sent for the first concentration control analyses toward the beginning of the administration period were also used to verify the homogeneity for the samples of the low and the high concentrations (100 and 1,000 mg/kg body weight/day). 6 samples (2 from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. The test substance suspensions were analyzed by HPLC .
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male / 1-4 female animals
- Length of cohabitation: 16.00 hours to about 7.30 hours on the following day
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear; referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 to 15 post coitum
Frequency of treatment:
once daily
Duration of test:
all animals were sacrificed on day 20 post coitum
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the doses were selected due to the results of a range-finding study
- Rationale for animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day #20 post coitum
- Organs examined: uterus, ovaries
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes
Statistics:
The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft. DUNNETT's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses. FISHER's Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Historical control data:
yes
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
see remarks
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
see remarks
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

No signs of maternal toxicity were observed. There were no substance-related effects in the dams with regard to food consumption, body weight, body weight gain, uteri weights and clinical or autopsy findings. The reproduction data (conception rate, mean number of corpora lutea and implantation sites, pre- and post-implantation loss, number of resorptions, number of viable fetuses) revealed no biologically relevant differences between the control and treated groups.  

 

No embryo/foetotoxicity was induced by the treatment with isobutylidene diurea. The sex ratio did not differ significantly between the treated groups and controls and the mean placental and foetal weights were unaffected. Weight of placentae and weight of fetuses were comparable to the actual control values.

 

The examination of the foetuses for external malformations revealed brachygnathia and aglossostoma, both in one low-dose foetus. This was regarded as spontaneous occurrence since it is also present in the historical control data of this rat strain at a low frequency. No external variations were found in any group. 

 

Soft tissue examination showed malformations such as hydrocephaly together with microphthalmia (left) and anophthalmia (right) in one foetus of the low dose group and dextrocardia in one foetus of the intermediate dose group. These were regarded as spontaneous occurrences since there was no dose response relationship and they are also present in the historical control data of this rat strain at a low frequency.  Soft tissue variations (dilated renal pelvis  and/or hydroureter) were seen throughout, without  statistically significant differences between the groups (dilated renal  pelvis incidence: 29/21/16/18; hydrourether incidence: 10/8/6/12).   

 

Foetal skeletal analyses showed various malformations of the skull (mandible fused or various skull abnormalities), the vertebral column, the sternum, and/or the ribs (fused ribs). However, only the incidences of two types of skeletal malformations (thoracic vertebral body/bodies dumbbell-shaped (incidence: 0/5/10/5) and bipartite sternebrae with dislocated ossification centres (incidence: 0/1/4/2) and the overall number of malformations of the skeleton (incidence: 2/9/15/7) were significantly increased in the treated groups, but without a clear dose-response relationship. Moreover, the frequency of dumbbell-shaped thoracic vertebral bodies and bipartite sternebra(e) was unusually low in  the concurrent controls. Hence, the observed malformations were not considered to be related to the treatment with the substance. 

 

Retardation of foetal skeletal development (incomplete or missing ossification of vertebral bodies/arches, sternebra(e), skull and/or the hyoid bone) occurred in all groups without biologically relevant differences between the groups.  

 

Hence, the incidence and type of the foetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations observed in the treated foetuses were similar to the concurrent and/or historical control data.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP and guideline study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity studies via the oral route


N,N' (isobutylidene)diurea was evaluated for its developmental effects on Wistar rats and New Zealand White rabbits in studies according to OECD guideline 414 (BASF SE, 2017; BASF AG, 1993; Hellwig, Klimisch and Beth-Hübner, 1997) and in a screening test according to OECD guideline 422 (BASF AG, 2003). Additionally, an Extended One-Generation Reproduction Toxicity test is available covering some developmental parameters (CompoExpert, 2022).


 


Rat:


In the developmental study with Wistar rats (BASF AG, 1993; Hellwig, Klimisch and Beth-Hübner, 1997), the test substance was administered by gavage on days 6 through 15 of gestation at dose levels of 0, 100, 400 or 1000 mg/kg bw/day. No signs of maternal toxicity were observed. There were no substance- related effects in the dams with regard to food consumption, body weight, body weight gain, uterine weight and clinical or autopsy findings. The reproduction data revealed no biologically relevant differences between the control and treated groups. No effects on viability and weight of fetuses or other signs of developmental toxicity were induced by the treatment with the test substance. The incidence and type of the fetal external, soft tissue and skeletal findings, which were classified as malformations, variations and/or retardations observed in the treated fetuses were similar to the concurrent and/or historical control data.


 


The following “No Observed Adverse Effect Levels” (NOAELs) were derived from this study:


NOAEL, maternal toxicity: 1000 mg/kg bw/day (highest tested dose)


NOAEL, developmental toxicity: 1000 mg/kg bw/day (highest tested dose)


 


In the screening test (BASF AG, 2003), there were no effects on pup mortality, and no notable clinical signs in the pups during the 4 day observation period. Pup body weights were similar in all groups on day 1 and day 4 post-partum. The treatment had no influence on the sex ratio (NOAEL, developmental toxicity: 1000 mg/kg bw/day; NOAEL maternal toxicity: 300 mg/kg bw/day (reduced body weight gain in females during pregnancy and lactation at 1000 mg/kg bw/day).


 


Additionally, an Extended One-Generation Reproduction Toxicity Test (CompoExpert, 2021) according to OECD guideline 443 (2018) - basic test design (Cohorts 1A and 1B without extension). The test substance was administered by oral gavage to groups of 24 male and 24 female to Sprague-Dawley rats at dose levels 100, 300 and 1000 mg/kg bw/day, plus one concurrent vehicle control (0.5% carboxymethylcellulose). For the P-generation, the treatment period started two weeks before mating, was continued during mating and until at least 6 weeks after mating for males, or throughout gestation and up to Day 20 of lactation for females. The F1-generation was treated from Day 21 to Day 90 of age (100 weeks) for Cohort 1A animals, and from Day 21 - Day 97 of age (11 weeks) for Cohort 1B animals. While there were no detailed examinations performed on the offspring regarding malformations and abnormalities, nor developmental neurotoxicity, developmental immunotoxicity was assessed by recording lymph node weights, conducting a splenic lymphocyte sub-population analysis, and taking bone marrow smears. 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.Furthermore, 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. Lastly, 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. Thus, there was no evidence on developmental toxicity in this study, and the following NOAEL was derived:



NOAEL (F1; developmental toxicity): 1000 mg/kg bw/day (highest dose tested)

 


 


Rabbit:


The prenatal developmental toxicity of the test substance was determined in rabbits as a second species (BASF SE 2017). The test substance was adminstered as an aqueous suspension (in 0.5% carboxymethylcellulose) to groups of 25 inseminatedd female New Zealand White rabbits orally by gavage in doses of 100, 300 and 1000 mg/kg bw/day on gestation days (GD) 6 through 28. The vehicle control group was dosed with the vehicle (0.5% carboxymethylcellulose suspension in drinking water) in parallel. On GD 29 all females were sacrificed. The following test substance-related adverse effects/findings were noted:


In test group 3 (1000 mg/kg bw/day), does showed reduced mean food consumption (16% below control on GD 6-28) and reduced mean body weight gain (69% below control on GD 6-28). The corrected body weight gain was distinctly below the control (-410.6 g vs. -209.4 g in control). Similarly, the carcass weight was lower (about 5% below controls). The fetuses at the same dose presented with a reduced fetal weight (about 13% below control) and increased incidences of one fetal variation indicating developmental delay. In test group 2 (300 mg/kg bw/day, does showed a reduced mean food consumption (8% below control on GD 6-28), reduced mean body weight gain (33% below control on GD 6-28 ) and corrected body weight gain below control (-267.2 g vs. -209.4 g in control). Fetuses at this dose level presented with a reduced fetal weight (about 11% below control). In test group 1 (100 mg/kg bw/day), no test substance-related adverse effects on does, gestational parameters or fetuses occurred.


In conclusion, the NOAEL for maternal toxicity and for prenatal developmental toxicity is 100 mg/kg bw/day in this prenatal developmental toxicity study in rabbits. However, reduced fetal body weights (at 300 and 1000 mg/kg bw/day) as well as a temporary delay of prenatal development of offspring (at 1000 mg/kg bw/day only) were associated with congenerous effects in their mothers and thus not considered as an independent effect. Under the conditions of this test, there is no evidence for selective developmental toxicity of the test substance. The test substance is not teratogenic in rabbits at the tested dose levels.


 


Developmental toxicity studies via the inhalation route


No studies are available addressing develomental toxicity via inhalation exposure to the test substance.


 


Developmental toxicity studies via the dermal route


No studies are available addressing develomental toxicity via dermal exposure to the test substance.


 


Conclusion on develomental toxicity:


Developmental effects associated with the treatment of the test substance were only observed in one study in the rabbit (BASF SE, 2017), where reduced fetal weight (300 and 1000 mg/kg bw/day) and a temporary delay in the development of the offspring were observed (1000 mg/kg bw/day). However, these effects were attributable to congenerous maternal toxicity and considered a secondary effect thereof. In the other studies assessing developmental toxicity, no adverse effects were observed up to the highest doses tested. Therefore, it was concluded that there was no evidence for developmental toxicity.


Justification for classification or non-classification


The combined evidence from a reproductive/developmental toxicity screening test (BASF 2003), a modern one generation study (BASF 2003), and the recently performed key study, which was an Extended One-Generation Reproduction Toxicity test (CompoExpert, 2022) suggest that N,N' (isobutylidene)diurea had no effects on the reproductive function of rats. The NOAEL was based on the dosages of the key study, i.e. 1000 mg/kg bw/day, the highest dose tested. 


No developmental effects were seen in a screening test in rats (BASF 2003), nor in the key studies assessing the developmental toxicity studies in Wistar rats (BASF 1993). In the key study performed in rabbits (BASF 2017) signs of developmental toxicity in the offspring occurred, but were due to congenerous maternal toxicity and not regarded as independent effects. Developmental immunotoxicity parameters investigated in the Extended One-Generation Reproduction Toxicity Test (CompoExpert, 2022) did not find any effects attributable to the test substance administration either. The NOAEL for developmental toxicity was based on the dosages of the rabbit key study, i.e. 100 mg/kg bw/day, but as described before, the effects noted are unlikely to be a result of the developmental toxicity potential of the test substance. Also they only occurred in one species (rabbit, but not rats). Therefore, based on the available data, no classification and labeling for effects on fertility or developmental toxicity will be required for N,N' (isobutylidene)diurea.


Additional information