Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproductive/developmental toxicity screening test, and in a modern one generation study, IBDU 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.

Link to relevant study records

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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:
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 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
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few macroscopic observations recorded were those commonly encountered in the laboratory rat of this strain and age and thus considered to be of no toxicological importance.
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:
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:
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 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
Description (incidence and severity):
When compared with their respective controls, higher absolute and relative adrenal weights were observed for treated males and lower absolute and relative ovary and uterus weights were noted for treated females. Considering that the differences in the adrenal weights were not dose-related and observed in the males only, they were considered to be without relationship to the treatment.
The differences in the weights of the reproductive organs, particulary for uterus and ovaries were considered not to be treatment-related. Indeed, most of individual values of ovaries weight at 600 and 1200 mg/kg/day were within the range of control group values. Concerning the slight decrease observed in uterus weight, this latter change was only due to the contribution of a few individual values (females Z29644 and Z29628). In addition, the differences (% from control group) in the ratio of uterus weight/number of fetuses and of ovaries weight/number of corpora lutea between the treated and control animals was minor and not dose-related. In conclusion, these minor differences from the control group in ovaries and uterus weights were considered not to be related to the treatment with the test item.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few macroscopic findings recorded were those which are commonly observed spontaneously in the untreated laboratory rat of this strain and age and thus considered to be of no toxicological importance.
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:
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 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
Description (incidence and severity):
Absolute organ weight:
The absolute organ weights are listed in table 1 in section “any other information on results incl. tables”. The decreased absolute mean brain and epididymis weights in male animals of test group 3 (1000 mg/kg bw/d) were regarded as incidental as there were no changes in relative weights and no histopathological correlate. The decreased mean absolute and relative pituitary gland weights in female animals of test group 2 (300 mg/kg bw/d) were regarded as incidental as there was no dose-response and weights in test group 3 were identical to control values (100%).

Relative organ weights:
The relative organ weights are listed in table 2 in section “any other information on results incl. tables”. Relative weights of the liver in male animals of test group 3 (2.307%) were slightly above historical controls (2.110-2.299%). This was assumed to be caused by a change in mean body weight in this group rather than an effect on the liver, i.e. weight increase. Historical controls for terminal body weights of male animals range from 352.84 to 386.52 g. The terminal body weights of test group 3 animals in this study were slightly below this value (352.77 g). This was assumed to be the reason for the slightly increased relative liver weights in test group 3 animals (Absolute liver weights were within historical controls. Historical controls: 7.611-8.493 g, test group 3: 8.135 g). Furthermore, there was no dose-response and no histopathological correlate in test group 3. The increased relative liver weights in male animals of test group 3 were therefore regarded as incidental. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
no effects observed
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
1 200 mg/kg bw/day
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Additional information

IBDU was tested in two 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 few or very few seminiferous tubules in very few treated males 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 in all groups. There were no notable clinical signs in the pups, and the pup body weights were similar in all groups. 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 guideline 416 and beeing similar to an OECD 443 lacking the 2nd generation and the DIT and DNT cohorts. The study focuses on fertiliy and reproduction endpoints and lacks a 2nd generation with only limited assessment of the 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 there 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 (not 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 substance-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 (females), or because of the lack of a dose-response (males). Seminology findings in the treated groups were not different from controls. 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. 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. 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 also not effects 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).

Short description of key information:  Data from a screening test (BASF AG, 2003) and a one generation study (BASF AG, 2003) indicates that the test substance has no effects on male or female fertility parameters.

Further, the repeated dose toxicity of IBDU was assessed in a study according to OECD guideline 408 (BASF SE, 2017). The test substance was administered by 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 the limit dose of 1000 mg/kg bw/day. For further details please refer to the repeated dose toxcicity section.

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.

Effects on developmental toxicity

Description of key information

Data from a screening test (BASF AG, 2003) and a two developmental toxicity studies (BASF AG, 1993, BASF SE, 2017) indicates that the test substance has no effects on the development of the offspring attributable to the substance and no teratogenic effects were observed.

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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 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:
other: OECD Guideline 422
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on test animals 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

IBDU 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).

 

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. 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 IBDU. 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).

Furthermore, in another study 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 insemented female New Zealand White rabbits orally by gavage in doses of 100, 300 and 1000 mg/kg 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:

Test group 3 (1000 mg/kg bw/d):
Does
-  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), low-er carcass weight (about 5% below controls).

Fetuses
- Reduced fetal weight (about 13% below control)
- Increased incidence of one fetal variation indicating developmental delay

Test group 2 (300 mg/kg bw/d):

Does
- 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),

Fetuses
- Reduced fetal weight (about 11% below control)

Test group 1 (100 mg/kg bw/d):
- No test substance-related adverse effects on does, gestational parameters or fetuses.

In conclusion, the NOAEL for maternal toxicity and for prenatal developmental toxicity is 100 mg/kg bw/d in this prenatal developmental toxicity study in rabbits. 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.
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.

Justification for classification or non-classification

In a reproductive/developmental toxicity screening test, and in a modern one generation study, IBDU had no effects on the reproductive function of rats (NOAEL: 1200 mg/kg bw/day; highest dose tested).

No developmental effects were seen in a screening test in rats (NOAEL developmental toxicity: 1000 mg/kg bw/day; NOAEL maternal toxicity: 300 mg/kg bw/day) and in the developmental toxicity studies performed in Wistar rats (NOAEL developmental toxicity: 1000 mg/kg bw/day; NOAEL maternal toxicity: 1000 mg/kg bw/day), and in rabbits (NOAEL developmental toxicity: 100 mg/kg bw/day; NOAEL maternal toxicity: 100 mg/kg bw/day).

Therefore, based on the available data, no classification and labeling for effects on fertility or developmental toxicity will be required.