Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 443 (GLP; Sprague Dawley rats; 20, 60, 180 mg/kg bw/day):

- increased kidney weights, enlarged kidneys and histopathological findings in the kidney at 180 mg/mg bw/d

- no adverse substance-related effects on fertility and reproductive performance

- NOAEL (general, systemic toxicity) = 60 mg/kg bw/d; NOAEL (fertility and reproductive performance) = 180 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018 (study initiation to sacrifice of rearing animals)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 Jul 2011, incl. correction of 2 Oct 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: The doses were selected based on signs of toxicity noted at dose levels of 100 and 300 mg/kg bw/d in a previously conducted dose range-finding reproduction / developmental toxicity study in Sprague-Dawley Rats which preceded this definitive extended one-generation reproduction toxicity study.
- Inclusion of extension of Cohort 1B
- Termination time for F2: PND 4 after culling or PND 21 after weaning
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B
- Inclusion of developmental immunotoxicity Cohort 3
- Route of administration: Orally by gavage.
- Choice of species: The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 03508136W0
- Purity: 99.9 g / 100 g
- Physical state / appearance: solid / white
- Expiry date: 13 Mar 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in 1% Sodium carboxymethyl cellulose suspension in drinking water over a period of 4 days at room temperature and over a period of 7 days in a refrigerator has been verified prior to the start of the study in a comparable batch. Before and during administration, the preparations will be kept homogeneous with a magnetic stirrer.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the test substance preparations, the specific amount of test substance will be weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD
Details on species / strain selection:
The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH / Charles River Laboratories, Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males about 6 weeks, females about 5 weeks
- Housing: 2 - 3 animals per cage in polysulfonate cages type 2000P; from delivery to randomization (F0 animals), during mating, gestation, lactation, females after weaning, for functional observational battery and motor activity measurements housing in polycarbonate cages type III, 1 animal per cage except during mating (1 male / 1 female per cage) and during rearing up to PND 21 / 22 (1 dam with her litter)
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse / rat "GLP"; ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70% (till 09.11.2017) and 45 - 65% (from 10.11.2017)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6 am to 6 pm illumination)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guaranteed that the test substance concentrations in the vehicle remained stable.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: overnight until there is evidence of copulation or a maximum period of 14 days has elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory during the study period (at the beginning, towards the middle and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (20 and 180 mg/kg bw/d). Three samples (one 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 samples of the middle of the study were not analyzed because no imprecision occurs during the analysis of the samples from the start and end of the study.
Duration of treatment / exposure:
After an acclimatization period of at least 5 days, the F0 animals, with the exception of the controls, received the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor) for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 6 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 3) received the test substance daily by gavage from PND 21 until one day before sacrifice.
Frequency of treatment:
once daily
Details on study schedule:
F0 generation parental animals were mated to produce F1 generation pups. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (= F1 generation parental animals) was selected to produce F2 pups.
On arrival and shortly before the beginning of the administration period, all F0 animals were examined for signs of illness. Only healthy animals were used for the study. F0 females were nulliparous and non-pregnant at the beginnng of the study.
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- F0 generation parental animals: 24
- F1 rearing animals, cohort 1A (Reproductive PND 90): 20
- F1 rearing animals, cohort 1B (= F1 generation parental animals): 24
- F1 rearing animals, cohort 2A (Neurotoxicity PND 75 - 90): 10
- F1 rearing animals, cohort 2B (Neurotoxicity PND 22): 10
- F1 rearing animals, cohort 3 (Immunotoxicity): 10
- F1 rearing animals, positive control animals: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on signs of toxicity noted at dose levels of 100 and 300 mg/kg bw/d in a previously conducted dose range-finding reproduction/developmental toxicity study in Sprague Dawley Rats which preceded this definitive extended one-generation reproduction toxicity study.
In the dose range-finding reproduction/developmental toxicity study, clinical signs of parental toxicity were observed in the high-dose F0 animals (300 mg/kg bw/d), such as mildly reduced food consumption and body weight gain during several sections of the study. Males were more affected than females. In addition, dilated cecum, enlarged and discolored kidneys, enlarged livers, reduced terminal body weights and distinctly increased absolute/relative kidney weights were noted in the 300 mg/kg bw/d F0 males at necropsy. Kidney weights were still significantly increased in F0 males at 100 mg/kg bw/d. Histopathology revealed dose-dependently increased incidences of findings in the cecum (thickening of wall, increased apoptosis), kidneys (tubular degeneration/regeneration, tubular dilation, medulla mineralization, lymphoid infiltration) and liver (lymphoid infiltration) in F0 males and females at 100 and 300 mg/kg bw/d.
F0 parental females (300 mg/kg bw/d) had a prolonged estrous cycle. All 8 pregnant females had a significantly lower average number of implants compared to the concurrent control (10.4 vs. 15.8). In addition, post-implantation loss was significantly increased (34.6 vs 3.6%) and 2 of the 8 pregnant females had complete intrauterine litter lasses. These effects resulted in a significantly lower live litter size (10.3 vs. 15.0). Newborn pups developed normally.
The selected high dose for the present study was expected to evoke moderate systemic toxic effects in the parental animals. The present dose selection, however, also considered the need of generating a sufficient number of F1 offspring to serve the purpose of an extended one-generation study which includes the full set of reproductive (extended to F2), neurotoxicity and immunotoxicty investigations along with additional learning/memory testing and histopathological investigations of the mammary gland. Thus, excessive impairment of reproductive performance in the F0/F1 parental animals was avoided. This procedure to select the high dose in a regulatory study meets the principles of guideline OECD 443 (adopted 2011), as well as ECHA practical guide 10 ("how to avoid unnecessary testing on animals"; chapter 4 "animal welfare"; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 86/609/EEC on animal protection.

- Rationale for animal assignment: Male and female Sprague Dawley Rats were randomized according to their weight and allocated to the dose groups before the beginning of the administration period.

Positive control:
Developmental immunotoxicity examinations in cohort 3 animals:
10 male and 10 female surplus offspring derived from test group 00 (control group) were selected at weaning to become a positive control group in this study.

- Name of positve control substance: Cyclophosphamide monohydrate
- Batch number: MKBX1822V
- CAS No.: 6055-19-2
- Purity: 100%
- Identity: Confirmed
- Homogeneity: given
- Expiry date: 19 Dec 2018
- Physical state: solid, white
- Storage conditions: Refrigerator
- Oral administration of 10 mL/kg bw once daily by gavage for about 4 weeks; for immunization intraperitoneal treatment of 1mL per animal split into 2 portions of 0.5 ml
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily; during the administration period all animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and / or signs of overt toxicity

MORTALITY: Yes
- Time schedule: twice daily on working days; once daily on Saturdays, Sundays or public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the administration period on day 0 and subsequently once per week

BODY WEIGHT: Yes
- Time schedule for examinations: generally once a week at the same time of the day (in the morning)
- During pregnancy: weekly on GD 0, 7, 14, and 20
- During lactation: PND 1, 4, 7, 10, 14, 17, 19, and 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- During pregnancy: weekly for GD 0-7, 7-14, and 14-20
- During lactation: PND 1-4, 4-7, 7-10, 10-14, 14-17, 17-19, and 19-21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: generally once a week

OTHER:
- Mortality: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays
- Urinalysis
- Clinical pathology (Hematology, clinical chemistry, hormones, sperm parameters)
- T-cell dependent antibody response (all animals of cohort 3)
- Splenic lymphocyte subpopulation analysis
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated preparing vaginal smears for all F0 and F1 female (= cohort 1B) parental animals during a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75.
Additionally, on the day of scheduled sacrifice, the estrous status was determined for each F0 and F1 female and cohort 1A female.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation and in all cohort 1A males:
testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology, spermatic head count in the testis
- sperm motility, sperm morphology, sperm head count (cauda epididymis, testis)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, body weight, anogenital distance (AGD), presence of nipples/areolae in male pups, puberty onset, sex ratio

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
- Auditory startle response habituation in cohort 2A animals
- Functional observational battery (FOB) in cohort 2A animals
- Motor activity measurement in cohort 2A animals
- Learning and memory test (Morris water maze) in cohort 2A animals

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
- Cyclophosphamide dependent immune system response in cohort 3 animals
Postmortem examinations (parental animals):
SACRIFICE
- All F0 parental animals will be sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues (see below) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 77 (cohort 2A), PND 22 (cohort 2B) (no information about days of age of cohorts 1A and cohort 3)

GROSS NECROPSY
- Gross necropsy consisted of organ weights, length and width of brain

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues (see below) were prepared for microscopic examination and weighed, respectively. The brain, spleen and thymus were weighed in one surplus F2 weanling per sex per litter
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- DUNNETT-Test (two-sided)
- Fisher's exact test
- KRUSKAL-WALLIS and WILCOXON test (two-sided)
- WILCOXON test (one-sided)
Reproductive indices:
- mating index
- fertility index
- gestation index
- Live birth index
- Postimplantation loss
Offspring viability indices:
- Viability index (pups)
- Lactation index (pups)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent temporary salivation was assumed to be test substance-induced, however, was considered to be no sign of systemic toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female was sacrificed moribund on Day 63. Histopathological findings were consistent with a gavage error.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights were statistically significantly increased in mid-dose females during premating days 7 – 14, in mid-dose male animals during the weeks 3 - 4 after the premating period (study weeks 14 - 15), and in mid-dose females during premating days 0 - 7. Body weight change of high-dose males was statistically significantly below the concurrent control values during premating days 56 - 63 (about 26%). All findings were considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 females was statistically significantly increased during major parts of the premating period and during GD 14 - 20 (up to 36% and 8%, respectively). Food consumption of the mid-dose F0 females was statistically significantly increased during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6%, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increase in males during major parts of the pre-mating period (up tp 22%) in 180 mg/kg bw/day and females of the high dose group (180 mg/kg bw/day) during major parts of the pre-mating and gestation period (up to 25% and 28%, respectively).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- Mean corpuscular hemoglobin (MCH) significantly increased in males at the end of administration calculated from measured hemoglobin and red blood cell counts. Both measured parameters were not statistically significantly altered. Therefore, the MCH change was regarded as incidental and not treatment-related.

20 mg/kg bw/day
- In males relative basophil cell counts significantly lower compared to controls, but values were not dose-dependently changed. Thus, alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
60 mg/kg bw/day
- In females, cholesterol values significantly increased. However, change was not dose-dependent and, thus, was regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males and females, urine pH values decreased (not statistically significant in females) which may be treatment-related but was regarded as non-adverse due to absence of other findings in the urine.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- in 21 out of 24 males, increased mineralization noted in the transition from medulla to cortex (minimal to severe)
- Nuclear crowding (accumulation of nuclei protruding into tubular lumen) in the kidneys in 22/24 male animals (minimal to moderate)
- Tubular dilation in the kidneys in 13/24 male animals (minimal to slight)

In males, the absolute and relative weights of the adrenal glands were significantly increased in test groups 02 and 03, which was regarded to be treatment-related. No further findings were noted macroscopically or histopathologically in the adrenal glands. This effect was regarded to be not adverse.
In male animals, the absolute and relative weights of the kidneys were significantly increased in test group 03. There were correlating macroscopic (enlarged) and histopathological findings in test group 03: increased mineralization, nuclear crowding and dilation of tubules in the outer zone of the outer medulla. Findings in the kidney in test group 03 were assessed as treatment-related and adverse.
The increased relative liver weight in test group 03 females were assumed to be treatmentrelated, but in the absence of any other findings (histopathology or clinical chemistry) regarded as non-adverse.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- No treatment-related alterations of T4 and TSH levels in males and females of all test groups
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Overall regular cycles in all females. However, mean estrous cycle duration was slightly but statistically significantly increased in the high dosed females (4.1 days vs 3.9 days of control).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
- incidence of abnormal sperm in the cauda epididymidis: no treatment-related effects
- sperm head counts in the testis and in the cauda epididymidis: no treatment-related effects
- motility of the sperm: significantly lower in males of test groups 01, 02 and 03 (20, 60 and 180 mg/kg bw/d) compared to controls; however, the lowest value (84% motile sperm) exactly matched the control value in the F1 generation and the overall span of sperm motility in this study reflects a normal range of biological variation in rat (multi)generation studies; no dose-dependency
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male mating index: 100%
- Male fertility index: between 91% and 100% (one control male, two males of 20 mg/kg bw/day and one male of 180 mg/kg bw/day did not generate F1 pups) reflecting a normal range of biological variation
- Female mating index: 100% (mean duration of copulaton varied between 2.0 and 2.3 days)
- Female fertility index: between 91% and 100% (one control female, two females of 20 mg/kg bw/day and one female of 180 mg/kg bw/day did not become pregnant) reflecting a normal range of biological variation
- Female gestation index: 100%
Delivery data: effects observed, treatment-related
- Mean duration of gestation: 22.0 days in all test groups
- Implantation unaffected
- Mean number of resorptions (0.5 / 0.8 / 1.3* / 1.5**) and postimplantation loss (3.1% / 5.9% / 9.4%* / 10.5%**) statistically significantly above control values in 60 and 180 mg/kg bw/day groups (control/low/mid/high; *=p≤0.05; **=p≤0.01)
- F1 pups delivered per dam lower in mid-and high dose groups, although not statistically significant (14.9 / 14.0 / 13.5 and 12.7 pups/dam, respectively in control/low/mid/high dose group)
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
Treatment related:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Transient salivation noted for several males and females in 180 mg/kg bw/day which was considered to be test substance-induced, however not considered a sign of systemic toxicity
- one female showed vaginal hemorrhage during study weeks 4-5 (not treatment-related)
- No substance-related findings in F1B females of all dose groups during gestation period for F2 litter
- one female showed vaginal hemorrhage on GD 23 (incidential)
- one female of 60 mg/kg bw/day and one female of 180 mg/kg bw/day group did not deliver F2 pups despite being sperm positive (not associated with treatment)
- no clinical findings during lactation period for F2 litter
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One female animal was found dead on pre-mating day 3. As the animal was partly cannibalized and no histopathological examination was conducted the cause of the death was not determined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body weights of high- and mid-dose females were statistically significantly increased during major parts of the premating period (up to 10% and 9%, respectively) and for mid-dose females additionally on PND 14 (about 6%).
- Body weight change of high-dose females was statistically significantly increased during premating days 0 - 21 and 0 – 70 (up to 18% and 10%, respectively) and for mid-dose females statistically significantly increaseds during premating days 0 - 21 (up to 20%).

- Body weights of the high- and mid-dose male F1B rats were statistically significantly decreased on premating day 14 (about 9% and 7%, respectively). This was a single event which is unlikely to be treatment-related.
- Decreased body weight changes of high-dose males during pre-mating days 7-14, 42-49 and 63-70 (up to 27%), mid-dose males during pre-mating days 7-14 (about 29%), and low-dose males during pre-mating days 7-14 (about 18%). Statistically significantly increased body weight change in the high-dose male animals during premating days 14 - 21 and in the mid-dose males during premating days 14 - 21, 35 - 42 and study weeks 3 - 4. - 21, 35 - 42. All of these changes were inconsistent in terms of timing and direction of the apparent effect. Thus, they are not considered to be test substance-related.
- Statistically significantly decreased body weight change in the high-dose female animals during GD 14 - 20, 0 - 20 and PND 14 - 17 as well as mid-dose females during PND 14-17 were considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- High dose females showed statistically significantly increased food consumption during pre-mating days 28-49 and 0-70 (up to 22% and 13%, resp.)
- During pre-mating days 7-14, food consumption was statistically significantly increased in mid-dose females (about 13%)
- Statistically significantly decreased food consumption in the high-dose males during premating days 7 - 14 was considered as spontaneous in nature.
- Statistically significantly decreased food consumption in the high-dose females during PND 4 - 7 was considered as spontaneous in nature.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- High dose females showed increased water consumption during major parts of the pre-mating period (up to 26%)
- Mid dose females showed increased water consumption during pre-mating days 14-17 and 42-45 (about 14% and 15%, resp.)
- Statistically significantly increased water consumption in the mid-dose males during premating days 0 - 17 was considered as spontaneous in nature.
- Statistically significantly decreased water consumption in the high-dose females during PND 4 - 5 was considered as spontaneous in nature.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males, absolute (not statistically significant) and relative increased adrenal gland weights (108% and 113%, resp.)
- In males, absolute and relative increase in kidney weights (126% and 132%, resp.)
- In males, abolute and relative decrease in liver weights (90% and 94%, resp.). Possibly treatment-related.
- In females, increased absolute kidney weight (107%).

60 mg/kg bw/day
- In males, absolute and relative increased adrenal gland weights (113% and 111%, resp.)
- In males, absolute and relative increased kidney weights (113% and 110%, resp.)
- In females, increased absolute terminal body weight (104%)

In females, the terminal body weight was statistically significantly increased in 60 mg/kg bw/d and without statistical significance in 180 mg/kg bw/d (106%). This possibly explains the increased absolute kidney weights in 180 mg/kg bw/d as no changes were seen in relative weights. Additionally, there were no histopathological findings in the kidneys of female animals of test group 180 mg/kg bw/d.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males, enlarged kidneys found in 10/24 animals

The female animals, which were not pregnant as well as the male mating partners did not show relevant gross findings consistent with impaired fertility.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related effects on mammary gland and mammary fat pad were seen in cohort 1B male animals.
The female animals, which were not pregnant as well as the male mating partners did not show relevant histopathological findings consistent with impaired fertility.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- 3.9 days in control, 4.0 in the low- and mid-dose group and 4.1 days in the high-dose group (statistically comparable to control)
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male mating index: 100%
- Male fertility index: between 96% and 100% (one mid-dose male and one high-dose male did not generate F2 pups). Normal range of biological variation
- Female mating index: 100%
- Mean duration until sperm was detected: 2.4 - 3.0 days without any relation to dose
- All females delivered pups except one mid-dose female and one high-dose female which did not become pregnant
- Female fertility index: between 96% and 100%. Normal range of biological variation
- Gestation index: 100%, 100%, 95%, 91% for control, low-, mid-, and high-dose groups, resp.
Delivery data: effects observed, treatment-related
- Mean duration of gestation: 21.9 days and 22.0 days without any relation to dose
- mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (15.2 / 14.6 / 15.4 and 13.7 implants/dam)
- Number of resorptions and postimplantation loss were statistically significantly increased in the high-dose group (0.9 / 0.8 / 1.1 / 3.3** [*= p ≤ 0.05 / **= p ≤ 0.01] and 6.4% / 5.3% / 11.1% and 24.6%** in control/low/mid/high dose group, respectively). Two high-dose females had a complete litter loss.
- Mean number of F2 pups was statistically significantly decreased in the high-dose group (14.3 / 13.8 / 14.9 and 11.4** pups/dam, respectively for control/low/mid/high dose)
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
Treatment related:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
- Lower mean number of F1 pups (14.9 / 14.0 / 13.5 and 12.7 pups/dam, respectively in control/low/mid/high dose group, resp.) in mid-and high dose groups (subsequent to higher resorption rate), no statistical significance
- Number of liveborn pups statistically significantly below control in high dose group (340 / 289/ 322 / 285*, respectively in test groups 00 - 03).
- Rate of liveborn pups indicated by live birth indices of 99% / 98% / 99% and 97% in control/low/mid/high dose group showed no significant differences
- Number of stillborn pups statistically significantly above control in high dose group (2 / 5 / 3 / 8*, respectively in test groups 00 - 03). Eight, however, does not seem unusually compared to 6 stillborn pups in F2 control

REARING ANIMALS
Cohort 1A
- Transient salivation noted for several males and females in 180 mg/kg bw/day which was considered to be test substance-induced. However, no sign of systemic toxicity.

Cohort 2A
- Transient salivation was noted for a few high-dose (180 mg/kg bw/d) male and female animals during several parts of the treatment period. This dose-dependent temporary salivation was considered to be test substance-induced. However, no sign of systemic toxicity.

Cohort 3
- no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
PUPS/LITTER
- The number of cannibalized and dead F1 pups were evenly distributed about the groups.
- Viability index: 99%, 97%, 99%, and 99% (during early lactation, PND 0-4) for control, low, mid, and high dose, resp.
- The lactation index indicating pup survival on PND 4 - 21 was 100% in all test groups.

REARING ANIMALS
Cohort 1A
- one female animal showed abdominal position and gaspin gon study day 0 and was found dead thereafter on day 0. No association to test substance assumed.

Cohort 2A
- no adverse effects observed

Cohort 3
- one animal of the low-dose was found dead on study day 18. Not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- mean body weights of male and female pups statistically significantly increased during PND 1-4 (up to 10%)
- mean body weight change of male pups statistically significantly increased during PND 1-4 (about 16%)

60 mg/kg bw/day
- mean body weights of male and female pups statistically significantly increased during PND 1-7 and on PND 21 (up to 12%, 11% and 6%, respectively).
- mean body weight change of male and female pups during PND 1-4 (about 19% and 16%, resp.)

20 mg/kg bw/day
- no effects observed on body weight
- mean body weight change of female pups during PND 14-21 (about 7%)

Per se, these effects do not constitute an adverse effect. Increased weights may reflect the advantageous nutritional condition of the pups in the smaller mid- and high-dose litters.

REARING ANIMALS
Cohort 1A
- Body weights of high and mid-dose females statistically significantly increased on study days 14 and 28 (up to 7%)
- Body weight change of high dose males statistically significantly decreased during study days 56-63 (up to 52%). As this had no impact on average body weights this is considered as an incidental finding.
- Body weight change of high and mid dose females statistically significantly increased during study days 7-14 and 0-7 (about 19% and 9%, respectively).
- The statistically significantly decreased body weight change in the mid-dose females during study days 56 - 63 was considered as spontaneous in nature.

Cohort 2A
- Comparable body weight and body weight changes of all treated groups to control

Cohort 3
- Comparable body weight and body weight changes of all treated groups to control
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- Females showed increased food consumption during major parts of the study period (up to 21%)

REARING ANIMALS
Cohort 1A
- Females of the high-dose showed increased food consumption during major parts of the study period (up to 21%)

Cohort 2A
- no effects observed

Cohort 3
- no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- Females showed increased water consumption during major parts of the study period (up to 25%)

60 mg/kg bw/day
- Females showed increased water consumption during days 7-17 (up to 20%)

REARING ANIMALS
Cohort 1A
- High-dose females showed statistically significant increase during major parts of the study period (up to 25%)
- Females of the mid-dose showed increased water consumption during days 7-17 (up to 20%)

Cohort 2A
- no effects observed

Cohort 3
- no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
180 mg/kg bw/day
- In males, prothrombin time (HQT, Hepatoquick's test) was prolonged at the end of adminstration period. Because this was the only measured plasma coagulation parameter besides platelet counts which can be affected by a dysregulated coagulation, this alteration has to be regarded as treatment-related and adverse.
- In males, hemoglobin values significantly higher. However, this was the only changed red blood cell parameter (i.e., hemoglobin, hematocrit and red blood cell (RBC) counts). Therefore, the hemoglobin change alone, was regarded as maybe treatment-related, but non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
- No alterations in absolute and relative lymphocyte subpopulation cell counts in spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) in both sexes
180 mg/kg bw/day
- In females, total protein, albumin and calcium levels were significantly increased at the end of administration period which was regarded as treatment-related and adverse.

- Sodium levels in all treated groups were significantly increased. However, mean and median values of sodium in the test groups were not changed dose-dependently and sodium was the only altered electrolyte parameter among these individuals. In males of the low-dose group, calcium levels were significantly decreased, but again these values were not dose-dependently altered. Therefore, the mentioned alteration in this paragraph were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In females, urine volume significantly higher and urine specific gravity significantly lower reflecting the physiological function of the kidneys towards higher fluid income and thus, without any other alterations in the urine, it was regarded as adaptive and non-adverse finding.
Sexual maturation:
no effects observed
Description (incidence and severity):
- Vaginal opening: Statistically significantly later puberty in low dose females was spontaneous in nature. Normal range of biological variation. At 20, 60 and 180 mg/kg bw/d test groups, 32.3, 33.2* (* = p≤0.05), 32.3 and 31.8 days, respectively.
- Preputial separation: No toxicologically relevant effect noted. At 20, 60 and 180 mg/kg bw/d test groups, 43.1, 43.0, 42.5 and 43.3 days, respectively.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
PUPS/LITTER
- no effects observed
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
PUPS/LITTER
- no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
180 mg/kg bw/day
- In males, increased absolute and relative kidney weights (112% and 117%, resp.)
- In males, decreased absolute liver, prostate, spleen, and thymus weights (86%, 90%, 83%, and 81%, resp.)
- In males, increased relative adrenal glands (113%)
- In males, decreased relative liver and spleen weights (91% and 87%, resp.)

60 mg/kg bw/day
- In males, decreased absolute prostate and absolute and relative spleen weight (91%, 91%, and 92%, resp.)

Absolute and relative spleen weights did not follow conspicious histopathological examination and no findings in clinical chemistry were found. Thus, this was regarded as equivocal and as not adverse. No findings in clinical chemistry were found regarding decreased absolute thymus weight in the highest dose group and thus, this was regarded to be an equivocal finding and as not adverse. The absolute weights of the prostate in test groups 60 and 180 mg/kg bw/day were decreased but no significant change was seen in relative weights, therefore the decrease in absolute weights was regarded as incidental. Decreased absolute and relative liver weights in 180 mg/kg bw/d group was regarded as possibly treatment-related.

Cohort 2A
- No mean absolute and relative weights were significantly changed in any test group

Cohort 2B
- No mean absolute and relative weights were significantly changed in any test group

Cohort 3
180 mg/kg bw/day
- In males, significantly decreased relative thymus weight (81%). However, similar weight changes occurred in cohort 1A without histopathological correlations and clinical chemistry findings. Thus, the decrease of the relative thymus weight was regarded as equivocal and non-adverse.

- Relative spleen weights were decreased in all treated groups, however, without reaching statistical significance. There were no correlates in clinical chemistry and there was no statistical significance, therefore, this was regarded as incidental.
- The positive control (Cyclophosphamide mynohydrate) showed expected results.

SURPLUS F1 GENERATION (F1 WEANLINGS NOT SELECTED FOR COHORTS)
- In males at 60 mg/kg bw/d, increased absolute brain and thymus weight (105% and 117%, resp.)
- In males at 20 mg/kg bw/d, increased absolute and relative thymus weight (124% and 120%, resp.)
- In males at 20 mg/kg bw/d, increased relative spleen weight (119%)
- In females at 60 mg/kg bw/d, decreased relative brain weight (91%)

All observed significant weight changes were assumed to be secondary to the increased terminal body weights in test groups 20 and 60 mg/kg bw/d (not significant) in both males and females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PUPS/LITTER
- A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored eye(s), empty stomach and hemorrhagic testis. Without any relation to dosing, these findings were considered to be not treatment-related.

REARING ANIMALS
Cohort 1A
- no effects observed

Cohort 2A
- no effects observed

Cohort 2B
- no effects observed

Cohort 3
- no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
REARING ANIMALS
Cohort 1A
180 mg/kg bw/day
- Increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate)
- Nuclear crowding in the kidneys in 6/20 male animals (minimal to slight)
- Tubular dilation in the kidneys in 7/20 male animals (minimal to slight)
- Increased incidence of atrophy in the mammary gland (7/20) and mammary fat pad (7/20)

Cohort 2A
- No treatment-related findings were seen

Cohort 2B
- No treatment-related findings were seen

Cohort 3
- no histopathology performed
Other effects:
no effects observed
Description (incidence and severity):
PUPS/LITTER
- Sex ratio: no substantial differences between control and treated groups; slight differences regarded to be spontaneous in nature

REARING ANIMALS
Cohort 1A
- Estrous cycle regular (4.1 days in all test groups)
- No treatment-related alterations of T4 and TSH levels in all test groups. Significant decrease of the TSH values in female PND4 pups of 20 and 180 mg/kg bw/d. However, these changes were not dose-dependent and therefore, they were regarded as incidental and not treatment-related.
- Spermanalysis: no effects observed
- No differences observed in differential ovarian follicle count
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 2A
- No influence of the test substance on auditory startle habituation (maximum amplitude and latency) observed
- FOB: Home cage observations without findings, open-field observations did not reveal any test-substance related findings, sensorimotor tests/reflexes without findings, quantitative parameter revealed no impaired parameters
- Motor activity measurement: not influenced by the test compound. There were statistically significant increases in activity during interval 11 in low-, mid- and highdose males. This isolated finding was not related to the dose and did neither influence the total session beam interruptions significantly, nor did it impair habituation. Thus, it was not considered to be related to the test substance.
- Learning and memory test: No influence in the aibility of rats to acquire position of a hidden platform. Memory was also considered to be unaffected.
- All length and width measurements were without any findings
- All morphometric brain measurements were without any findings. Only some single parameters in male (Nucleus caudatus width left, Corpus callosum width) or female (Nucleus caudatus width left) animals of test group 180 mg/kg bw/d showed a statistically significant increase or decrease. As no other values were changed, this minimal width in-/decrease is assumed as incidental and not related to treatment.

Cohort 2B
- All length and width measurements were without any findings
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 3
- No changes in SRBC IgM titer found in males and females. Positive control (Cyclophosphamide) vaild.
ANIMALS OF THE POSITIVE CONTROL
A significant decrease in absolute and relative weights of the spleen and a not statistically significant decrease of these weight parameters in the thymus occurred in the positive control male and female animals. The decreased weights of spleen and thymus were the expected result.
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1 (cohort 1B)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased post-implantation loss in F1 progeny
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
other: Cohort 2A and 2B
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
mammary gland
Treatment related:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Mean number of F2 pups delivered per dam (average litter size) was statistically significantly decreased in the high-dose group (14.3 / 13.8 / 14.9 and 11.4** pups/dam, respectively in control/low/mid/high dose group, resp.).
- Rate of liveborn pups not affected (live birth indices: 98%, 99%, 99%, and 98% for control, low, mid, and high-dose, resp.)
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- The number of cannibalized and dead F2 pups were evenly distributed about the groups.
- Viability index: 98%, 100%, 98%, 99% for control, low, mid, and high-dose, resp.
- Lactation index (indicating pup survival): 100%, 100%, 100%, and 99% for control, low, mid, and high-dose, resp.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of the high-dose female pups were statistically significantly above the concurrent control values on PND 1 (about 7%). However, this isolated finding was considered to be incidental.
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
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F2 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach, dilated renal pelvis and small testis. These findings occurred without any relation to dosing. Thus, all these findings were not considered to be associated to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio: no substantial differences; slight differences were regarded to be spontaneous in nature.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased post-implantation loss in F1 progeny
Critical effects observed:
no
Reproductive effects observed:
no
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Treatment related:
no

Table 1: Summary of absolute organ weights of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

01

(20)

02

(60)

03

(180)

Adrenal glands

104%

109%*

112%*

Kidneys

96%

104%

116%**

Thymus

113%

113%*

93%

* : p <= 0.05, **: p <= 0.01

Table 2: Summary of relative organ weights of F0 parental animals

F0

Male animals

Female animals

Test group (mg/kg bw/d)

01

(20)

02

(60)

03

(180)

01

(20)

02

(60)

03

(180)

Adrenal glands

106%

109%*

116%**

 

 

 

Kidneys

97%

104%

120%**

 

 

 

Liver

 

 

 

100%

102%

107%**

Thymus

116%

112%*

96%

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 3: Summary of gross pathology of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Cecum

 

 

 

 

·       Enlarged

0

0

0

3

Kidneys

 

 

 

 

·       Enlarged

0

0

0

6

Table 4: Summary of histopathology of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Mineralization, medulla, (m)f

0

0

1

21

·       Grade1

 

 

1

11

·       Grade2

 

 

 

7

·       Grade3

 

 

 

1

·       Grade4

 

 

 

2

Nuclear crowding

0

0

0

22

·       Grade1

 

 

 

11

·       Grade2

 

 

 

8

·       Grade3

 

 

 

3

Dilation, tubular

0

0

0

13

·       Grade1

 

 

 

7

·       Grade2

 

 

 

6

Table 5: Summary of female reproductive data of F0 parental animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals (N)

23

21

24

23

Postimplantation loss

 

 

 

 

·       Total

11

16

32

35

·       Mean

0.5

0.8

1.3*

1.5**

·       SD

0.67

0.89

0.92

1.62

% Postimplantation loss

 

 

 

 

·       Mean

3.1

5.9

9.4*

10.5**

·       SD

4.31

7.70

7.23

10.86

·       N

23

21

24

23

* : p <= 0.05, **: p <= 0.01

Table 6: Summary of absolute organ weights of F1A animals

F1A

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Kidneys

97%

103%

112%**

Liver

102%

99%

86%**

Prostate

96%

91%*

90%**

Spleen

93%

91%*

83%**

Thymus

96%

100%

81%*

* : p <= 0.05, **: p <= 0.01

Table 7: Summary of relative organ weights of F1A animals

F1A

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Adrenal glands

98%

98%

113%**

Kidneys

98%

103%

117%**

Liver

104%

100%

91%**

Spleen

94%

92%*

87%**

* : p <= 0.05, **: p <= 0.01

Table 8: Summary of histopathology of F1A animals

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

20

20

20

20

Mineralization, medulla, (m)f

0

0

1

7

·       Grade1

 

 

1

2

·       Grade2

 

 

 

2

·       Grade3

 

 

 

3

Nuclear crowding

1

1

0

6

·       Grade1

1

1

 

5

·       Grade2

 

 

 

1

Dilation, tubular

0

0

2

7

·       Grade1

 

 

2

4

·       Grade2

 

 

 

3

 

Mammary gland

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

20

18

20

20

Atrophy, (multi)focal

1

0

2

7

·       Present

1

0

2

7

 

Mammary gland fat pad

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

10

10

10

10

Atrophy,(multi)focal

1

0

2

7

·       Present

1

0

2

7

Table 9: Summary of absolute organ weights of F1B animals

F1B

Male animals

Female animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

11

(20)

12

(60)

13

(180)

Terminal body weight

 

 

 

98%

104%*

106%

Adrenal glands

105%

113%**

108%

 

 

 

Kidneys

102%

113%**

126%**

98%

103%

107%*

Liver

104%

99%

90%*

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 10: Summary of relative organ weights of F1B animals

F1B

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Adrenal glands

105%

111%*

113%**

Kidneys

103%

110%**

132%**

Liver

105%

97%

94%*

* : p <= 0.05, **: p <= 0.01

Table 11: Summary of gross pathology of F1B animals

F1B

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Kidneys

 

 

 

 

·       Enlargedd

0

0

1

10

Table 12: Summary of female reproductive data of F1B animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals (N)

24

24

22

23

Postimplantation loss

 

 

 

 

·       Total

22

18

25

76

·       Mean

0.9

0.8

1.1

3.3**

·       SD

0.97

0.79

1.28

3.94

% Postimplantation loss

 

 

 

 

·       Mean

6.4

5.3

11.1

24.6**

·       SD

6.43

5.71

21.25

30.10

·       N

24

24

22

23

* : p <= 0.05, **: p <= 0.01

Conclusions:
Under the conditions of the present modified extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 60 mg/kg bw/d for the F0 and F1 parental as well as adolescent animals, based on evidence for kidney toxicity, as well as corresponding effects on clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 180 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 180 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 and F2 progeny is 20 mg/kg bw/d, based on increased postimplantation loss in the F1 progeny, which was observed at the LOAEL (Lowest Observed Adverse Effect Level) of 60 mg/kg bw/d. The NOAEL for developmental neurotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental immunotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.
Executive summary:

The test substance was administered to groups of 24 male and 24 female healthy young Sprague-Dawley rats for test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 24 males and 24 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

RESULTS

The following test substance-related adverse effects/findings were noted:

180 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the premating and gestation period (up to 25% and 28% above control, respectively)

• Increased food consumption in females during major parts of the premating period and during GD 14 - 20 (up to 36% and 8% above control, respectively)

• Increased postimplantation loss (10.5% vs. 3.1% in control)

• Increased absolute (+16%) and relative (+20%) weights of the kidneys in males

• Macroscopically enlarged kidneys in 6/24 males

• Increased mineralization in the kidneys in the transition from medulla to cortex in 21/24 male animals (minimal to severe)

• Nuclear crowding in the kidneys in 22/24 male animals (minimal to moderate)

• Tubular dilation in the kidneys in 13/24 male animals (minimal to slight)

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• Decreased number of liveborn pups subsequent to higher postimplantation loss

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the study period (up to 25% above control)

• Increased food consumption in females during major parts of the study period (up to 21% above control)

• Increased body weights in females on study days 14 and 28 (up to 7% above control, respectively)

• Increased body weight change in females during study days 7 - 14 (about 19% above control, respectively)

• Prolonged prothrombin time in males

• Increased total protein, albumin and calcium values in females

• Increased absolute (+12%) and relative (+17%) weights of the kidneys in males

• Increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate)

• Nuclear crowding in the kidneys in 6/20 male animals (minimal to slight)

• Tubular dilation in the kidneys in 7/20 male animals (minimal to slight)

• Increased incidence of atrophy in the mammary gland and mammary fat pad

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the premating period (up to 26% above control)

• Increased food consumption in females during premating days 28 - 49 and 0 - 70 (up to 22% and 13% above control, respectively)

• Increased body weights in females during major parts of the premating period (up to 10% above control)

• Increased body weight change in females during premating days 0 - 21 and 0 - 70 (up to 18% and 10% above control, respectively)

• Increased postimplantation loss (24.6% vs. 6.4% in control)

• Increased absolute (+26%) and relative (+32%) weights of the kidneys in males.

• Macroscopically enlarged kidneys in 10/24 males

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• Decreased mean pups delivered subsequent to higher postimplantation loss

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

60 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during GD 17 - 20 (up to 22% above control)

• Increased food consumption in females during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6% above control, respectively)

• Increased postimplantation loss (9.4% vs. 3.1% in control)

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during study days 7 - 17 (up to 20% above control)

• Increased body weights in females on study days 14 and 28 (up to 7% above control, respectively)

• Increased body weight change in females during study days 0 - 7 (about 9% above control, respectively)

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during premating days 14 - 17 and 42 - 45 (about 14% and 15% above control, respectively)

• Increased food consumption in females during premating days 7 - 14 (about 13% above control)

• Increased body weights in females during major parts of the premating period and on PND 14 (up to 9% and 6% above control, respectively)

• Increased body weight change in females during premating days 0 – 21 (up to 20% above control)

• Increased absolute (+13%) and relative (+10%) weights of the kidneys in males

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

20 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study which was performed according to OECD guideline 421 (Reproduction / Developmental Toxicity Screening Test), however with limited documentation, is available (OECSEH, 2001). 12 Crj:CD (SD) rats per sex per dose were orally administered with the test substance 4,4’-sulphonyldiphenol at daily dose levels of 0, 10, 60 and 300 mg/kg bw.

Male rats were treated from 14 days before mating to the day before necropsy (including the mating period; 45 days in total) and female rats from 14 days before mating to day 3 of lactation (including the mating period, gestation period, and delivery; a total of 40 to 46 days). The animals were terminally sacrificed on day 45 (males) and on day 4 of lactation (females).

 Distinct indications of maternal toxicity were observed in the mid and high dose group (60, 300 mg/kg bw). A suppression in body weight gain in the high dose group is resulting from a decrease in food consumption. Furthermore, cecum distention including histopathological lesions in the cecum epithelium occurred in the mid and high dose and a liver enlargement accompanied by centrilobular hypertrophy was identified in the high dose group.

With respect to the observed cecum effects in rats and in accordance to the subchronic assays, the relevance for humans is unclear. Significant anatomical and functional differences of the cecum exist for rodents in comparison to humans. In rodents this anatomical structure is large and in comparison to humans has a significant function in digestion. Nevertheless, it is apparent, that these disturbances in the content and structure of the gastrointestinal tract in rats have a significant impact e.g. on body weight development.

In parent animals in the 300 mg/kg bw/day group, there were no test article-related changes in the copulation index, delivery index, gestation index, number of corpora lutea, length of gestation period, delivery condition, or lactation behavior. However, at a systemic toxic dose (300 mg/kg bw/day) there were significant prolongation of estrous cycle, increase in the number of animals that showed prolonged diestrus period, and decrease in the implantation index at 300 mg/kg bw/day. A tendency toward decrease in the fertility index was also observed at 300 mg/kg bw/day.

In offspring in the 300 mg/kg bw/day group, the total number of offspring delivered, number of live offspring and the number of offspring alive on day 4 of lactation tended to be low, but these effects were considered to be due to decreased implantation index. In the 300 mg/kg bw/day group, there were no test article-related changes in the live-birth index, sex ratio, body weight, viability index on day 4 after birth, anogenital distance, general condition, external findings or necropsy findings. In the 10 and 60 mg/kg bw/day groups, there were no test article-related changes in parent animals or offspring.

 

Since the relevance of rat cecum effects is questionable for humans and no further adverse effect was observed at 60 mg/kg bw, this dose is considered to be a NOAEL for parental toxicity. Based on a prolongation of estrous cycle and decreased implantation index the NOAEL for reproductive toxicity was considered to be 60 mg/kg bw/day.

 

The final Decision on substance evaluation acknowledges the possibility of strain specificities, as“…the same rat strain used for the OECD 421 (Sprague Dawley) shall be tested … to avoid any difference in sensitivity between rat strains”. Due to the change in the testing rat strain prescribed in the Decision on substance evaluation pursuant to Article 46(1) of Regulation (EC) No 1907/2006, a 28-day range finding experiment (bridging study) was performed. For 4,4’-sulphonyldiphenol no control data in non-mated and mated SD rats was available in the executing laboratory. In addition, rat strain or source specific differences in sensitivity could not be excluded based on the existing data, and the existing data for SD rats from an unknown source is of limited transferability to the present testing campaign. For a reliable identification of the dose levels and an unambiguous interpretation of the test results from a definitive study it appears inevitable to generate lab internal control data for 4,4'-sulphonyldiphenol in non-mated and mated SD rats from the specific supplier.

 

Therefore, a Repeated-dose 28-day toxicity study in SD rats (bridging study) was performed (BASF, 2017). 5 male and female Sprague Dawley rats were dosed at 0, 100, 300 and 600 mg/kg bw/ day by gavage. A 0.5% Carboxymethylcellulose suspension in drinking water was used as vehicle. The following parameters were investigated: detailed clinical examinations, food and drinking water consumption, body weight, organ weights and organ/tissue fixation of the target organs adrenal glands, kidneys, liver, all other organs were weighted and fixed (adrenal glands, brain, epididymides, heart, ovaries, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix, lymph nodes (axillary and mesenteric), mammary gland incl. inguinal lymph nodes), histopathological examination of all gross lesions, adrenal glands, cecum, liver, kidneys, mammary gland, spleen and uterus.

 

Salivation was observed in all test groups treated with test substance (22/30 animals). Clinical chemistry showed reduced hemoglobin in males at 600 mg/kg bw, no relevant findings were observed in females.

Changes in body and organ weights were the following:

-    dose dependent and statistically significant decrease of terminal body weight in males (-9% at 300mg/kg bw, -12% at 600 mg/kg bw).

-    increase of mean relative weights of the kidneys in males (+33% at 300 and 600 mg/kg bw, statistically significant at 600 mg/kg bw) and females (+12% at 300 mg/kg bw and 600 mg/kg bw, statistically significant at 600 mg/kg bw).

-    not statistically significant increase in relative weights of the adrenal glands in males (+18 and +39% at 300 and 600 mg/kg bw, respectively)

-    dose dependent and statistically significant increase of the mean relative weights of the liver in females (+9 and 16% at 300 and 600 mg/kg bw, respectively); not statistically significant increase of the mean relative weight of the liver in males (+12%)

-    decrease in relative weights of prostate and seminal vesicles at 600 mg/kg bw but not to a statistical significance (-15% and -16%, respectively).

-     decrease in thymus weights in males and females, but not statistically significant

The following gross lesions were observed:

- Enlarged kidneys in 4/5 male animals at 600 mg/kg bw and 3/5 male animals at 300 mg/kg bw as well as cecum dilation in in 2/5 male animals at 600 mg/kg bw were observed.

The following histopathology findings were observed:

-    tubular degeneration/regeneration in the kidney of all male animals at 300 and 600 mg/kg bw graded minimal to moderate and in 2/5 male animals at 100 mg/kg bw graded minimal or slight.

- Tubular hypertrophy in the kidney in 5/5 males at 600 mg/kg bw graded mostly moderate, in 5/5 males at 300 mg/kg bw graded mostly minimal and in 1/5 male at 100 mg/kg bw graded mostly minimal.

-    minimal hypertrophy/hyperplasia in the adrenal gland cortex in 3/5 male animals at 600 mg/kg bw

-    centrilobular hypertrophy of the liver

at 600 mg/kg bw in all male animals graded up to moderate and in all female animals graded mostly slight

at 300 mg/kg bw in 4/5 male animals graded slight,

at 100 mg/kg bw in 1/5 male animal graded minimal.

-    dilation of the cecum in 5/5 male and 2/5 female animals at 600 mg/kg bw

-    diffuse atrophy of the mammary gland in 4/5 male animals at 600 mg/kg bw and in 3/5 male animals at 300 mg/kg bw.

The target organs identified in the existing repeated dose studies for 4,4’-sulphonyldiphenol were confirmed (28-day gavage study, Reproduction / Developmental Toxicity Screening Test in SD rats, OECSEH 1999, 2001; 90 day gavage study in Wistar rats, BASF, 2015). The kidney appears to be more susceptible to DHDPS related effects in SD-rats compared to Wistar rats.

 

 In a Reproduction/Developmental Toxicity Screening Study which was performed as a Range-Finding Study for an Extended One Generation Reproductive Toxicity study (EOGRTS), 4,4'-sulphonyldiphenol was orally administered via gavage to groups of 10 male and 10 female Sprague-Dawley rats (F0 animals) at doses of 0, 30, 100 and 300 mg/kg bw/d (BASF, 2017). The duration of treatment covered a 6-week premating and a 14-days mating period in both sexes, approximately 4 weeks post-mating in males, the entire gestation and lactation periods in F0 females and their pups, as well as about one week after weaning in selected F1 offspring.

 

Clinical signs of parental toxicity included salivation in F0 males and females at 300 mg/kg bw. Food consumption was mildly increased in F0 females of all dose groups (13-16%) in the pre-mating phase of the study. No clear trend could be observed for food consumption of F0 males. In F0 males and females at the high dose level body weights were decreased by 7.1 and 5.8% at the end of the pre-mating period, respectively. 

In males, treatment related target organ specific toxicity was identified by macroscopic and histopathological examinations in the cecum, liver and kidneys. Examination of the liver and kidney showed macroscopic enlargement which was correlated with an increase in liver and kidney weight in males. Histopathology further identified a diffuse atrophy in mammary glands of the high dose group.

Indications for target organ specific toxicity were less pronounced in females. In the high dose group, histopathological examinations identified substance related effects in the kidney (tubular degeneration/regeneration and tubular dilation in males, and lymphoid infiltration and medullar mineralization in females) and liver (lymphoid infiltration sin females).

High-dose F0 parental females (300 mg/kg bw/d) had a statistically significant and distinctly prolonged estrous cycle (5.16 vs. 4.02 days). All 8 pregnant females had a significantly lower average number of implants compared to the concurrent control (10.4 vs. 15.8). In addition, post-implantation loss was significantly increased (34.6 vs 3.6%) and 2 of the 8 pregnant females had complete intrauterine litter losses. These effects resulted in a significantly lower live litter size (10.3 vs. 15.0). Newborn pups developed normally.

  

According to the final Decision on substance evaluation an Extended One Generation Reproductive Toxicity Study in Sprague-Dawley rats, oral route, according to test method OECD 443, with the developmental neurotoxicity and immunotoxicity (DNT/DIT) cohorts and with the conditional extension of Cohort 1B to mate the F1 animals to produce an F2 generation has been conducted (BASF, 2019).

The test substance was administered to groups of 24 male and 24 female healthy young Sprague-Dawley rats for test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (= F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 24 males and 24 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Administration period for F0 parental animals was four months in total, F1B parental animals approximately four months, cohort F1A two months, cohort 2A one and a half months, and cohort 3 just over one month in total. Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

 

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

Water consumption of the F0 and F1 parents and F1 rearing animals was determined regularly once weekly and weekly during gestation days (GD) 0-1, 3-4, 7-8, 10-11, 14-15, 17-18, 19-20 and lactation days (PND) 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21. Food consumption of the F0 and F1 parents and F1 rearing animals was determined regularly once weekly and weekly during GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10, 10-14, 14-17, 17-19 and 19-21.

In general, body weights of F0 and F1 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0/F1 females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 17, 19 and 21.

A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A, 1B, 2A and 3 at weekly intervals.

Estrous cycle data were evaluated for F0 and cohort 1B (= F1 generation) females over a three weeks period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

An auditory startle response test was carried out in all animals of cohort 2A on PND 24.

Learning and memory was tested using a Morris water maze in all animals of cohort 2A from PND 60 onwards.

A functional observational battery (FOB) was performed in all animals of cohort 2A on PND 75.

Motor activity was measured in all animals of cohort 2A on PND 75.

The F1 and F2 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter).

Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 parental generation was recorded.

All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully reexamined one PND 20 (F1) or PND 21 (F2).

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live F1 and F2 pups on PND 1.

Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group.

Further blood samples were taken from 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 and 1A males at scheduled sacrifice or after appropriate staining.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 parental females. All F1 rearing animals were assessed by different pathological, neuro- and histopathological examinations.

 

Test substance-related adverse effects/findings were noted in the F0 parental animals at 180 mg/kg bw/day regarding kidneys. Increased absolute (+16%) and relative (+20%) weights of the kidneys in males, macroscopically enlarged kidneys in 6/24, increased mineralization in the kidneys in the transition from medulla to cortex ranging from minimal to severe in 21/24 males, nuclear crowding in the kidneys ranging from minimal to moderate in 22/24 males, and tubular dilation in the kidneys ranging from minimal to slight in 13/24 males were observed. F1 pups in the 180 mg/kg bw/day showed a decreased number of liveborn pups subsequent to higher postimplantation loss. Cohort F1A animals in the 180 mg/kg bw/day had increased water consumption in females during major parts of the study period (up to 25% above control) and increased food consumption in females during major parts of the study period (up to 21% above control). In addition, increased body weights in females on study days 14 and 28 (up to 7% above control, respectively) and increased body weight change in females during study days 7 - 14 (about 19% above control, respectively) were observed. Furthermore, prolonged prothrombin time in males and increased total protein, albumin and calcium values in females were reported. In males, increased absolute (+12%) and relative (+17%) weights of the kidneys as well as increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate), nuclear crowding in the kidneys in 6/20 male animals (minimal to slight), tubular dilation in the kidneys in 7/20 male animals (minimal to slight), and increased incidence of atrophy in the mammary gland and mammary fat pad were observed.

In F1 parental animals (cohort 1B) at 180 mg/kg bw/day, increased water consumption in females during major parts of the premating period (up to 26% above control) and increased food consumption in females during premating days 28 - 49 and 0 - 70 (up to 22% and 13% above control, respectively) were reported. Additionally, increased body weights in females during major parts of the premating period (up to 10% above control) and increased body weight change in females during premating days 0 - 21 and 0 - 70 (up to 18% and 10% above control, respectively) were observed. In females, increased postimplantation loss (24.6% vs. 6.4% in control) was revealed. Males showed increased absolute (+26%) and relative (+32%) weights of the kidneys and macroscopically enlarged kidneys in 10/24 animals.

In F2 pups, decreased mean pups delivered were subsequent to higher postimplantation loss in F1B maternal animals.

Adverse effects noted in F0 parental animals at 60 mg/kg bw/day were increased water consumption in females during GD 17 - 20 (up to 22% above control), increased food consumption in females during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6% above control, respectively), and increased postimplantation loss (9.4% vs. 3.1% in control).

Cohort F1A animals showed increased water consumption in females during study days 7 - 17 (up to 20% above control), increased body weights in females on study days 14 and 28 (up to 7% above control, respectively), and increased body weight change in females during study days 0 - 7 (about 9% above control, respectively) at 60 mg/kg bw/day.

At 60 mg/kg bw/day F1B parental animals showed increased water consumption in females during premating days 14 - 17 and 42 - 45 (about 14% and 15% above control, respectively) and increased food consumption in females during premating days 7 - 14 (about 13% above control). In addition, increased body weights in females during major parts of the premating period and on PND 14 (up to 9% and 6% above control, respectively) and increased body weight change in females during premating days 0 - 21 (up to 20% above control) were observed. Increased absolute (+13%) and relative (+10%) weights of the kidneys in males were noted.

No treatment-related adverse findings were observed at 20 mg/kg bw/day in any treatment group, either parental or progeny.

 

Under the conditions of the present modified extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 60 mg/kg bw/d for the F0 and F1 parental as well as adolescent animals, based on evidence for kidney toxicity, as well as corresponding effects on clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 180 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 180 mg/kg bw/d, the highest dose tested.

Effects on developmental toxicity

Description of key information

OECD 414 (GLP; Wistar rats; 30, 100, 300 mg/kg bw/d):

- a dose of 300 mg/kg bw/d caused evidence of maternal toxicity (reduced food consumption and (net) body weight gain)

- no toxicologically relevant adverse fetal findings evident

- NOAEL (maternal toxicity) = 100 mg/kg bw/d; NOAEL (prenatal developmental toxicity) = 300 mg/kg bw/d

 

OECD 443 (GLP; Sprague Dawley rats; 20, 60, 180 mg/kg bw/day):

- increased kidney weights, enlarged kidneys and histopathological findings in the kidney at 180 mg/mg bw/d

- increased post-implantation loss in F0 and F1B parental animals at 60 and 180 mg/kg bw/d

- NOAEL (general, systemic toxicity) = 60 mg/kg bw/d; NOAEL (developmental toxicity) = 20 mg/kg bw/day; NOAEL (developmental neurotoxicity) = 180 mg/kg bw/d; NOAEL (developmental immunotoxicity) = 180 mg/kg bw/d 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep 2013 - 28 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 11-13 weeks
- Weight at study initiation:
- Housing: Makrolon cages type M III (1 animal per cage)
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): yes
- Acclimation period: from GD 0 (day of supply) to the beginning of administration (GD 6), the animals were accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C;
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % suspension in drinking water
Details on exposure:
Gavage exposure from gestation day (GD) 6 through GD 19.

PREPARATION OF DOSING SOLUTIONS:
10 ml/kg body weight were administered. Test substance preparations were suspended in 1 % Carboxymethylcellulose in drinking water.
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability. The preparations were kept in a refrigerator.
For the test substance preparations, the specific amount of test substance were weighed, topped up with 1% 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:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations of the test substance preparations, and verification of stability of the test substance in 1 % Carboxymethylcellulose suspension in drinking water over a period of a maximum of 7 days prior to the start of the study (Project No.: 01Y0066/05Y009).
Details on mating procedure:
The animals were paired by the breeder (time-mated animals) and were supplied at noon on the day of evidence of mating. This day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
Gavage exposure from gestation day (GD) 6 through GD 19.
On GD 20, blood samples were obtained from dams by retrobulbar venous puncture.
Following blood sampling on GD 20, all dams were sacrificed and examined. Fetuses were removed from the opened uterus.
Frequency of treatment:
daily
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, for GD 0-1 , 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17,
17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes, on GD 20, following anesthetisia with isoflurane, sacrificion by decapitation.
Fetuses were be removed from the uterus.
- Organs examined: Adrenal glands, Kidneys, Liver, Spleen. Organ / Tissue fixation for all gross lesions and organs examined.

HEMATOLOGY /CLINICAL CHEMISTRY: Yes
Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
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
- Site of implantations in the uterus
Fetal examinations:
- Weight of each fetus
- Sex
- Weight of the placentas
- Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord);
- fetuses wer sacrificed by subcutaneous injection of pentobarbital, and about half of the fetuses of each dam were skinned, fixed in ethyl alcohol and, after fixation, stained according to a modified method (KIMMEL and TRAMMELL) to show the skeleton and the cartilage. The other half of the fetuses of each dam were fixed in Harrison's fluid.
- examination of skeletons/cartilage and soft tissues.
Statistics:
Food consumption, body weight, organ weights - DUNNETT's test.
Implantations, resorptions and live fetuses - DUNNETT's test.
Number of pregnant animals, mortality and number of litters with fetal findings - FISHER's exact test.
Proportion of fetuses with findings per litter - WILCOXON test.
Clinical pathology parameters - KRUSKAL-WALLIS and WILCOXON test.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no test substance-related or spontaneous mortalities in any females of all test groups.
Seven (out of 25) high-dose females showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 20 minutes) and was initially observed on GD 10.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of the low- and the mid-dose groups during the entire study period.

The mean food consumption of the high-dose dams was statistically significantly reduced at the beginning of the treatment period (GD 6-13; up to 15% below control), but recovered afterwards.
If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the high-dose dams consumed 8% or 6%, respectively, less food in comparison to the concurrent control group.
The mean food consumption of the dams in test groups 1 and 2 (30 or 100 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. The only exception was a slightly, but statistically significantly lower mean food consumption of the low- and high-dose dams on GD 0-1, which was a solitary event and considered to be accidental.

The mean body weights of the high dose dams were in general comparable to the controls throughout the entire study period.
The body weight change of the high-dose dams was statistically significantly reduced on GD 8-10 (approx. 29% below control) and if calculated for the entire treatment period (GD 6-19; 8% below control).
The mean body weights and the average body weight gains of the low- and mid-dose dams were in general comparable to the controls throughout the entire study period.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of high dose was clearly lower than the concurrent control value (approx. 10% below control), but without attaining statistical significance.
The corrected body weight gain of low and mid dose groups revealed no difference of any biological relevance to the corresponding control group.
Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Mean gravid uterus weights of the animals of all test groups were not influenced by the test substance.

No necropsy findings which could be attributed to the test substance were seen in any dam.
In the high dose spontaneous findings were noted in individual females of the high dose group (a diaphragmatic hernia (this female was not pregnant), a dilated renal pelvis, and a hemometra).

The conception rate varied between 96% in the high dose group 3 and 100% in the other test groups. With these rates, a sufficient number of
pregnant females were available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences 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.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The sex distribution of the fetuses in all dose groups was comparable to the control fetuses.
The mean placental weights of all dose groups were comparable to the corresponding control group.
The mean fetal weights of all dose groups were not influenced by the test substance and did not show any biologically relevant differences in
comparison to the control group.

No soft tissue malformations were recorded.
Some soft tissue variations were detected in all test groups including the control, i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant. Most of them can be found in the historical control data at comparable incidences.
No unclassified soft tissue observations were recorded.

One mid-dose fetus with multiple external malformations was seen (misshapen head and absent face). The overall incidences of external malformations were comparable to those found in the historical control data. An association of these findings to the treatment is not assumed.
No external variations were recorded.
One unclassified external observation, i.e. blood coagulum around placenta, was recorded in two fetuses of the high-dose group. This finding was not considered biologically relevant.

Skeletal malformations were detected in all test groups exept low dose group (0, 100 and 300 mg/kg bw/d) affecting the skull, sternum and forelimbs.
One fetus of the mid dose group (same animal as external malformation) was multiple-malformed (malformations affected the skull, vertebral column, ribs, pelvic girdle and forelimbs) and had associated external findings. The average rate of affected fetuses per litter showing skeletal malformations was statistically significantly increased in the high-dose group. However, each of the findings leading to that increased rate ist present in the historical control data and no abnormality pattern became obvious. The total incidences of skeletal malformations in the low- and mid-dose groups were comparable to the concurrent control group. These scattered observations in individual fetuses of these groups are are common for this rat strain.

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 and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data, and no dose dependency was observed.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.
Key result
Dose descriptor:
NOAEC
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no observed effects at highest dose level
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Terminal body weights, food consumption and relative organ weights [g].

dose [mg/kg bw/day]

0

30

100

300

food consumption (0-20)

19.3

19.1

19.3

18.2

absolute terminal bw

295.9

302.4

297.8

291

corrected bw gain

40.9

43.7

40

36.9

carcass weight

236.8

242.8

239.1

235.2

bw change 6 -19

85.2

89.8

84.3

78.6*

 

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of DHDPS to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 300 mg/kg bw/d caused evidence of maternal toxicity, such as reduced food consumption and (net) body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d.
There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d.
Executive summary:

In a prenatal developmental toxicity study the test substance DHDPS was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

The test substance caused no mortality nor clinical symptoms of systemic toxicity in any of the exposed groups receiving 30, 100 or 300 mg/kg bw/d DHDPS. Some females (7 out of 25) of the high-dose group (300 mg/kg bw/d) showed transient salivation after treatment. This salivation persisted in the respective females for a few minutes immediately after each administration. It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The high-dose of the test substance (300 mg/kg bw/d) caused a significant decrease in food consumption (mainly at the beginning of treatment) and body weight gain as well as a distinct decrease in the corrected (net) body weight gain. These effects are considered to be treatment-related and adverse. No toxicologically relevant effect on food consumption and body weight gain was noted for the animals exposed to 30 or 100 mg/kg bw/d DHDPS.

No differences of toxicological relevance between the control and the treated groups (30, 100 or 300 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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

A prenatal developmental toxicity study was performed with 4,4’-sulfonyldiphenol in accordance to the OECD 414 guideline. The test substance was administered to pregnant Wistar rats by daily gavage from implantation to one day prior to the expected day of parturition (GD 6-19) (BASF, 2014).

The test substance caused no mortality nor clinical symptoms of systemic toxicity in any of the exposed groups receiving 30, 100 or 300 mg/kg bw/d 4,4’-sulfonyldiphenol. Signs of maternal toxicity included transient salivation after treatment of the high dose (300 mg/kg bw/d). In addition, a significant decrease in food consumption (mainly at the beginning of treatment) and a decrease in body weight gain as well as a distinct decrease in the corrected (net) body weight gain were observed in the high dose group. These effects are considered to be treatment-related and adverse. No toxicologically relevant effect on food consumption and body weight gain was noted for the animals exposed to 30 or 100 mg/kg bw/d DHDPS.

No differences of toxicological relevance between the control and the treated groups (30, 100 or 300 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

 

 

According to the final Decision on substance evaluation an Extended One Generation Reproductive Toxicity Study in Sprague-Dawley rats, oral route, according to test method OECD 443, with the developmental neurotoxicity and immunotoxicity (DNT/DIT) cohorts and with the conditional extension of Cohort 1B to mate the F1 animals to produce an F2 generation has been conducted (BASF, 2019).

The test substance was administered to groups of 24 male and 24 female healthy young Sprague-Dawley rats for test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg bw/d). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 24 males and24 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Administration period for F0 parental animals was four months in total, F1B parental animals approximately four months, cohort F1A two months, cohort 2A one and a half months, and cohort 3 just over one month in total.

Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

 

Test substance-related adverse effects/findings were noted in the F0 parental animals at 180 mg/kg bw/day regarding kidneys. Increased absolute (+16%) and relative (+20%) weights of the kidneys in males, macroscopically enlarged kidneys in 6/24, increased mineralization in the kidneys in the transition from medulla to cortex ranging from minimal to severe in 21/24 males, nuclear crowding in the kidneys ranging from minimal to moderate in 22/24 males, and tubular dilation in the kidneys ranging from minimal to slight in 13/24 males were observed. F1 pups in the 180 mg/kg bw/day showed a decreased number of liveborn pups subsequent to higher postimplantation loss.

Cohort F1A animals in the 180 mg/kg bw/day had increased water consumption in females during major parts of the study period (up to 25% above control) and increased food consumption in females during major parts of the study period (up to 21% above control). In addition, increased body weights in females on study days 14 and 28 (up to 7% above control, respectively) and increased body weight change in females during study days 7 - 14 (about 19% above control, respectively) were observed. Furthermore, prolonged prothrombin time in males and increased total protein, albumin and calcium values in females were reported. In males, increased absolute (+12%) and relative (+17%) weights of the kidneys as well as increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate), nuclear crowding in the kidneys in 6/20 male animals (minimal to slight), tubular dilation in the kidneys in 7/20 male animals (minimal to slight), and increased incidence of atrophy in the mammary gland and mammary fat pad were observed.

In F1 parental animals (cohort 1B) at 180 mg/kg bw/day, increased water consumption in females during major parts of the premating period (up to 26% above control) and increased food consumption in females during premating days 28 - 49 and 0 - 70 (up to 22% and 13% above control, respectively) were reported. Additionally, increased body weights in females during major parts of the premating period (up to 10% above control) and increased body weight change in females during premating days 0 - 21 and 0 - 70 (up to 18% and 10% above control, respectively) were observed. In females, increased postimplantation loss (24.6% vs. 6.4% in control) was revealed. Males showed increased absolute (+26%) and relative (+32%) weights of the kidneys and macroscopically enlarged kidneys in 10/24 animals.

In F2 pups, decreased mean pups delivered were subsequent to higher postimplantation loss in F1B maternal animals.

Effects noted in F0 parental animals at 60 mg/kg bw/day were increased water consumption in females during GD 17 - 20 (up to 22% above control), increased food consumption in females during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6% above control, respectively), and increased postimplantation loss (9.4% vs. 3.1% in control).

Cohort F1A animals showed increased water consumption in females during study days 7 - 17 (up to 20% above control), increased body weights in females on study days 14 and 28 (up to 7% above control, respectively), and increased body weight change in females during study days 0 - 7 (about 9% above control, respectively) at 60 mg/kg bw/day.

At 60 mg/kg bw/day F1B parental animals showed increased water consumption in females during premating days 14 - 17 and 42 - 45 (about 14% and 15% above control, respectively) and increased food consumption in females during premating days 7 - 14 (about 13% above control). In addition, increased body weights in females during major parts of the premating period and on PND 14 (up to 9% and 6% above control, respectively) and increased body weight change in females during premating days 0 – 21 (up to 20% above control) were observed. Increased absolute (+13%) and relative (+10%) weights of the kidneys in males were noted.

No treatment-related adverse findings were observed at 20 mg/kg bw/day in any treatment group, either parental or progeny.

 

Under the conditions of the present modified extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 60 mg/kg bw/d for the F0 and F1 parental as well as adolescent animals, based on evidence for kidney toxicity, as well as corresponding effects on clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 180 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 and F2 progeny is 20 mg/kg bw/d, based on increased postimplantation loss in P0 and P1 animals, which was observed at the LOAEL (Lowest Observed Adverse Effect Level) of 60 mg/kg bw/d. The NOAEL for developmental neurotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested. The NOAEL for developmental immunotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

 

 

In a Reproduction/Developmental Toxicity Screening Study which was performed as a Range-Finding Study for an Extended One Generation Reproductive Toxicity study (EOGRTS), 4,4'-sulphonyldiphenol was orally administered via gavage to groups of 10 male and 10 female Sprague-Dawley rats (F0 animals) at doses of 0, 30, 100 and 300 mg/kg bw/d (BASF, 2017). The duration of treatment covered a 6-week premating and a 14-days mating period in both sexes, approximately 4 weeks post-mating in males, the entire gestation and lactation periods in F0 females and their pups, as well as about one week after weaning in selected F1 offspring.

 

Clinical signs of parental toxicity included salivation in F0 males and females at 300 mg/kg bw. Food consumption was mildly increased in F0 females of all dose groups (13-16%) in the pre-mating phase of the study. No clear trend could be observed for food consumption of F0 males. In F0 males and females at the high dose level body weights were decreased by 7.1 and 5.8% at the end of the pre-mating period, respectively. 

In males, treatment related target organ specific toxicity was identified by macroscopic and histopathological examinations in the cecum, liver and kidneys. Examination of the liver and kidney showed macroscopic enlargement which was correlated with an increase in liver and kidney weight in males. Histopathology further identified a diffuse atrophy in mammary glands of the high dose group.

Indications for target organ specific toxicity were less pronounced in females. In the high dose group, histopathological examinations identified substance related effects in the kidney (tubular degeneration/regeneration and tubular dilation in males, and lymphoid infiltration and medullar mineralization in females) and liver (lymphoid infiltration in females).

High-dose F0 parental females (300 mg/kg bw/d) had a statistically significant and distinctly prolonged estrous cycle (5.16 vs. 4.02 days). All 8 pregnant females had a significantly lower average number of implants compared to the concurrent control (10.4 vs. 15.8). In addition, post-implantation loss was significantly increased (34.6 vs 3.6%) and 2 of the 8 pregnant females had complete intrauterine litter losses. These effects resulted in a significantly lower live litter size (10.3 vs. 15.0). Newborn pups developed normally.

A reproduction/ developmental toxicity screening test was performed with 4,4´-sulphonyldiphenol according to OECD 421 (OECSEH, 2001). 12 Dawley rats per sex per dose were orally administered with the test substance at daily dose levels of 0, 10, 60 and 300 mg/kg bw for 45 days in males and from 14 days before mating to day 3 of lactation in females. The animals were terminally sacrificed on days 45 (males) and on day 4 of lactation (females).

Distinct indications of maternal toxicity were observed in the mid and high dose group (60, 300 mg/kg bw). A suppression in body weight gain in the high dose group is resulting from a decrease in food consumption. Furthermore, cecum distention including histopathological lesions in the cecum epithelium occurred in the mid and high dose and a liver enlargement accompanied by centrilobular hypertrophy was identified in the high dose group. With respect to cecum effects in rats the relevance for humans is unclear. Significant anatomical and functional differences of the cecum exist for rodents and humans. In rodents this anatomical structure is large and in comparison to humans has a significant function in digestion. Nevertheless, these disturbances in the content and structure of the gastrointestinal tract in rats may have a significant impact e.g. on body weight development. Therefore, the NOAEL for parental toxicity (repeated dose) was considered to be 60 mg/kg bw/day.

In parent animals in the 300 mg/kg bw/day group, there were no test substance-related changes in the copulation index, delivery index, gestation index, number of corpora lutea, length of gestation period, delivery condition, or lactation behavior. However, at a severely systemic toxic dose (300 mg/kg bw/day) there were significant prolongation of estrous cycle, increase in the number of animals that showed prolonged diestrus period, and decrease in the implantation index at 300 mg/kg bw/day. A tendency toward decrease in the fertility index was also observed at 300 mg/kg bw/day.

In offspring in the 300 mg/kg bw/day group, the total number of offspring delivered, number of live offspring and the number of offspring alive on day 4 of lactation tended to be low, but these effects were considered to be due to decreased implantation index. In the 300 mg/kg bw/day group, there were no test article-related changes in the live-birth index, sex ratio, body weight, viability index on day 4 after birth, anogenital distance, general condition, external findings or necropsy findings. In the 10 and 60 mg/kg bw/day groups, there were no test article-related changes in parent animals or offspring.

 

Under the conditions of the present reproduction/ developmental toxicity screening test the NOAEL (no observed adverse effect level) for parental general, systemic toxicity was considered to be 60 mg/kg bw/day. Based on a prolongation of estrous cycle and decreased implantation index the NOAEL for reproductive toxicity was considered to be 60 mg/kg bw/day.

Kolla et al. investigated if BPS exposure alters the development of the male mouse mammary gland and sensitizes it to a peripubertal estrogen challenge.

Therefore, mated female CD-1 mice were treated in two experiments orally via feed (experiment 1) or via pipet (experiment 2) either from pregnancy day 9 through lactation day 2 or until lactation day 20 in experiment 2 and 1, respectively. Pups were weaned at PND21 (experiment 1) and mammary glands were collected at PND24 or 9 weeks of age. In experiment 2 at PND21, two males from each litter were randomly selected. One pup was assigned to receive vehicle and one received an estrogen challenge (1µg/kg bw/day EE2) orally via pipet for 10 days. On PND31, body weight, anogenital distance, seminal vesicle weight, and mammary glands were collected.

In experiment 1, treated males at 2 and 200 µg/kg bw/day had more space located between cells due to lumen formation. Males exposed to 200 µg/kg bw/day showed significantly smaller ductal trees in the left mammary gland. At week 9, males at 200 µg/kg bw/day had significantly larger left epithelial trees. At PND24, a significant increase in size of the mammary glands in males treated with 2 and 200 µg/kg bw/day were observed. In experiment 2, mice perinatally exposed were non-responsive to EE2 challenge conserning seminal vesicle weight leading to the assumption that treated males are less responsive to their own endogeneous hormones. At 200 and 2000 g/kg bw/day, significantly increased growth parameters were reported, i.e. ductal area and branching points.

Results suggest that developmental exposure of the test substance alters the response of males to postnatal estrogens, but the responses are complex; low doses diminish the effects and high doses enhance the effects of postnatal estrogens. The authors questioned if the effects seen in this study would be evaluated as adverse by risk assessors. If so, they concluded an NOAEL, based on the results, of 200 µg/kg bw/day or lower.

Justification for classification or non-classification

For 4,4'-sulphonyldiphenol adverse effects on sexual function were described in Reproduction/Developmental Toxicity Screening Studies, by an increased incidence of irregular estrus cycles. Adverse effects on fertility were described by a decrease in fertility index, a decrease in the number of implantation sites and post-implantation loss. This evidence was consistently made in two studies with rats of good quality, at equivalent dose levels and with a statistical significance. Effects on fertility occurred together with signs of maternal toxicity in dams (e.g. salivation, GI-tract- and kidney-toxicity).

Under the conditions of the present modified extended 1-generation reproduction toxicity study, there were no indications from clinical examinations as well as gross and histopathology, that the test substance adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including the highest administered dose of 180 mg/kg bw/d.

The main findings were increases of mean number of resorptions and of postimplantation loss in the high- and mid-dose groups. These findings were noted in both F0 and F1 parental animals.

In addition, estrous cycle length was increased in the high-dose group in the F0 and F1 generations: the increase in the F0 generation was statistically significant with 4.1 days vs. 3.9 days in the control group, in the F1 generation the increase was not significant with 4.1 days vs. 3.9 days in the control group .

No evidence of immunotoxicity or neurotoxicity was noted. 

 The NOAEL (no observed adverse effect level) for general, systemic toxicity is 60 mg/kg bw/d for the F0 and F1 parental as well as adolescent animals, based on evidence for kidney toxicity, as well as corresponding effects on clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 180 mg/kg bw/d.

The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 180 mg/kg bw/d, the highest dose tested.

The NOAEL for developmental toxicity was established at 20 mg/kg (based on increased postimplantation loss in F1 progeny).

 

Currently, 4,4’- sulphonyldiphenol is self-classified and labelled as Repr. Cat. 2 H361f.

 

In October 2019 the Belgium FPS Public Health, Food Chain Safety and Environment submitted a “CLH report - Proposal for Harmonised Classification and Labelling of 4,4’-sulphonyldiphenol (CAS 80-09-1)”. The CLH dossier submitter concluded based on the available data that classification and labelling as

Repr.Cat 1B H360FD is warranted.