Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jun 2018 - 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objectives of this study were to determine the potential toxic effects of 4,4’-methylenebis-N-secbutylaniline when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate its potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jun 2018 - 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objectives of this study were to determine the potential toxic effects of 4,4’-methylenebis-N-sec-butylaniline when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate its potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(2016)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
Remarks:
(
Qualifier:
according to guideline
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (2000)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No correction factor applied.
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-11 weeks (males), 13-14 weeks (females)
- Weight at study initiation: 285-342 gr (males) or 211-251 gr (females)
- Fasting period before study: no (only males were fasted overnight before necropsy).
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity measurements (maximum 2 hours).
- Water: Free access to tap water, except during motor activity measurements (maximum 2 hours). Periodic analysis of the water was performed by the test facility.
- Acclimation period: At least 8 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (main study): From: 23 Aug 2018 to 18 Oct 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Adjustment was made for specific gravity of the vehicle and test item.

Storage conditions of formulations: The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Justification for use and choice of vehicle: Choice of vehicle was based on trial preparations performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All analyses were performed using a validated analytical procedure (Charles River Study No. 20145350). Concentration of the formulations were analysed in week 1 (all groups), homogeneity was analysed also in week 1 (low and high dose only). Concentration and homogeneity of the formulations were analysed in duplicate. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Charles River Study No. 20145350) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finder with oral administration of 4,4’-methylenebis-N-sec-butylaniline in rats, and in an attempt to produce graded responses to the test item. In the dose range finder study 3 females/group were exposed for 7 (300 mg/kgbw/day) or 10 (150 mg/kgbw/day) consecutive days. Daily formulation portions were prepared freshly (300 mg/kg bw/day) or within 8 days prior to the corresponding administration day, and stored in the refrigerator protected from light (150 mg/kg bw/day). The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Mortality was monitored twice daily throughout the study. Clinical observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights were measured on Day 1 prior to dosing and on Days 5 and 10. In order to monitor the health status, all animals dosed at 300 mg/kg bw/day were also weighed on Day 7 and Day 8 of treatment. Food consumption was measured over Days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy) or sooner (decedents). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and all gross lesions were recorded.
- Animal selection:
Five animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS:
Yes, for general health and moribundity
- Time schedule: once daily. During the dosing period, these observations were performed directly after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finder). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.

BODY WEIGHT:
Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Males were fasted overnight with a maximum of approximately 24.5 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Males were fasted overnight with a maximum of approximately 24.5 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5 animals/sex/group (except for thyroid hormone levesl, which were analysed for all rats/group)
- Parameters checked were: According to test guidelines (including coagulation parameters (prothrombin time and activated partial thromboplastin time) and thyroid hormone levels(thyroxine (T4) and thyroid stimulating hormone (TSH)))

URINALYSIS: No

FUNCTIONAL TESTS:
Yes
- Time schedule for examinations: Selected males were tested during Week 4 of treatment and the selected females were tested during lactation (PND 6-13).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal), locomotor activity (recording period: 1-hour under normal laboratory light conditions), total movements and ambulations.

ESTROUS CYCLE:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals surviving to scheduled necropsy and all moribund animals underwent necropsy, and specified tissues were retained but not weighed.
Full post mortem examination was performed on all surviving rats, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

ORGAN WEIGHTS
- The organs as specified in the guidance were weighed at necropsy (5 animals/sex/group). Sex organs and related tissues were weighed for all males.

HISTOPATHOLOGY: Yes
Performed on 5 rats/group, all tissues and organs as defined in the guidances.

Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose groups vs control group, mid dose group vs control group, high dose group vs control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation observed after dosing among animals exposed to test item during multiple consecutive days from the second week of the treatment period onwards. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This effect was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. Prior to early sacrifice, these females were lean, had a hunched posture and were noted with piloerection from approximately Day 25 of treatment onwards. For both females, main macroscopic findings were present in the liver: pale discoloration, thickened, enlarged and/or accentuated lobular pattern. Correlating microscopic findings were moderate to marked hepatocellular vacuolation and moderate to marked hepatocellular hypertrophy. No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain were statistically significantly reduced in males dosed at 75 mg/kg bw/day from Day 15 of the treatment period onwards with up to 13% lower by the end of treatment when compared to the control group. Body weight gain was lower in females at 75 mg/kg bw/day during post-coitum (up to 8% lower compared to concurrent controls). Body weight gain during lactation was considered to be within the same range as the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In females in the high dose group, relative food consumption was increased during post-coitum, reaching statistical significance over Days 14-17 and 17-20 post-coitum (10 and 17% compared to concurrent controls). Normal values for food consumption and relative food consumption were noted in females during the rest of the treatment period. As the increase in food consumption was probably due to the slightly lower body weight at 75 mg/kg bw/day and as all values remained within the range of the historical controls, this finding was considered not toxicologically relevant. In males at 75 mg/kg bw/day, a trend towards a lower absolute and relative food consumption was noted during the first two weeks of treatment (up to 17 and 12% lower than levels in concurrent controls, respectively). Values were on the low end or just outside the historical control range. Historical control data are included below under "overall remarks".
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males a slight increase was seen in white blood cell count in the high dose group (8.7, 10.6, 8.2 and 11.3 *10E9/L for the control group and the groups exposed to 3, 15 and 75 mg/kg bw/day, respectively), which was mainly related to increased monocyte concentration (0.2, 0.2, 0.1 and 0.3 *10E9/L for the control group and the groups exposed to 3, 15 and 75 mg/kg bw/day, respectively). Haemoglobulin and haematocrit concentrations were statistically significantly decreased for the high dose males (haemoglobulin: 10.1 mmol/L for controls versus 9.2 mmol/L for the high dose; haematocrit: 0.487 L/L for controls versus 0.451 L/L for the high dose). Furthermore reduced mean corpuscular volume (MCV) and Reduced mean corpuscular haemoglobin (MCH) were noted in high dose males (53.4 fL for controls versus 50.8 fL for the high dose (MCV) and 1.11 fmol for controls versus 1.04 fmol for the high dose (MCH), both decreases statistically significant).
Due to the fact that the changes were minimal and a dose-response relationship was absent these effects were considered not toxicologically relevant. Furthermore none of these effects were seen in females. In female rats, the platelet concentrations were statistically significantly decreased in all dose groups (861, 756, 737 and 653 *10E9/L for the controls and the females exposed to 3, 15 and 75 mg/kg bw/day, respectively). Although a dose-related response was observed, the platelet concentrations were still within historical control data (historical control data (non-fasted) female Wistar Han rats (period 2015 - 2018): Platelets (10E9/L) Mean = 749; P5 – P95 = 552 – 932 (n = 144)) and therefore these values were not considered toxicologically relevant.

Coagulation parameters in males and females were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In high dose males, the concentration bilirubin was statistically significantly increased compared to the controls (2.6 µmol/L in controls versus 4.2 µmol/L in high dose males). Urea levels were decreased in all dosed males (8.1, 6.0, 6.4 and 5.7 mmol/L for controls and low, mid and high dose group males, respectively (statistically significant for low and high dose rats)). The urea values in treated males were considered to have arisen as a result of slightly high control values and in the absence of a treatment-related distribution considered to be of no toxicological significance. The effects on bilirubin and urea were not seen in female rats. Cholesterol levels were statistically significantly increased for high dose males (1.79, 1.77, 2.18 and 2.93 mmol/L for controls and low, mid and high dose group males, respectively) and also for high dose females (2.26, 2.25, 2.17 and 2.92 mmol/L for controls and low, mid and high dose group males, respectively). The increase in cholesterol levels was statistically significant for high dose rats in both sexes. A dose-dependent decrease in bile acids was observed (not statistically significant; 39.5, 37.6, 24.2 and 23.4 µmol/L for controls and low, mid and high dose group males, respectively). Other statistically significant changes in clinical biochemistry parameters (inorganic phosphatase in males) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

A decrease in total T4 was observed for males at 15 and 75 mg/kg bw/day (0.72 and 0.57x of control, respectively). A corresponding increase in TSH was observed at 75 mg/kg bw/day (1.99x of control), although values did not reach statistical significance. All values remained within the historical control range. In females, a similar trend towards a decrease in total T4 and an increase in TSH was observed at 75 mg/kg (0.79x and 1.59x of control for respectively total T4 and TSH), although statistical significance was not achieved and values remained within the historical control range. Historical control data included under "overall remarks".
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In females, grip strength was similar between treated and control animals. In males, foreleg grip strength was similar between treated and control animals. Hindleg grip strength however was statistically significantly decreased in males at 3 and 75 mg/kg (22 and 20% lower compared to concurrent controls, respectively). This finding was considered not to be toxicologically relevant, as no dose-related trend was observed and all values remained within the historical control range (historical control data included in the report). Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 75 mg/kg bw/day group males and females and test item-related lower thymus (absolute and relative to body weight) and spleen (only absolute) weights were noted in 75 mg/kg bw/day males. These results are summarized in a table under "overall remarks". For males treated at 15 and 75 mg/kg bw/day , the remainder of the organ weight changes were in line with the lower terminal body weight. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A test item-related macroscopic finding was noted in the liver of males and females: Enlarged liver was observed in 4/10 males and in 3/8 females treated at 75 mg/kg bw/day. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver, lung, thyroid gland, kidneys (males only), thymus and spleen. A summary of the results is included under “overall remarks”. Hepatocellular vacuolation was observed in males at 15 mg/kg bw/day at minimal degree and in males and females treated at 75 mg/kg bw/day up to moderate degree. The vacuolation was mainly midzonal at 75 mg/kg bw/day and was multifocal/scattered at 15 mg/kg bw/day. Hepatocellular hypertrophy, mainly centrilobular was present in males and females treated at 75 mg/kg bw/day up to a moderate degree. This correlated with the macroscopic enlarged livers and with the increased liver weight.
In lung tissue, alveolar macrophage aggregation was present at increased incidence and severity in males and females treated at 75 mg/kg bw/day up to moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in males and females treated at 75 mg/kg bw/day up to moderate degree. An increased incidence and severity of hyaline droplet accumulation was present in kidneys of males treated at 75 mg/kg bw/day up to moderate degree.
Lymphoid depletion was present in the thymus of males treated at 75 mg/kg bw/day at minimal degree. This correlated with decreased thymus weight. This was not seen in females.
An increased incidence and severity of extramedullary hematopoiesis was present in the spleen of males and females treated at 75 mg/kg bw/day up to slight degree.
An increased incidence and severity of pigmentation (likely hemosiderin) was present in females treated at 15 and 75 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Analysis of dose preparations:

The concentrations analyzed in the formulations of the low, mid and high dose group were in agreement with target concentrations (i.e. mean accuracies between 93% and 96%).

A small response at the retention time of the test item was observed in the chromatograms of the control group formulation prepared for use in Week 1. The maximum contribution to the low dose group samples was 0.12%.

The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤ 2.6%).

Summary results dose range finding study:

At 300 mg/kg bw/day, all animals were euthanized for humane reasons at Day 8 of treatment. Hunched posture and piloerection was observed in 3/3 animals from Day 3 or 4 onwards, respectively. From Day 4 onwards these clinical signs were noted up to 3 hours after dosing. Other clinical signs included chromodacryorrhoea, noted in 1/3 animals from Day 5 onwards. This animal was also noted to be lean from Day 5 onwards. Body weight loss was noted in all 3 animals

of 7-13% between Days 1-5 and of 12-26 % between Days 5-8. Reduced food consumption was noted during days 1-5. At necropsy pale discoloration of the liver was seen in all three rats, one rat was found to have a spleen with reduced size.

At 150 mg/kg bw/day, no mortality occurred. Hunched posture was seen in 3/3 animals on Day 4, one hour after dosing and from Day 6 until Day 8. Piloerection was noticed in 1/3 animals on Day 7 and in 3/3 animals on Day 8. For one animal, piloerection was observed on Day 9. Salivation was noted on a single Day for 2/3 animals. Flat gait was noted on a few days for 3/3 animals. Body weight loss of 1-4% was measured in 3/3 animals between Days 1-5 and 4-8% in 3/3 animals between Days 5-10. Food consumption was slightly reduced during Days 1-5 and reduced during Days 5-10. Macroscopic examination revealed pale discoloration and enlargement of the livers of all animals. Liver weights were increased (approximately 50-68% compared to the mean of the historical control data). Furthermore kidney weights were also increased (approximately 6-14% compared to the mean of the historical control data).

Conclusions:
In an oral OECD 422 screening study with rats, the NOAEL for repeated dose toxicity was determined to be 15 mg/kg bw/day, based on severe effects at 75 mg/kg bw/day (mortality, reduced weight (gain) and adverse liver effects).
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. 4,4’-Methylenebis-N-sec-butylaniline was administered by daily oral gavage to male and female rats at dose levels of 0, 3, 15 and 75 mg/kg bw/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days. Formulation analysis showed that formulations were prepared accurately and homogeneously. Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. In these early deceased females liver effects were found ( livers were found to be pale, thickened, enlarged and/or accentuated lobular pattern, this correlated with microscopic findings consisting of moderate to marked hepatocellular vacuolation and moderate to marked hepatocellular hypertrophy. For the surviving rats, body weight and body weight gain were statistically significantly reduced in rats dosed at 75 mg/kg bw/day for females from Day 15 of the treatment period onwards and for females post-coitum (- 13% and -8% at the end of treatment compared to the control group for males and females, respectively). Functional observation parameters were considered not to be affected by treatment. Heamatology in females was not affected in a toxicologically relevant way, in males haemoglobulin and haematocrit concentrations were statistically significantly decreased at the highest dose. In high dose males, the concentration bilirubin was statistically significantly increased compared to the controls (2.6 µmol/L in controls versus 4.2 µmol/L in high dose males). Cholesterol levels were statistically significantly increased for all rats dosed at 75 mg/kg bw/day. A decrease in total T4 was observed for males and females at 75 mg/kg bw/day, however since statistical significance was not achieved and values remained within the historical control range, this effect was not considered toxicologically relevant. Increased liver weights (absolute and relative to body weights) were noted in the 75 mg/kg bw/day group males and females and test item-related lower thymus (absolute and relative to body weight) and spleen (only absolute) weights were noted in high dose males. Histopathology revealed hepatocellular hypertrophy, mainly centrilobular in males and females treated at 75 mg/kg bw/day up to a moderate degree. In lung tissue, alveolar macrophage aggregation was present at increased incidence and severity in males and females treated at 75 mg/kg bw/day up to moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in males and females treated at 75 mg/kg bw/day up to moderate degree. An increased incidence and severity of hyaline droplet accumulation was present in kidneys of males treated at 75 mg/kg bw/day up to moderate degree. Lymphoid depletion was present in the thymus of males treated at 75 mg/kg bw/day at minimal degree. This correlated with decreased thymus weight. This was not seen in females. An increased incidence and severity of extramedullary hematopoiesis was present in the spleen of males and females treated at 75 mg/kg bw/day up to slight degree. Based on mortality, reduced weight (gain) and adverse liver effects observed at 75 mg/kg bw/day, a No Observed Adverse Effect Level (NOAEL) for 4,4’-Methylenebis-N-sec-butylaniline of 15 mg/kg bw/day was established.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jun 2018 - 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objectives of this study were to determine the potential toxic effects of 4,4’-methylenebis-N-secbutylaniline when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate its potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support re
gulatory submissions. Acceptable models which do not use live animals currently do not exist. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(2016)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (2000)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No correction factor applied.
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-11 weeks (males), 13-14 weeks (females)
- Weight at study initiation: 285-342 gr (males) or 211-251 gr (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany), except during motor activity measurements (maximum 2 hours).
- Water: Free access to tap water, except during motor activity measurements (maximum 2 hours).
Periodic analysis of the water was performed by the test facility.
- Acclimation period: At least 8 days
ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES (main study): From: 23 Aug 2018 to 18 Oct 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Adjustment was made for specific gravity of the vehicle and test item.
Storage conditions of formulations: The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Justification for use and choice of vehicle: Choice of vehicle was based on trial preparations performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All analyses were performed using a validated analytical procedure (Charles River Study No. 20145350). Concentration of the formulations were analysed in week 1 (all groups), homogeneitywas analysed also in week 1 (low and high dose only). Concentration and homogeneity of the formulations were analysed in duplicate. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Charles River Study No. 20145350) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w
Details on study schedule:
- Age at mating of the animals in the study: Approximately 15-16 weeks
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
-The dose levels were selected based on the results of a 10-day dose range finder with oral administration of 4,4’-methylenebis-N-sec-butylaniline in rats, and in an attempt to produce graded responses to the test item. Details on the dose range finder study are included in section 7.5.1.
Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS:
Yes, for general health/mortality and moribundity
- Time schedule: once daily. During the dosing period, these observations were performed directly after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finder). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity. Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.
BODY WEIGHT:
Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION:
Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Males were fasted overnight with a maximum of approximately 24.5 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines
CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Males were fasted overnight with a maximum of approximately 24.5 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5 animals/sex/group (except for thyroid hormone levesl, which were analysed for all rats/group)
- Parameters checked were: According to test guidelines (including coagulation parameters (prothrombin time and activated partial thromboplastin time) and thyroid hormone levels(thyroxine (T4) and thyroid stimulating Hhormone (TSH)))
URINALYSIS: No
FUNCTIONAL TESTS:
Yes
- Time schedule for examinations: Selected males were tested during Week 4 of treatment and the selected females were tested during lactation (PND 6-13).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal), locomotor activity (recording period: 1-hour under normal laboratory light conditions), total movements and ambulations.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of
estrous. This was done for all females, except for females that had to be euthanized in extremis.
Sperm parameters (parental animals):
For the testes of five males of the control and the high dose group and all males that failed to sire, a detailed qualitative examination was made at necrpsy, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily detailed clinical observations were made in all animals. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Body weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Sex: Sex was externally determined for all pups on PND 1 and 4.
- Anogenital distance was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Animals surviving to scheduled necropsy and all moribund animals underwent necropsy, and specified tissues were retained but not weighed.
Full post mortem examination was performed on all surviving rats, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
ORGAN WEIGHTS
- The organs as specified in the guidance were weighed at necropsy (5 animals/sex/group). Sex organs and related tissues were weighed for all males.
HISTOPATHOLOGY: Yes
Performed on 5 rats/group, all tissues and organs as defined in the guidances.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

GROSS NECROPSY
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter per sex at sacrifice.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose groups vs control group, mid dose group vs control group, high dose group vs control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-ttest). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females with living pups on Day 1/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
Offspring viability indices:
- Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100)
- Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation observed after dosing among animals exposed to test item during multiple consecutive days from the second week of the treatment period onwards. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This effect was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. Prior to early sacrifice, these females were lean, had a hunched posture and were noted with piloerection from approximately Day 25 of treatment onwards. For both females, main macroscopic findings were present in the liver: pale discoloration, thickened, enlarged and/or accentuated lobular pattern. Correlating microscopic findings were moderate to marked hepatocellular vacuolation and moderate to marked hepatocellular hypertrophy. No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain were statistically significantly reduced in males dosed at 75 mg/kg bw/day from Day 15 of the treatment period onwards with up to 13% lower by the end of treatment when compared to the control group. Body weight gain was lower in females at 75 mg/kg bw/day during post-coitum (up to 8% lower compared to concurrent controls). Body weight gain during lactation was considered to be within the same range as the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In females in the high dose group, relative food consumption was increased during post-coitum, reaching statistical significance over Days 14-17 and 17-20 post-coitum (10 and 17% compared to concurrent controls). Normal values for food consumption and relative food consumption were noted in female s during the rest of the treatment period. As the increase in food consumption was probably due to the slightly lower body weight at 75 mg/kg bw/day and as all values remained within the range of the historical controls, this finding was considered not toxicologically relevant. In males at 75 mg/kg bw/day, a trend towards a lower absolute and relative food consumption was noted during the first two weeks of treatment (up to 17 and 12% lower than levels in concurrent controls, respectively). Values were on the low end or just outside the historical control range. Historical control data were included in the report.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males a slight increase was seen in white blood cell count in the high dose group (8.7, 10.6, 8.2 and 11.3 *10E9/L for the control group and the groups exposed to 3, 15 and 75 mg/kg bw/day, respectively), which was mainly related to increased monocyte concentration (0.2, 0.2, 0.1 and 0.3*10E9/L for the control group and the groups exposed to 3, 15 and 75 mg/kg bw/day, respectively).
Haemoglobulin and haematocrit concentrations were statistically significantly decreased for the high dose males (haemoglobulin: 10.1 mmol/L for controls versus 9.2 mmol/L for the high dose; haemat
ocrit: 0.487 L/L for controls versus 0.451 L/L for the high dose). Furthermore reduced mean corpuscular volume (MCV) and Reduced mean corpuscular haemoglobin (MCH) were noted in high dose m
ales (53.4 fL for controls versus 50.8 fL for the high dose (MCV) and 1.11 fmol for controls versus 1.04 fmol for the high dose (MCH), both decreases statistically significant).
Due to the fact that the changes were minimal and a dose-response relationship was absent these effects were considered not toxicologically relevant. Furthermore none of these effects were seen
in females. In female rats, the platelet concentrations were statistically significantly decreased in all dose groups (861, 756, 737 and 653 *10E9/L for the controls and the females exposed to 3, 15 and 7
5 mg/kg bw/day, respectively). Although a dose-related response was observed, the platelet concentrations were still within historical control data (historical control data (non-fasted) female Wistar Han rat
s (period 2015 - 2018): Platelets (10E9/L) Mean = 749; P5 – P95 = 552 – 932 (n = 144)) and therefore these values were not considered toxicologically relevant. Coagulation parameters in males and females were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In high dose males, the concentration bilirubin was statistically significantly increased compared to the controls (2.6 μmol/L in controls versus 4.2 μmol/L in high dose males). Urea levels were decreased in all dosed males (8.1, 6.0, 6.4 and 5.7 mmol/L for controls and low, mid and high dose group males, respectively (statistically significant for low and high dose rats)). The urea values in treated males were considered to have arisen as a result of slightly high control values and in the absence of a treatment-related distribution considered to be of no toxicological significance. The effects on bilirubin and urea were not seen in female rats. Cholesterol levels were statistically significantly increased for high dose males (1.79, 1.77, 2.18 and 2.93 mmol/L for controls and low, mid and high dose group males, respectively) and also for high dose females (2.26, 2.25, 2.17 and 2.92 mmol/L for controls and low, mid and high dose group males, respectively). The increase in cholesterol levels was statistically significant for high dose rats in both sexes. A dose-dependent decrease in bile acids was observed (not statistically significant; 39.5, 37.6, 24.2 and 23.4 μmol/L for controls and low, mid and high dose group males, respectively). Other statistically significant changes in clinical biochemistry parameters (inorganic phosphatase in males) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. A decrease in total T4 was observed for males at 15 and 75 mg/kg bw/day (0.72 and 0.57x of control, respectively). A corresponding increase in TSH was observed at 75 mg/kg bw/day (1.99x of control), although values did not reach statistical significance. All values remained within the historical control range. In females, a similar trend towards a decrease in total T4 and an increase in TSH was observed at 75 mg/kg (0.79x and 1.59x of control for respectively total T4 and TSH), although statistical significance was not achieved and values remained within the historical control range. Historical control data included under "overall remarks" in section 7.5.1.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In females, grip strength was similar between treated and control animals. In males, foreleg grip strength was similar between treated and control animals. Hindleg grip strength however was statistically significantly decreased in males at 3 and 75 mg/kg (22 and 20% lower compared to concurrent controls, respectively). This finding was considered not to be toxicologically relevant, as no dose-related trend was observed and all values remained within the historical control range (historical control data included in the report) . Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
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):
Test item-related microscopic findings were noted in the liver, lung, thyroid gland, kidneys (males only), thymus and spleen. A summary of the results is included under “overall remarks” in the study summary in section 7.5.1. Hepatocellular vacuolation was observed in males at 15 mg/kg bw/day at minimal degree and in males and females treated at 75 mg/kg bw/day up to moderate degree. The vacuolation was mainly midzonal at 75 mg/kg bw/day and was multifocal/scattered at 15 mg/kg bw/day. Hepatocellular hypertrophy, mainly centrilobular was present in males and females treated at 75 mg/kg bw/day up to a moderate degree. This correlated with the macroscopic enlarged livers and with the increased liver weight. In lung tissue, alveolar macrophage aggregation was present at increased incidence and severity in males and females treated at 75 mg/kg bw/day up to moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in males and females treated at 75 mg/kg bw/day up to moderate degree. An increased incidence and severity of hyaline droplet accumulation was present in kidneys of males treated at 75 mg/kg bw/day up to moderate degree. Lymphoid depletion was present in the thymus of males treated at 75 mg/kg bw/day at minimal degree. This correlated with decreased thymus weight. This was not seen in females. An increased incidence and severity of extramedullary hematopoiesis was present in the spleen of males and females treated at 75 mg/kg bw/day up to slight degree. An increased incidence and severity of pigmentation (likely hemosiderin) was present in females treated at 15 and 75 mg/kg bw/day up to slight degree. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. An acylic cycle was noted for one of the females dosed at 3 mg/kg bw/day (with normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic profiling did not reveal substance-related effects.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The mating indices were considered not to be affected by treatment. The mating indices were 100, 100, 90 and 100% for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. There were 2/10 females treated at 75 mg/kg bw/day that were euthanized in extremis on Day 10 and 18 post-coitum. In addition, there were 4/10 couples treated at 15 mg/kg bw/day and 1/10 at 3 mg/kg bw/day, that failed to deliver healthy pups. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. Precoital time (i.e. days before mating) was considered not to be affected by treatment. All mated females showed evidence of mating within four days, except for two females at 75 mg/kg (5 and 7 days, respectively). This slightly delayed mating was within the normal range of biological variation. At the single incidence it was considered to be unrelated to treatment with the test item. Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring. The gestation index was 100% for all groups. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

The fertility indices were 100, 90, 67 and 90 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. The fertility index of 67% at 15 mg/kg was outside the historical control range. A total of 1 female at 3 mg/kg bw/day, 3 females at 15 mg/kg bw/day and 1 female at 75 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, this was not considered to be related to treatment.

Live litter sizes were 11.6, 11.4, 10.3, and 7.5 living pups/litter for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. A statistically significantly lower mean number of living pups was recorded at 75 mg/kg bw/day. As live litter sizes for controls were comparable to the mean of the historical control data and the live litter size at 75 mg/kg bw/day was 35% lower compared to control values, this finding was considered treatment-related.
A summary of reproduction data is included as attached background material.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. For the pup of a female dosed at 3 mg/kg bw/day who was found dead on Day 2, absence of milk in the stomach was noted at first litter check. For the pup of one female dosed at 75 mg/kg bw/day who was sacrificed in extremis on PND 11, a lean appearance was noted on PND 9 and 10. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 98, 91 and 91 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively.
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100, 100, 100 and 98 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. One pups at 75 mg/kg bw/day was found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 99, 100 and 100% for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. One pup at 3 mg/kg bw/day was found dead on PND 2. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 99, 100, 100 and 95% for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. One pup of the control group and one pup at 75 mg/kg bw/day were found missing on PND 7 or 9. Pups missing were most likely cannibalised. One pup at 75 mg/kg bw/day was sacrificed in extremis on PND 11. This female pup was extremely small: body weight of this pup on PND1 and 4 was respectively 25 and 37% lower than the mean body weight of the others female pups in her litter. Moreover, a lean appearance was noted for this pup on PND 9 and 10 and. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of male pups and combined pup weights were statistically significantly increased at 75 mg/kg bw/day on PND 1 (with 12 and 11%, respectively). This finding was most likely due to the lower litter size at 75 mg/kg bw/day. From PND 7 onwards, body weights of pups were decreased at 75 mg/kg bw/day, reaching statistical significance for male pups on PND 13 (9% decreased compared to concurrent controls). Total body weight gain for male and female pups combined from PND 1 to 13 was 24.7 g in controls compared to 21.8 g at 75 mg/kg bw/day (12% lower at 75 mg/kg bw/day compared to concurrent controls). Considering the magnitude of the effect, this finding was regarded treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. The statistically significantly lower mean anogenital distance of female pups at 15 mg/kg bw/day occurred without a dose related-trend. These variations were not considered to be related to treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 75 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
A summary of reproduction data is included as attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Generation:
other: P
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Significant reduction of the number of implantation sites at 75 mg/kg bw/day
Remarks on result:
other: Effects were seen at a dose level at which severe toxicity was seen in the parental rats.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Details on the results of the dose range finding study and on the analysis of dose preparations are included in section 7.5.1.

Conclusions:
In an oral OECD 422 screening study with rats, the parental NOAEL for repeated dose toxicity was determined to be 15 mg/kg bw/day, based on severe effects at 75 mg/kg bw/day (mortality, reduced weight (gain) and adverse liver effects). At this dose the number of implantation sites was significantly reduced, therefore the reproduction NOAEL was considered to be 15 mg/kg bw/day.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. 4,4’-Methylenebis-N-sec-butylaniline was administered by daily oral gavage to male and female rats at dose levels of 0, 3, 15 and 75 mg/kg bw/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days. Formulation analysis showed that formulations were prepared accurately and homogeneously.

Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. For the surviving rats, body weight and body weight gain were statistically significantly reduced in rats dosed at 75 mg/kg bw/day. Increased liver weights (absolute and relative to body weights) were noted in the 75 mg/kg bw/day group males and females and test item-related lower thymus (absolute and relative to body weight) and spleen (only absolute) weights were noted in high dose males. Histopathology revealed hepatocellular hypertrophy, mainly centrilobular in males and females treated at 75 mg/kg bw/day up to a moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in males and females treated at 75 mg/kg bw/day up to moderate degree.

Reproduction toxicity was observed at 75 mg/kg bw/day. At this dose, the number of implantation sites was significantly reduced. No treatment-related toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Based on mortality, reduced weight (gain) and adverse liver effects observed at 75 mg/kg bw/day, a No Observed Adverse Effect Level (NOAEL) for 4,4’-Methylenebis-N-sec-butylaniline of ≥ 15 mg/kg bw/day was established. At 75 mg/kg bw/day the number of implantation sites was significantly reduced, therefore the reproduction NOAEL was considered to be 15 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
(2016)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
(July 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
Principles of method if other than guideline
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (2000)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenebis[N-sec-butylaniline]
EC Number:
226-122-6
EC Name:
4,4'-methylenebis[N-sec-butylaniline]
Cas Number:
5285-60-9
Molecular formula:
C21H30N2
IUPAC Name:
4,4'-methylenebis[N-sec-butylaniline]
Test material form:
liquid
Details on test material:
Appearance: Slight yellow liquid
Storage conditions: At room temperature protected from light

Specific details on test material used for the study:
No correction factor applied.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 13-14 weeks
- Weight at study initiation: 211-251 g
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Females were individually housed in Macrolon cages.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity measurements (maximum 2 hours).
- Water: Free access to tap water, except during motor activity measurements (maximum 2 hours).
Periodic analysis of the water was performed by the test facility.
- Acclimation period: At least 8 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES (main study): From: 23 Aug 2018 to 18 Oct 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Method of formulation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Adjustment was made for specific gravity of the vehicle and test item.
Storage conditions of formulations: The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Justification for use and choice of vehicle: Choice of vehicle was based on trial preparations performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All analyses were performed using a validated analytical procedure (Charles River Study No. 20145350). Concentration of the formulations were analysed in week 1 (all groups), homogeneitywas analysed also in week 1 (low and high dose only). Concentration and homogeneity of the formulations were analysed in duplicate. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Charles River Study No. 20145350) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Duration of treatment / exposure:
Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w
Duration of test:
40 - 56 days
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 10-day dose range finder with oral administration of 4,4’-methylenebis-N-sec-butylaniline in rats, and in an attempt to produce graded responses to the test item. Details on the dose range finder study are included in section 7.5.1.
Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible
from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes,
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS:
Yes, for general health/mortality and moribundity
- Time schedule: once daily. During the dosing period, these observations were performed directly after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finder). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity. Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.
BODY WEIGHT:
Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17,
and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION:
Yes
Food consumption was quantitatively measured weekly, except for females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: No
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines
CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: No
- How many animals: 5 animals/sex/group (except for thyroid hormone levesl, which were analysed for all rats/group)
- Parameters checked were: According to test guidelines (including coagulation parameters (prothrombin time and activated partial thromboplastin time) and thyroid hormone levels(thyroxine (T4) and thyroid stimulating Hhormone (TSH)))
URINALYSIS: No
FUNCTIONAL TESTS:
Yes
- Time schedule for examinations: Selected females were tested during lactation (PND 6-13).
- Dose groups that were examined: all (5 animals/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb grip strength (recorded as the mean of three measurements per animal), locomotor activity (recording period: 1-hour under normal laboratory light conditions), total movements and ambulations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily detailed clinical observations were made in all animals. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
- Body weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Sex: Sex was externally determined for all pups on PND 1 and 4.
- Anogenital distance was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- All male pups in each litter were examined for the number of areola/nipples on PND 13.
- On PND 4 and PND 14-16 T4 and TSH levels were determined in pooled blood from 2 pups/litter
GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose groups vs control group, mid dose group vs control group, high dose group vs control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-onettest). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
For each group, the following calculations were performed:
- Mating index (%): (Number of females mated/Number of females paired) x 100
- Precoital time (%): Number of days between initiation of cohabitation and confirmation of mating
- Fertility index (%): (Number of pregnant females/Number of females paired) x 100
- Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
- Duration of gestation (%): Number of days between confirmation of mating and the beginning of parturition
- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
- Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100
- Viability index (%): (Number of live pups on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100)
- Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Historical control data:
Included in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation observed after dosing among animals exposed to test item during multiple consecutive days from the second week of the treatment period onwards. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This effect was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. Prior to early sacrifice, these females were lean, had a hunched posture and were noted with piloerection from approximately Day 25 of treatment onwards. For both females, main macroscopic findings were present in the liver: pale discoloration, thickened, enlarged and/or accentuated lobular pattern. Correlating microscopic findings were moderate to marked hepatocellular vacuolation and moderate to marked hepatocellular hypertrophy. No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was lower in females at 75 mg/kg bw/day during post-coitum (up to 8% lower compared to concurrent controls). Body weight gain during lactation was considered to be within the same range as the controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In females in the high dose group, relative food consumption was increased during post-coitum, reach ing statistical significance over Days 14-17 and 17-20 post-coitum (10 and 17% compared to concurre nt controls). Normal values for food consumption and relative food consumption were noted in females during the rest of the treatment period. As the increase in food consumption was probably due to the slightly lower body weight at 75 mg/kg bw/day and as all values remained within the range of the historical controls, this finding was considered not toxicologically relevant. In males at 75 mg/kg bw/day, a trend towards a lower absolute and relative food consumption was noted during the first two weeks of treatment (up to 17 and 12% lower than levels in concurrent controls, respectively). Values were on the low end or just outside the historical control range. Historical control data were included in the report.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Platelet concentrations were statistically significantly decreased in females in all dose groups (861, 756, 737 and 653 *10E9/L for the controls and the females exposed to 3, 15 and 75 mg/kg bw/day, respectively). Although a dose-related response was observed, the platelet concentrations were still within historical control data (historical control data (non-fasted) female Wistar Han rats (period 2015 - 2018): Platelets (10E9/L) Mean = 749; P5 – P95 = 552 – 932 (n = 144)) and therefore these values were not considered toxicologically relevant. Coagulation parameters were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol levels were statistically significantly increased for high dose females (2.26, 2.25, 2.17 and 2.92 mmol/L for controls and low, mid and high dose group males, respectively). The increase in cholesterol levels was statistically significant for high dose rats in both sexes. A dose-dependent decrease in bile acids was observed (not statistically significant; 39.5, 37.6, 24.2 and 23.4 μmol/L for controls and low, mid and high dose group males, respectively). A decrease in total T4 and an increase in TSH was observed at 75 mg/kg bw/day (0.79x and 1.59x of control for respectively total T4 and TSH), although statistical significance was not achieved and values remained within the historical control range. Historical control data ar included in section 7.5.1.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In females, grip strength was similar between treated and control animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 75 mg/ kg bw/day group females. These results are summarized in a table under "overall remarks" in section 7.5.1. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged liver was observed in 3/8 females treated at 75 mg/kg bw/day. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver, lung, thyroid gland, thymus and spleen. A summary of the results is included under “overall remarks” in section 7.5.1. Hepatocellular vacuolation was observed in females treated at 75 mg/kg bw/day up to moderate degree. The vacuolation was mainly midzonal. Hepatocellular hypertrophy, mainly centrilobular was present in females treated at 75 mg/kg bw/day up to a moderate degree. This correlated with the macroscopic enlarged livers and with the increased liver weight. In lung tissue, alveolar macrophage aggregation was present at increased incidence and severity in females treated at 75 mg/kg bw/day up to moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in females treated at 75 mg/kg bw/day up to moderate degree. An increased incidence and severity of extramedullary hematopoiesis was present in the spleen of females treated at 75 mg/kg bw/day up to slight degree. An increased incidence and severity of pigmentation (likely hemosiderin) was present in females treated at 15 and 75 mg/kg bw/day up to slight degree. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The number of implantation sites was significantly reduced at 75 mg/kg bw/day (8.6 vs 12.4 in concurrent controls). As values for the control group are comparable to the mean of the historical control data and the number of implantation sites at 75 mg/kg bw/day are 31% lower compared to control values, this finding was considered treatment-related.
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 98, 91 and 91 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively.For three females, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the (14-16) days of) lactation. No toxicological relevance was attached to this finding in the current study.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100, 100, 100 and 98 % for the control, 3, 15 and 75 mg/kg groups, respectively. One pups at 75 mg/kg was found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a doserelated trend and remained within the range considered normal for pups of this age.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring. The gestation index was 100% for all groups.
Other effects:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Remarks:
Development
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 98, 91 and 91 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100, 100, 100 and 98 % for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. One pups at 75 mg/kg bw/day was found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Live litter sizes were 11.6, 11.4, 10.3, and 7.5 living pups/litter for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. A statistically significantly lower mean number of living pups was recorded at 75 mg/kg bw/day. As live litter sizes for controls were comparable to the mean of the historical control data and the live litter size at 75 mg/kg bw/day was 35% lower compared to control values, this finding was considered treatment-related.
Body weights of male pups and combined pup weights were statistically significantly increased at 75 mg/kg bw/day on PND 1 (with 12 and 11%, respectively). This finding was most likely due to the lower litter size at 75 mg/kg bw/day. From PND 7 onwards, body weights of pups were decreased at 75 mg/kg bw/day, reaching statistical significance for male pups on PND 13 (9% decreased compared to concurrent controls). Total body weight gain for male and female pups combined from PND 1 to 13 was 24.7 g in controls compared to 21.8 g at 75 mg/kg bw/day (12% lower at 75 mg/kg bw/day compared to concurrent controls). Considering the magnitude of the effect, this finding was regarded treatment-related.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 99, 100 and 100% for the control, 3, 15 and 75 mg/kg groups, respectively. One pup at 3 mg/kg was found dead on PND 2. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 99, 100, 100 and 95% for the control, 3, 15 and 75 mg/kg bw/day groups, respectively. One pup of the control group and one pup at 75 mg/kg bw/day were found missing on PND 7 or 9. Pups missing were most likely cannibalised. One pup at 75 mg/kg bw/day was sacrificed in extremis on PND 11. This female pup was extremely small: body weight of this pup on PND1 and 4 was respectively 25 and 37% lower than the mean body weight of the others female pups in her litter. Moreover, a lean appearance was noted for this pup on PND 9 and 10. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of a female dosed at 3 mg/kg bw/day which was found dead on Day 2, absence of milk in the stomach was noted at first litter check. For the pup of a female dosed at 75 mg/kg bw/day which was sacrificed in extremis on PND 11, a lean appearance was noted on PND 9 and 10. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
A summary of developmental data is included as attached background material.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
Development (pups)
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Effects were seen at a dose level at which severe toxicity was seen in the parental rats.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

As one female was not mated and three females were not pregnant, developmental data is available of only 6 females at 15 mg/kg bw/day. Although the guidelines requires at least 7 pregnant females for evaluation, the available data from the mid dose group was considered to be sufficient for the determination of the NOAEL as the litters available did not show any indication of developmental toxicity at 15 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study with rats, the parental NOAEL for repeated dose toxicity was determined to be 15 mg/kg bw/day, based on severe effects at 75 mg/kg bw/day (mortality, reduced weight (gain) and adverse liver effects). Based on reduced total body weight gain for male and female pups combined from PND 1 to 13 at 75 mg/kg bw/day compared to concurrent controls, the developmental NOAEL was considered to be 15 mg/kg bw/day.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. 4,4’-Methylenebis-N-sec-butylaniline was administered by daily oral gavage to male and female rats at dose levels of 0, 3, 15 and 75 mg/kg bw/day. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 40-54 days. Formulation analysis showed that formulations were prepared accurately and homogeneously.

Two females at 75 mg/kg bw/day were sacrificed in extremis on Day 30 and 34 of treatment (Day 10 and 18 of post-coitum), respectively. For the surviving rats, body weight and body weight gain were statistically significantly reduced in rats dosed at 75 mg/kg bw/day. Increased liver weights (absolute and relative to body weights) were noted in the 75 mg/kg bw/day group females. Histopathology revealed hepatocellular hypertrophy, mainly centrilobular in females treated at 75 mg/kg bw/day up to a moderate degree. Follicular cell hypertrophy in thyroid glands was present at increased incidence and/or severity in females treated at 75 mg/kg bw/day up to moderate degree.

Total body weight gain for male and female pups combined from PND 1 to 13 was 12% lower at 75 mg/kg bw/day compared to concurrent controls. Considering the magnitude of the effect, this finding was regarded as treatment-related and adverse. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Based on mortality, reduced weight (gain) and adverse liver effects observed at 75 mg/kg bw/day, a No Observed Adverse Effect Level (NOAEL) for 4,4’-Methylenebis-N-sec-butylaniline of ≥ 15 mg/kg bw/day was established. Based on reduced total body weight gain for male and female pups combined from PND 1 to 13 at 75 mg/kg bw/day compared to concurrent controls, the developmental NOAEL was considered to be 15 mg/kg bw/day.