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Description of key information

In an oral OECD422 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).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A reliable study is available (Klimisch 1 study).
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.

Justification for classification or non-classification

Based on the fact that severe effects (mortality in females, reduced body weight (gain) and hepatocellular hypertrophy in males and females) were seen at 75 mg/kg bw/day in a sub-acute repeated dose study, it is concluded that 4,4'-methylenebis-N-sec-butylaniline has to be classified STOT RE 2 according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.

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