Registration Dossier

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Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on oral exposure:
The administration volume was 5 ml/kg body weight per day.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability of the test substance in the formulations was assumed as 4 hours (The determination of the stability/homogeneity of the test item M 530 in different solvents was not possible with the analytical methods currently available at the analytical laboratory). The time of preparation as well as the time of dosing (at least time of dosing of first animal of the respective dose group and time of last dosing of respective dose group) was documented for each preparation (hour and minute). The relevant time point for preparation was the time point of admix of polyethylene glycol 400 to the test substance.
Twice (at study start and at the end of the study), the preparation of the test formulation was checked by a second person (4-eye-principle) and the adequacy of the preparation was documented. The preparation of the formulation was described in the raw data.
The test substance was administered as a solution in the vehicle. The formulations were stored at room temperature for maximum 4 hours.
Duration of treatment / exposure:
29 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100, 500, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

In a two-week pilot study five animals per sex and dose received 0, 100, 300 and 1000 mg/kg test substance. In-life data, necropsy, organ weight measurements and histopathology (stomach only) did not show any treatment-related changes up to 1000 mg/kg.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes (incl. Open Field Observations (OFO))
- Time schedule: once before start and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB: once, day 22-23 ); MA: once, day 22-23
- Dose groups that were examined: all dose groups incl. controls
- Battery of functions tested: Functional Observational Battery (FOB); Motor Activity (MA)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)

ORGAN Weights:
Brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides (both), testes (both), prostate, seminal vesicles with coagulation glands, ovaries/oviducts (both) and uterus/cervix.

Fixed organs:
Adrenals, aorta, brain (cerebrum, cerebellum, ponslmedulla), epididymides, esophagus, eyes, eyelids, extraorbital lachrymal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular, bronchial/hilus, and mesenteric), lung, mamma, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroids (including parathyroid glands), tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, Zymbal’s glands and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (high dose group and controls)
- Microscopic: Adrenals, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, eyes, femur, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, lung, ovaries, oviducts, prostate, sciatic nerve, skeletal muscle (thigh), spinal cord (cervical, thoracic, lumbar), sternum with bone marrow, testes, seminal vesicles (incl. coagulating glands), stomach, trachea and thyroids glands, urinary bladder, uterus with uterine cervix, vagina and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (all dose groups and controls)
-Microscopic: thyroids
Statistics:
Statistical tests on FOB, body weights and weight gain as well as on absolute organ weights were done using the Dunnett Exact Homogeneous Test. For relative organ weights the Dunnett Exact Homogeneous Test after log. Transformation was used.
If primary food and water intake data were recorded, the calculated food/water intake per animal was evaluated using adjusted Mann-Whitney U-tests.
The Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log. Transformation or the Bonferroni/Mann-Whitney U-test was used for clinical pathology parameters. Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. This included measures of general tendency (mean and median (median not given for food and water intake)) and general variability (standard deviation, minimum and maximum) as appropriate.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
ORGAN WEIGHTS:

At the high dose of 1000 mg/kg b.w., relative liver weights were increased in females, the same trend was observed in absolute liver organ weights of high dosed females.

HAEMATOLOGY:

At the high dose of 1000 mg/kg b.w., ERY, HB and HCT were slightly, but significantly reduced in females and the same trend, but not statistically significant, could be seen in high-dosed males.

HISTOPATHOLOGY:

In the thyroid gland, a minimal numerical increase of follicular cell hypertrophy was noticed in males at 1000 mg/kg b.w. (incidence: 0/1/0/3). In two males also the severity score was slightly higher (grade 2).
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Haematology: decrease of erythrocytes, hemoblobin and hematocrit
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Microscopic changes in thyroid gland
Critical effects observed:
not specified
Executive summary:

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), the substance was adimistered via gavage to 5 rats/sex/dose at 0, 100, 500, 1000 mg/kg bw in PEG 400 for 4 weeks. Up to and including 1000 mg/kg bw no mortality occured. The behavior and clinical appearance of the rats were not influenced by the treatment up to and including 1000 mg/kg in both sexes. The body weight gain, the food, water intake, clinical chemistry and organ weights were not affected up to and including 1000 mg/kg in males and females. The results from Functional Observational Battery (FOB) measurements in males and females receiving up to and including 1000 mg/kg bw did not differ from the control animals. Motor and Locomotor Activity(MA/LMA) tests did not indicate neurotoxicity up to and including 1000 mg/kg.

Adverse findings were observed at 1000 mg/kg b.w. and consisted of a decrease of erythrocytes, hemoblobin and hematocrit in males and females and histopathological findings in the thyroid gland of male rats.

Therefore, under the condition of the present study, the NOAEL (no-observed-adverse-effect-level) for M 530 after 4-week daily oral treatment by gavage is 500 mg/kg b.w. for male and female rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Feb 2021 to 22 Jul 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Ophthalmoscopy was performed in Week 12 instead of Week 13 of the study. Since ophthalmoscopy was performed in the second to last week of dosing it can be considered as End of Treatment. Therefore, this deviation had no impact on study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
Target Age at Initiation of Dosing: At least 6 weeks
Target Weight at Initiation of Dosing: 100 to 300 g (males) and 100 to 200 g (females)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals:
Age at initiation of dosing: 6-7 weeks old
Body weight range at initiation of dosing: 146-189 g (males) and 109-149 g (females)
Acclimatization period: 15 days

Food: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
Water: Municipal tap water, ad libitum
Caging: Up to 5 animals of the same sex and same dosing group together in Polycarbonate cages (Makrolon type IV, height 18 cm and Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.

Environmental conditions:
The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 42 to 57%. 12 h light, 12 h dark cycle with 10 or mor air changes/h
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400 (Merck, Darmstadt, Germany) Batch: PEG4009 PEG40010 Specific gravity: 1.125
Details on oral exposure:
The first day of dosing will be designated as Day 1 (exception: Alternate animals used for replacement after Day 1 will assume the day of the animal being replaced). The dose formulations will be stirred continuously during dosing. The doses will be given using a plastic feeding tube. Dose pot identification via Provantis will be used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation was prepared on a daily base. The concentrations analyzed in the dose formulations prepared for use in Weeks 1, 6 and 12, were found to be within the acceptance criteria (measured concentrations at ±10% of the theoretical concentrations), except in Week 6, Group 2, which showed an accuracy of +34.8%
during the first analysis and +36.3% during the second analysis. No reason for this deviation was found during an out of specification test and based on the results we can state that the Group 2 animals were dosed with at least 20 mg/mL (~27.0-27.3 mg/mL) in Week 6. No test item was found in the Group 1 formulation. Dose formulation homogeneity was also demonstrated as the Relative Standard Deviation (RSD) was found to be <= 10% for each tested group.
Duration of treatment / exposure:
7 days a week for a minimum of 13 weeks.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle only
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes
Positive control:
no
Observations and examinations performed and frequency:
Mortality: At least twice daily. Beginning upon arrival through termination. Except on days of receipt and necropsy where frequency will be at least once daily. Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.
Cageside Observations: At least once daily; from Day 1at 0 to 1 hours postdose. Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. For observations that cannot be attributed to an individual animal due to social housing (e.g., watery feces), the observation will be
recorded to each animal in the socialized group.
Detailed Clinical Observations: Pretreatment and weekly; from Week 1 and throughout the study, and on the day of necropsy. Animals are removed from the cage.
Arena Observations: Pretreatment and weekly during the Treatment. Animals will be observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs will be recorded.
Individual Body Weights: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, animals may be weighed more often. Fasted weight on the day of necropsy.
Food Consumption: Weekly; from at least Day 1 and throughout the study Quantitatively measured per cage.
Water Consumption:Regular basis throughout the study. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.
Sacrifice and pathology:
Animals surviving until scheduled euthanasia will have a terminal body weight recorded and will be deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals will be fasted (overnight with a maximum of 24 hours) before their scheduled necropsy. Animals will be subjected to a complete necropsy examination, which will include evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The organs detailed in section 12 and ATTACHMENT B tables will be weighed at necropsy. Paired organs will be weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ weight as a percent of body weight (using the terminal body weight) will be calculated. Representative samples of tissues will be collected and preserved in 10% neutral buffered formalin or modified Davidson's solution as detailed in Test Facility SOPs. Additional tissue samples may be collected to elucidate abnormal findings. Tissues will be embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Tissues will be evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation will be communicated to the Study Director; tissues will be evaluated and reported. Special stains may be used at the discretion of the pathologist to further characterize lesions and changes identified during routine evaluation of individual animals. Any special stains will be documented in the individual animal data. Any additional stains or evaluations, if deemed necessary by the pathologist, may be added by Study Plan amendment following
discussion with the Study Director and in consultation the Sponsor. Efforts will be made to evaluate all Study Plan-required tissues microscopically; however, it is not always feasible for every Study Plan-required tissue to be present on every slide. Study Plan-required tissues that are not examined will be documented in the histopathology data and the impact of these missing tissues on the study will be documented in the Pathology Report.
Ophthalmic Examinations:
Frequency: Pretreatment Period - All animals once (including spare animals) Dosing Period - All Group 1 and 4 animals during Week 13. If treatment-related findings are noted, the other animals will also be examined.
Procedure: The eyes will be examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).
Functional Tests:
Frequency: Once during the Dosing Period. The first 5 animals per sex per group during Week 12-13. These tests will be performed after clinical observations (including arena observation, if applicable).
Procedure: The following tests will be performed:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
Estrous Stage Determination:
Frequency: End of Treatment - on the day of necropsy, a vaginal smear will be taken to determine the stage of estrus from all animals. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously. Procedure: Estrous stage will be evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.
Hematology: A blood smear will be prepared from each hematology sample. Blood smears are labeled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results. The smears may be subsequently evaluated and this evaluation will be described in a Study Plan amendment.
Clinical Chemistry: After receipt of the serum for T3, T4 and TSH analysis it will be divided in two aliquots. One aliquot will be used for measurement of thyroid hormones TSH using the IMMULITE® 1000 analyser. The aliquot for TSH will be stored in an ultra-low freezer (≤ -75°C) until analysis. Any remaining sample after TSH analysis will be discarded. The other aliquot will be used for measurement of T3 and T4 using LC-MS. The aliquot for T3 and T4 will be collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and stored in a freezer (≤ -15°C) until analysis. Measurement of T3 and T4 will be performed according to the bioanalytical method validated in Test Facility Study No. 20213516. Any samples remaining after the LC-MS analysis will be returned to storage for the retention period.
Statistics:
All statistical analyses will be performed within the respective study phase, unless otherwise noted. Statistics for data collected/processed in ToxData. All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% or 5% levels. Numerical data collected on scheduled occasions will be analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of pretreatment or control values when deemed appropriate. Inferential statistics will be performed according to the matrix below when possible, but will exclude semi-quantitative data, and any group with less than 2 observations.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental hunched posture and erected fur were noted in individual males and/or females treated at 300 and 1000 mg/kg/day. At the low incidence observed, these findings were considered not toxicologically relevant. Incidental abnormal breathing sounds were observed in individual males and/or females of all groups. These clinical signs were considered to be related to the method of dosing and not as test item-related. Salivation and/or incidental ploughing were observed after dosing in all groups (including the control group) and were considered not toxicologically relevant, taking into account the nature of the effect and its time of occurrence (i.e. after dosing). These signs were considered to be a physiological response rather than a sign of systemic toxicity. Any other clinical signs (bent tail, scabs and thin fur cover) noted during the Dosing 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 or occurred in the control group. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No effect on body weight and body weight gain was observed in males up to 300 mg/kg/day and in females up to 1000 mg/kg/day. In males treated at 1000 mg/kg/day, body weight was decreased from Day 36 of treatment onwards (down to 6-9% lower than control; not always statistically significant) and an overall lower body weight gain between Days 1-91 was noted. In females treated at 300 mg/kg/day, a lower body weight compared to the control was observed on Day 1 of treatment, which was not test item-related and recovered over time (with higher body weight gain compared to the control between Days 1-8). As findings were slight (-9%) and in absence of
corroborating findings, these findings were considered not adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No effect on food consumption was observed in males up to 300 mg/kg/day and in females up to 1000 mg/kg/day. In males treated at 1000 mg/kg/day, lower food consumption compared to the control was observed from Day 36 onwards (down to 5-9% lower than control; not statistically significant), except between Days 64-71 (8% higher than control). Overall food consumption between Days 1 and 91 was slightly lower compared to the control (2% lower than control).
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were considered unaffected by treatment with the test item in males and females up to 300 mg/kg/day. At 1000 mg/kg/day, decreased hemoglobulin concentration (0.95x of control; not statistically significant) and hematocrit (0.95x of control) were observed in females. Remaining differences in hematology parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions. Coagulation parameters were considered unaffected by treatment with the test item in males and females up to 1000 mg/kg/day. Females treated at 1000 mg/kg/day showed a shorter activated partial thromboplastin time (APTT). Since the control mean was considered to be slightly high compared to historical control data1, this finding was considered not test item related. In absence of a histopathological correlation these findings were considered to be not adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased alkaline phosphatase (ALP) activity was observed in males at 1000 mg/kg/day and in females at 300 and 1000 mg/kg/day. In addition, increased cholesterol, HDL and/or LDL cholesterol concentrations were observed in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day. Remaining differences in clinical chemistry parameters (including T3 and T4), regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions. Increased triglyceride concentrations were noted in all test item-treated males and increased calcium concentrations were observed in males at 1000 mg/kg/day. In absence of a histopathological correlation these findings were considered to be not adverse.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
An increased thyroid-stimulating hormone (TSH) concentration was observed in males at 1000 mg/kg/day and in females at 100 and 1000 mg/kg/day. A general overlap of individual values with the range of control values was observed and in absence of an effect on organ weight and a histopathological correlation these findings were considered to be not adverse.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. No effect on grip strength was observed in females. In males at 100 and
1000 mg/kg/day, lower foreleg grip strength was observed compared to the control, whereas males at 300 mg/kg/day showed lower hindleg grip strength. In absence of a dose-related response, these findings were considered not test item-related 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:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights. A few organ weight differences were statistically significant in males at 1000 mg/kg/day (heart, absolute weight; liver relative to body weight) but was interpreted to be related to the lower terminal body weights in that group (-9%).
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. The few recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No microscopic alterations were noted in males and females up to 300 mg/kg/day. A test item-related histiocytic infiltrates (increased incidence and severity) was noted in the mesenteric lymph node of males and females at 1000 mg/kg/day. In males and females at 1000 mg/kg/day, histiocytic infiltrates were present in the mesenteric lymph node at a higher incidence and severity than control animals (up to mild degree). This was characterized by the presence of small aggregates of histiocytes within the cortical/paracortical region of the lymph node. There were no inflammatory, degenerative, or necrotic changes associated with the presence of these small aggregates of histocytes therefore this observation was interpreted to be a non-adverse finding. Histiocytic infiltrates were noted in the mesenteric lymph nodes of some animals at 100 and 300 mg/kg/day but were of the same severity grade (minimal) as the control groups and at generally comparable incidences, therefore these findings were interpreted as being not test item-related. There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings and/or were within the range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Limit dose with no adverse effects.
Key result
Critical effects observed:
no
Conclusions:
In conclusion, administration of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2- yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated by once daily oral gavage for at least 90 days was well tolerated in Wistar Han rats at dose levels up to 1000 mg/kg/day in both males and females. Non-adverse changes to body weight (gain), food consumption, and hematology and clinical chemistry parameters were observed in males and females up to 1000 mg/kg/day. Also, non-adverse, test item-related increased incidence and severity (up to mild) of microscopic histiocytic infiltrates was noted in the mesenteric lymph node of males and females at 1000 mg/kg/day.
Based on these results, the No Observed Adverse Effect Level (NOAEL) was established to be at least 1000 mg/kg/day in males and females.
Executive summary:

The objective(s) of this study was to determine the potential toxicity of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated, when given orally by gavage for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated. The study design was as follows:

 Group No.  Test Item Id.  dose level (mg/kg/day)  dose volume (ml/kg)  dose concentration (mg/ml)  no. male  no. females
 1  Control  0 (vehicle)  5  0  10  10
 2

Reaction product of 4-

[2-(4 -hydroxyphenyl) -propan-2-yl]phenol, 2-(2 -hydroxyethylamino)

ethanol and form

aldehyde), propoxylated

 100  5 20   10  10
 3  300  5  60  10  10
 4 1000   5  200 10   10

Chemical analyses of formulations were conducted in Week 1, 6 and 12 to assess accuracy and homogeneity. Accuracy and homogeneity of formulations were within acceptance criteria, except for Group 2 formulations for use in Week 6 which showed an accuracy of approximately +35-36%. No reason for this deviation was found and based on the results it can be stated that the Group 2 animals were dosed with at least 100 mg/kg/day (~135 - 136 mg/kg/day) in Week 6. The following parameters and end points were evaluated in this study: clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrous stage determination, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations. At 100 mg/kg/day, increased triglyceride concentrations were observed in males. In absence of a histopathological correlation, this finding was considered to be not adverse. At 300 mg/kg/day, increased alkaline phosphatase activity in females and increased cholesterol, HDL cholesterol and triglyceride concentrations in males was observed. In absence of a histopathological correlation, these findings were considered not adverse. At 1000 mg/kg/day, decreased body weight (gain) and food consumption were observed in males. Changes in clinical pathology parameters comprised of decreased hemoglobulin concentration and hematocrit in females, increased triglyceride and calcium concentrations in males and increased alkaline phosphatase activity and (HDL and LDL) cholesterol concentrations in males and females. As these findings were slight and/or in absence of corroborating findings, they were considered to be non-adverse. Histopathological alterations consisted of test item-related, non-adverse increased incidence and severity (up to mild) of histiocytic infiltrates in the mesenteric lymph node in males and females. Thyroid hormones showed an increase in thyroid stimulating hormones (TSH) in males and females treated at 1000 mg/kg/day. A general overlap of individual values with the range of control values was observed and in absence of an effect on organ weight and a histopathological correlation, this findings was considered to be not adverse. No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, ophthalmoscopy, coagulation parameters, thyroid hormones (T3 and T4), macroscopic examination and organ weight). In conclusion, administration of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2 -yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated by once daily oral gavage for at least 90 days was well tolerated in Wistar Han rats at dose levels up to 1000 mg/kg/day in both males and females. Non-adverse changes to body weight (gain), food consumption, and hematology and clinical chemistry parameters were observed in males and females up to 1000 mg/kg/day. Also, non-adverse, test item-related increased incidence and severity (up to mild) of microscopic histiocytic infiltrates was noted in the mesenteric lymph node of males and females at 1000 mg/kg/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was established to be at least 1000 mg/kg/day in males and females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), the substance was adimistered via gavage to 5 rats/sex/dose at 0, 100, 500, 1000 mg/kg bw in PEG 400 for 4 weeks. Up to and including 1000 mg/kg bw no mortality occured. The behavior and clinical appearance of the rats were not influenced by the treatment up to and including 1000 mg/kg in both sexes. The body weight gain, the food, water intake, clinical chemistry and organ weights were not affected up to and including 1000 mg/kg in males and females. The results from Functional Observational Battery (FOB) measurements in males and females receiving up to and including 1000 mg/kg bw did not differ from the control animals. Motor and Locomotor Activity (MA/LMA) tests did not indicate neurotoxicity up to and including 1000 mg/kg.

Adverse findings were observed at 1000 mg/kg b.w. and consisted of a decrease of erythrocytes, hemoblobin and hematocritin males and females and histopathological findings in the thyroid gland of male rats.

Therefore, under the condition of the study, the NOAEL (no-observed-adverse-effect-level) for M 530 after 4-week daily oral treatment by gavage is 500 mg/kg b.w. for male and female rats.

In a second repeated dose oral toxicity study in rats (Wistar, OECD TG 408), the substance was adimistered via gavage to 10 rats/sex/dose at 0, 100, 300, 1000 mg/kg bw in PEG 400 for 90 days. No mortality occurred during the study period. In conclusion, administration of (Reaction product of 4-[2-(4-hydroxyphenyl)propan-2- yl]phenol, 2-(2-hydroxyethylamino)ethanol and formaldehyde), propoxylated by once daily oral gavage for at least 90 days was well tolerated in Wistar Han rats at dose levels up to 1000 mg/kg/day in both males and females. Non-adverse changes to body weight (gain), food consumption, and hematology and clinical chemistry parameters were observed in males and females up to 1000 mg/kg/day. Also, non-adverse, test item-related increased incidence and severity (up to mild) of microscopic histiocytic infiltrates was noted in the mesenteric lymph node of males and females at 1000 mg/kg/day. Based on these results, the No Observed Adverse Effect Level (NOAEL) was established to be at least 1000 mg/kg/day in males and females.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
OECD 407 with a NOAEL of 500 mg/kg/day as most sensitive threshold value.


Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: thyroids

Justification for classification or non-classification

No classification is required for repeated dose toxicity according to EU-Directive 67/548/EEC, Annex VI and according to Regulation (EC) No 1272/2008, Annex I.