Formaldehyde, reaction products with bisphenol A and diethanolamine, propoxylated

Endpoint summary

Description of key information

Repeated dose toxicity, 28 days, rat, oral: NOAEL = 500 mg/kg bw/day

Repeated dose toxicity 90 days, rat, oral: NOAEL > 1000 mg/kg bw/day

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

Reference 1

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.

Reference 2

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018-06-25

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: stable for 4 days in the refrigerator protected from light followed by at least 12 hours at room temperature protected from light; stable for 20 days in the freezer protected from light; stability in the vehicle PEG 400 was analyitically verified within the concentration range 1 to 800 mg/mL

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

- Condition: Outbred, SPF-Quality
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 146-189 g (males) and 109-149 g (females)
- Fasting period before study: No
- Housing: group-housed (max. 5 animals of the same sex and same dosing group) 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); during locomotor activity monitoring, animals were housed individually in Hi-temp polycarbonate cages (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water (for max. 2 hours)
- Diet (ad libitum): pellets; SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water (ad libitum): municipal tap water
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
- Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test
Facility
- Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
- It is considered that there are no known contaminants in the feead and water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 42 to 57
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-02-25 To: 2021-05-28

Route of administration:

oral: gavage

Details on route of administration:

- oral route of exposure was selected because it is a possible route of human exposure during manufacture, handling or use of the test item
- gavage was performed by using a plastic feeding tube

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- dosing solutions were prepared daily
- dose formulations were divided into aliquots to allow dispension on each dosing occasion, if required
- formulations were homogenized to visually acceptable levels

VEHICLE
- Justification for use and choice of vehicle (if other than water): PEG 400 was selected as vehicle due to low solubility of the test item in water.
- Amount of vehicle (if gavage):
- Lot/batch no.: PEG4009 and PEG40010 (Merck, Darmstadt, Germany)
- Specific gravity: 1.125

DOSING PROCEDURE:
- dose formulations were stirred continuously during dosing
- doses were given using a plastic feeding tube
- dose pot identification via Provantis was 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:

- dose formulations were analysed for preparations used in Weeks 1, 6 and 12
- doses were within 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; based on the results Group 2 animals were dosed with at least 20 mg/mL (~27.0-27.3 mg/mL) according to at least 100 mg/kg bw/day (~135.0-136.5 mg/kg bw/day) in Week 6.
- No test item was found in the Group 1 (control) formulation.
- Dose formulation homogeneity was confirmed and within Relative Standard Deviation (RSD)

Duration of treatment / exposure:

90 days

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
cf. Table in the Attachment named "Study design and animal assignment"

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected according to results of a 28-day study according to OECD 407 with dose levels of 100, 500 and 1000 mg/kg bw/day; the NOAEL in this study was 500 mg/kg bw/day and adverse findings were observed at 1000 mg/kg bw/day (decrease of erythrocytes, hemoblobin and hematocrit in males and females and histopathological findings in the thyroid gland of male rats)
- Rationale for animal assignment: random under consideration of body weight variation not exceeding +/- 20 % of the sex mean
- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Dose range finding studies: not conducted, since a 28-day study according to OECD 407 is available

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily; from day 1 at 0-1 hours post-dose
- Animals were 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 observations were recorded to each animal in the socialized group.
- Observation for mortality: At least twice daily beginning upon arrival through termination. Except on days of receipt and necropsy where frequency was once daily. Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pretreatment and weekly; from Week 1 and throughout the study and on day of necropsy
- Animals were removed from the cage.

ARENA OBSERVATIONS:
- Time schedule: Pretreatment and weekly during the Treatment.
- Animals were observed for clinical signs outside the home cage in a standard arena.
- Animals were observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study (Fasted weight on day of necropsy)
- Body weight gains calculated between each scheduled interval

FOOD CONSUMPTION:
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study
- Quantitatively measured per cage.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: On a regular basis throughout the study
- Water consumption was monitored by visual inspection of the water bottles. If inter group differences would have been noted, consumption would have been assessed by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once pretreatment; during Week 13 of dosing period. If treatment-related findings would have been noted, the other animals would have been also examined.
- Dose groups that were examined: Group 1 and 4
- The eyes were examined using an ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatmnet
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters:
White Blood Cell Count (WBC)
Neutrophils (absolute)
Lymphocytes (absolute)
Monocytes (absolute)
Eosinophils (absolute)
Basophils (absolute)
Large unstained cells (LUC) (absolute)
Red Blood Cell Count
Reticulocytes (absolute)
Red Blood Cell Distribution Width Gated (RDWG)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelets
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters:
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Triglycerides
HDL and LDL Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters:
Triiodothyronine (T3)
Thyroxine (T4)
Thyroid-Stimulating Hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during dosing period; the first 5 animals per sex per group during Week 12-13; performed after clinical observation
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex and static righting reflex) / grip strength (fore- and hind-limb grip strength) / motor activity (Total movements and ambulations were recorded)

IMMUNOLOGY: No

OTHER:
ESTROUS STAGE DETERMINATION:
- Time schedule for examinations: on the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all females
- Estrous stage was evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.

Sacrifice and pathology:

NECROPSY:
- animals were deeply anesthetized using isoflurane and were subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

ORGAN WEIGHTS:
- The organs were weighed at necropsy for all animals. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) were calculated.

GROSS PATHOLOGY: Yes
- Animals were subjected to a complete necropsy examination,which included 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.

- Complete postmortem examinations were performed on all animals. Animals were anesthetized using isoflurane and subsequently exsanguinated. At the time of necropsy, the following tissues and organs were collected and placed in 10% neutral-buffered formalin fixative unless otherwise noted in the tables in the Attachment (cf. below)

- Gross lesions were examined from all animals and correlated to microscopic findings if possible.

HISTOPATHOLOGY: Yes
- hematoxylin-eosin-stained paraffin sections were prepared for all tissues collected at necropsy (with exceptions as indicated in the tables in the attachement, cf. below) from all control group and 1000 mg/kg/day treated animals, slides of the mesenteric lymph nodes from all 100 and 300 mg/kg/day treated rats.

For details on organs and tissues examined please refer to the two tables in the Attachements named:
"Tissue Weighing, Collection, Processing and Evaluation Table 1 of 2"
"Tissue Weighing, Collection, Processing and Evaluation Table 2 of 2"

Optional endpoint(s):

Not applicable

Other examinations:

Not applicable

Statistics:

Numerical data collected on scheduled occasions were summarized and statistically analyzed as follows according to sex and occasion.

Inferential Statistical Methods:
- 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% and 5% levels.
- variable for inferential analysis with statistical method "parametric/ non-parametric": body weight, body weight gains, hematology variables, clinical chemistry variables, functional observation battery quantitative variable, organ weights and organ weights relative to body weight
- variable for inferential analysis with statistical method "incidence": functional observation battery qualitative variable

Parametric/ Non-parametric:
- Levene's test was used to assess the homogeneity of group variables
- groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively

Non-Parametric:
- groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

ANCOVA:
- data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

Incidence:
- A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- Incidental hunched posture and erected fur were noted in individual males and/or female. At the low incidence observed, these findings were considered not toxicologically relevant and not treatment-related.
- Incidental abnormal breathing sounds were observed in individual males and/or females. These clinical signs were considered to be related to the method of dosing and not as test item-related.

300 mg/kg bw/day:
- Incidental hunched posture and erected fur were noted in individual males and/or female. At the low incidence observed, these findings were considered not toxicologically relevant and not treatment-related.
- Incidental abnormal breathing sounds were observed in individual males and/or females. These clinical signs were considered to be related to the method of dosing and not as test item-related.

100 mg/kg bw/day:
- Incidental abnormal breathing sounds were observed in individual males and/or females. These clinical signs were considered to be related to the method of dosing and not as test item-related.


Non-treatment-related effects observed in all groups:
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, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males: body weight was decreased from Day 36 of treatment onwards (down to 6-9% lower than control; not always statistically significant)
- males: an overall lower body weight gain between Days 1-91 was noted

300 mg/kg bw/day
- females: 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 (body weight changes < 10 % and not statistically significant) 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):

1000 mg/kg bw/day:
- males: 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).
- males: overall food consumption between Days 1 and 91 was slightly lower compared to the control (2% lower than control).

As findings were slight (food consumption changes < 10 % and not statistically significant) and in absence of corroborating findings, these findings were considered not adverse and not test item related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

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:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- females: decreased hemoglobulin concentration (0.95x of control; not statistically significant)
- females: decreased hematocrit (0.95x of control; statistically significant p - females: shorter activated partial thromboplastin time (APTT) (significant p - In absence of a histopathological correlation these findings were considered to be not adverse.

Non-treatment-related effects observed:
- 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.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males increased alkaline phosphatase (ALP) activity (significant p - females: increased alkaline phosphatase (ALP) activity (not significant)
- males and females: increased cholesterol concentrations (significant p - males and females: increased HDL cholesterol concentrations (significant p - males and females: increased LDL cholesterol concentrations (significant p - males: increased calcium concentrations (significant p - males: increased triglyceride concentrations (not significant)

300 mg/kg bw/day:
- females: increased alkaline phosphatase (ALP) activity (not significant)
- males: increased cholesterol concentrations (significant p - males: increased HDL cholesterol concentrations (significant p - males: increased triglyceride concentrations (not significant)

100 mg/kg bw/day:
- males: increased triglyceride concentrations (not significant)

Non-treatment-related effects observed:
Remaining differences in clinical chemistry 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.


In absence of a histopathological correlation these findings were considered to be not adverse.

Please refer to the table in the Attachements named "Extract of clinical chemistry parameters".

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males and females: increased thyroid-stimulating hormone (TSH) concentration (not significant)

100 mg/kg bw/day:
- females: increased thyroid-stimulating hormone (TSH) concentration (not significant)


Non-treatment-related effects observed:
Remaining differences in TSH, 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. Furthermore, these findings were considererd non-adverse as no effects on organ weights or any histopathological correlation was observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males: lower foreleg grip strength was observed compared to the control

300 mg/kg bw/day:
- males: lower hindleg grip strength was observed compared to the control

100 mg/kg bw/day:
- males: lower foreleg grip strength was observed compared to the control

In absence of a dose-related response, these findings were considered not test item-related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males: A few organ weight differences were statistically significant (heart, absolute weight; liver relative to body weight) but was interpreted to be related to the lower terminal body weights in that group (-9%). Thus, these findings were considered secondary on non-adverse

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, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
- males and females: histiocytic infiltrates in the mesenteric lymph node with increased incidence (males: 3/10; females: 3/10 ) and severity (mild degree); 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.


Non-treatment-related effects observed:
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.


(Please refer to the table in the Attachements named "Summary Test Item-Related Microscopic Findings"

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No effects observed regarding the estrous cycle of females in all groups.

Details on results:

For details on results (raw data) please refer to the documents in the Attachements named as follows:
OECD408_body weight, body weight gain and food consumption
OECD408_Hematology and clinical chemistry
OECD408_macroscopic and microscopic pathology
OECD408_neurobehaviour and FOB
OECD408_organ weights

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

at the limit dose of 1000 mg/kg bw/day

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

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.