2,2',6,6'-Tetrabromo-4,4'-isopropylidenediphenol, oligomeric reaction products with Propylene oxide and n-butyl glycidyl ether

Endpoint summary

Description of key information

Repeated dose toxicity, oral: A 28-day study (OECD 407, GLP) was conducted resulting in a NOAEL of 5 mg/kg bw/day for females and 10 mg/kg bw/day for males

Repeated dose toxicity, oral: A 90-day study (OECD 408, GLP) was conducted resulting in a NOAEL of 40 mg/kg bw/day for males and females

Key value for chemical safety assessment

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

Specific details on test material used for the study:

- Substance type: brominated polyether polyol
- Stability under test conditions: It is very unlikely that the test substance would react with corn oil or petroleum jelly at ambient temperature in the time scale of storage of the solutions - less than 7.5 hours. The adjuvant is mainly paraffin oil and surfactant and no reaction would be anticipated with these ingredients. I therefore consider that the formulations of the test substance in these media used in this study were stable for their period of use.

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

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:

Analytical determination of stability and/or homogeneity of formulations were not possible due to technical reasons. For the same reason test item concentrations were not verified during the study (content check), too. Each stock formulation and dilutions of it were prepared daily and were stirred until visual homogeneity. Formulations were prepared in the presence of a second person (dual control) twice during the study.

Duration of treatment / exposure:

30 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
females: 0, 5, 15, 45 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
males: 0, 10, 30 and 90 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:
Dose levels were selected based on results of a previous dose range finding study with gavage administration (T2082796) in rats. Test item formulated in corn oil was first administered daily at 1000 mg/kg in a volume of 5 ml/kg body weight to 3 rats per sex. Animals lost weight, had diarrhea and had to be killed in moribund condition after 4 treatments. Gross pathologically nodal changes, discoloration and content changes of forestomach and/or cecum were seen.

Additional 3 rats per sex were dosed at 500 and 100 mg/kg daily (same formulation and administration volume). After second treatment rats showed comparable clinical symptoms at 500 mg/kg (both sexes) and at 100 mg/kg (one female).

After discontinuation of treatment for 2 days rats treated twice at 500 mg/kg were treated daily at 100 mg/kg. Rats treated twice at 100 mg/kg were treated at 30 mg/kg without discontinuation. One 500 mg/kg-female was killed in extremis on the second day of treatment discontinuation. Male rats treated at 500 then 100 mg/kg recovered, gained weight and showed no more clinical symptoms, while 100 then 30 mg/kg males stayed without symptoms. Females treated first at 500 then at 100 mg/kg lost further body weight. One of these females was killed in moribund condition, the other stopped losing body weight. Females first treated twice at 100 mg/kg then at 30 mg/kg gained weight after dose reduction and diarrhea disappeared.

Overall females were more sensitive than males. Therefore different dose schedules were chosen for the subacute study.

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 30/31
- How many animals: all dose groups incl. controls

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, day 30/31
- How many animals: all dose groups incl. controls

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB: once, day 28 (males), day29 (females); MA: once, day 28 (males), day 29 (females)
- 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)
-Adrenal gland * Oviduct*; Bone; sternum Parathyroid gland *; Bone marrow; sternum Peyer's patch; Brain (three regions) Prostate gland; Cecum Rectum; Cervix Sciatic nerve; Coagulating gland * Seminal vesicle *; Colon Skeletal muscle; Duodenum Spinal cord (three regions); Epididymis * Spleen Femur/knee joint (with bone marrow) Stomach (nonglandular and glandular); Heart Testis*; Ileum Thymus; Jejunum Thyroid gland *Kidney* Trachea. Liver Urinary bladder; Lung Uterus; Lymph node; iliac* Vagina; Lymph node; mesenteric; Macroscopic abnormalities; Ovary*
For paired organs(*); both sides were examined

HISTOPATHOLOGY: Yes (all dose groups and controls)
-Microscopic: duodenum, jejunum, ileum, cecum, colon, rectum, Peyer's patch and mesenteric lymph node

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:

effects observed, treatment-related

Description (incidence and severity):

Since no rat died during the study, survival was not affected by treatment with the test item.In all high dose rats (both sexes) treatment with the test item induced changed feces consistency (soft, pulpish).

Mortality:

mortality observed, treatment-related

Description (incidence):

Since no rat died during the study, survival was not affected by treatment with the test item.In all high dose rats (both sexes) treatment with the test item induced changed feces consistency (soft, pulpish).

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):

effects observed, treatment-related

Description (incidence and severity):

There was no dose and time dependent difference in water consumption in low dose rats and in mid dose females. Related to body weight mid and high dose males consumed more water. High dose females had a slightly higher water intake in the last 2 weeks.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

HAEMATOLOGY:

Mean neutrophile counts of high dose females marked as significantly higher are based on individual values within the
control rang ofrats of the same strain and age run some months before (T6082745). This
difference is thus considered as normal variation. All other means identified as significantly
different were without dose-relation and are hence considered irrelevant.

CLINICAL CHEMISTRY:
Mean ALA T activity of high dose males marked as significantly higher is without dose relation and thus considered a chance result. Creatinine concentration of mid dose males and high dose rats of both sexes was slightly but significantly lower with no dose correlation in males.


HISTOPATHOLOGY:
Minor inflammatory changes in the intestine (mainly cecum) were seen at dosages of 10, 30 and 90 mg/kg/day in males and at 15 and 45 mg/kg/day in females. Histologically, intestinal changes mostly in the cecum were seen in all mid dose group and most high dose animals. These comprised a minimall mild mononuclear cell infiltration of the cecal mucosa, in some animals associated with minimal/ mild diffuse mucosal hyperplasia. Mild mononuclear cell infiltration ofthe cecal mucosa was also observed in one single male of the low dose group. Moreover, the mucosa of other intestinal segments was also infiltrated in most high dose females and some mid and high dose males, without dose relationship. Possibly secondary to the intestinal inflammatory process a tendency towards minor unspecific histopathological changes were noted in mesenteric lymph nodes (all treated animals), although dose relationship was missing. In the Peyer´s patches of mid and high dose females vacuoles (minimal numbers, low incidence) without dose relation were seen. Because there were no surrounding reactions this finding is considered as non-adverse.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: inflammatory changes in the intestine

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: inflammatory changes in the intestine

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, 10, 30, 90 mg/kg bw (male) or 0, 5, 15, 45 mg/kg bw (female) in corn oil for 4 weeks. Up to and including 90 or 45 mg/kg bw no mortality occured. The behavior of the rats were not influenced by the treatment up to and including 90 mg/kg (males) or 45 mg/kg (females). There were no clinical findings induced by treatment with the test item in rats dosed at low and mid dose. In all high dose rats (both sexes) treatment with the test item induced changed feces consistency (soft, pulpish). Related to body weight mid and high dose males consumed more water. High dose females had a slightly higher water intake in the last 2 weeks.The body weight gain, the food intake, clinical chemistry and organ weights were not affected up to and including 90 mg/kg in males and 45 mg/kg in females. The results from Functional Observational Battery (FOB) measurements in males and females receiving up to and including 90 mg/kg bw or 45 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 90 mg/kg or 45 mg/kg.

At necropsy consistency changes of small and large intestine and/or cecum dilations were observed (mid and high dosed animals). Histopathological examinations revealed minor inflammatory changes in the intestine (mainly cecum, mid and high dose, both sexes, one low dosed male). Possibly related to this a tendency towards minor unspecific histopathological changes were noted in mesenteric

lymph nodes (all treated animals, no dose relation).

Therefore, under the condition of the present study, the NOAEL (no-observed-adverse-effect-level) for the test item after 4-week daily oral treatment by gavage is 10 mg/kg bw and 5 mg/kg bw for male and female rats.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jun 2022 to 11 Nov 2022

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:

adopted 25-06-2018

Deviations:

yes

Remarks:

dose levels were adjusted during study; mean maximum humidity was 75%: values that were outside the targeted humidity range occurred for 9 days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the 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: room temperature
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: corn oil used as vehicle, stability for at least 24 hours at room temperature (15 to 25°C) under normal laboratory light conditions, and stability for at least 8 days in the refrigerator (set to maintain 4°C) has been confirmed over the concentration range 1 to 200 mg/mL

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

- Condition: outbred, SPF-quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 132-173 g (males), 107-137 g (females
- Fasting period before study: no
- Housing: group-housed (max. 5 animals of the same sex and dose group) in Polycarbonate cages (Makrolon type IV, height 18 cm or 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. 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: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany; no access during motor activity measurements for max. 2 h.
- Water: ad libitum, munincipal tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
- Diet: no known contaminants according to analysis for nutritional components and environmental contaminants provided by the supplier
- Water: no known contaminants according to periodic analysis of the water conducted by the test facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C
- Humidity (%): 45-75% (The values that were outside the targeted humidity range, i.e. > 70 %, occurred for 9 days)
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21-06-2022 To: 21-09-2022

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:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- dosing solutions (w/w) prepared weekly by homogenisation to visually acceptable levels, adjustment for specific gravity of the vehicle made, storage of the dosing solutions at 4°C

VEHICLE
- Justification for use and choice of vehicle: Corn oil due to low water solubility
- Concentration in vehicle:
Group 2: 2 mg/mL (day 1-27) followed by 4 mg/mL (day 28-90)
Group 3: 8 mg/mL (day 1-27) followed by 16 mg/mL (day 28-90)
Group 4: 30 mg/mL (day 1-27) followed by 60 mg/mL (day 28-35) followed by 40 mg/mL (day 37-90)
- Amount of vehicle (if gavage): 5 mL/kg bw
- Specific gravity: 0.92

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:

ACCURACY:
- concentrations analyzed in the formulations of Groups 2-4 for use in Weeks 1 and 6, and Groups 3 and 4 for use in Week 12 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85-115% of target concentration)
- the formulation of Group 2 prepared for use in Week 12, the mean concentration was 84% of target. An Out of Specification investigation was performed, but no analytical reason could be found for the slightly lower mean recovery. As the deviation from the acceptance criteria (≥15%) was minimal, these results were accepted, and the formulation was considered to be prepared correctly
- No test material was detected in the Group 1 formulations (control group)
HOMOGENEITY:
- formulations of Group 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%)

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Group 1: vehicle control

Dose / conc.:

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

Remarks:

Group 2: 10 mg/kg bw/day (day 1-27) followed by 20 mg/kg bw/day (day 28-90): dose levels were increased by 2-fold, since the highest dose level did not produce definite signs of toxicity

Dose / conc.:

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

Remarks:

Group 3: 40 mg/kg bw/day (day 1-27) followed by 80 mg/kg bw/day (day 28-90): dose levels were increased by 2-fold, since the highest dose level did not produce definite signs of toxicity

Dose / conc.:

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

Remarks:

Group 4: 150 mg/kg bw/day (day 1-27) followed by 300 mg/kg bw/day (day 28-35) followed by 200 mg/kg bw/day (day 37-90): dose levels were increased by 2-fold after 27 days, since the highest dose level did not produce definite signs of toxicity. Dose level was reduced to 200 mg/kg bw/day from day 37 onwards, since deterioration of observed clinical signs, bw loss and mortality

No. of animals per sex per dose:

10
cf. Table in the Attachement named "Study design and animal assignment"

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale
14-day DRF, oral route:
Males and females treated in the dose range finder at 1000 mg/kg/day showed body weight loss and diarrhea and were sacrificed in moribund condition after four treatments. At 500 and 100 mg/kg/day, similar clinical signs were observed in males and females (at 100 mg/kg/day only in females), and dose levels were lowered to 100 mg/kg/day/day (after two-day discontinuation of treatment) and 30 mg/kg/day/day, respectively. At 100 mg/kg/day, males recovered and showed no further clinical signs. In females, at 100 mg/kg/day, body weight loss continued, and one female was sacrificed in moribund condition. Body weight of the second female at 100 mg/kg/day stabilized over time. At 30 mg/kg/day, no clinical signs were observed in males, and effects in females recovered over time.

28-day repeated dose toxicity study, oral route:
The dose levels for the 28-day repeated dose study were set at 10, 30 and 90 mg/kg/day in males and 5, 15 and 45 mg/kg/day in females. Up to and including 90 or 45 mg/kg/day no mortality occurred and behavior of the rats were not influenced by the treatment. There were no clinical findings at 10 and 30 mg/kg/day in males and at 5 and 15 mg/kg/day in females. All high dose rats (both sexes) showed changed feces consistency (soft, pulp). Males at 30 and 90 mg/kg/day, and females at 45 mg/kg/day showed a (slightly) higher water intake compared to the control. Body weight gain, food consumption, clinical chemistry, functional tests, motor activity and organ weights were not affected up to and including 90 mg/kg/day in males and 45 mg/kg/day in females. At necropsy consistency changes of small and large intestine and/or cecum dilations were observed (mid and high dosed animals). Histopathological examinations revealed minor inflammatory changes in the intestine (mainly cecum, mid and high dose, both sexes, one low dosed male). Based on these results, the NOAEL (no-observed-adverse-effect-level) for the test material after 4-week daily oral treatment by gavage was set at 10 and 5 mg/kg/day for males and females, respectively.

- Rationale for animal assignment: randomly assigned
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale: randomly assigned
- Dose range finding studies: not conducted, since a 28-day study according to OECD 407 is available
- Other: Dose levels were adjusted twice as follows:
Group 2: 10 mg/kg bw/day (day 1-27) followed by 20 mg/kg bw/day (day 28-90): dose levels were increased by 2-fold, since the highest dose level did not produce definite signs of toxicity
Group 3: 40 mg/kg bw/day (day 1-27) followed by 80 mg/kg bw/day (day 28-90): dose levels were increased by 2-fold, since the highest dose level did not produce definite signs of toxicity
Group 4: 150 mg/kg bw/day (day 1-27) followed by 300 mg/kg bw/day (day 28-35) followed by 200 mg/kg bw/day (day 37-90): dose levels were increased by 2-fold after 27 days, since the highest dose level did not produce definite signs of toxicity. Dose level was reduced to 200 mg/kg bw/day from day 37 onwards, since deterioration of observed clinical signs, bw loss and mortality

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily, from Day 1 at 0 to 1-hour 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 the 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).
- In order to monitor the health status, animals of Group 4 were weighed also on Days 32, 35 and 39, Group 4 Female No. 80 on Day 41 and Group 2 Male No. 14 on Day 79.

FOOD CONSUMPTION: Yes
- Time schedule for examination: 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: Regular basis throughout the study
- Water consumption was monitored by visual inspection of the water bottles. When inter group differences were noted, consumption was assessed by weight.
- Due to visual differences, water consumption was recorded from Day 50 onward every other day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals once prior initial dosing and all Group 1 and 4 animals, and Group 3 females, during Week 13
- Dose groups that were examined: Group 1 and 4 animals and Group 3 females
- The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol, tropicamide 5 mg/mL solution, THEA Pharma, Wetteren, Belgium).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight with maximum of 24 h)
- How many animals: all animals surviving until end of treatment
- Blood collected from the retro-orbital sinus or alternatively from the aorta prior necropsy if collection from retro-orbital sinus was unsuccessful
- Parameters examined for haematology and coagulation:
White blood cells (WBC),
Neutrophils (absolute),
Lymphocytes (absolute),
Monocytes (absolute),
Eosinophils (absolute),
Basophils (absolute),
Large unstained cells (LUC) (absolute),
Red blood cells (RBC),
Reticulocytes (absolute),
Red blood cell distribution width (RDW),
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 (overnight with maximum of 24 h)
- How many animals: all animals surviving until end of treatment
- Blood collected from the retro-orbital sinus or alternatively from the aorta prior necropsy if collection from retro-orbital sinus was unsuccessful
- Parameters examined for clinical chemistry:
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/LIPIDS: Yes
- Time of blood sample collection: end of treatment
- Animals fasted: Yes (overnight with maximum of 24 h)
- How many animals: all animals surviving until end of treatment
- Blood collected from the retro-orbital sinus or alternatively from the aorta prior necropsy if collection from retro-orbital sinus was unsuccessful
- Parameters examined:
Triiodothyronine (T3),
Thyroxine (T4),
Thyroid-Stimulating Hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12-13
- Dose groups that were examined: 5 animals per dose group examined, execpt for the high dose group (Group 4), since only two animals survived until neurobehavioural examination
- 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 reported)

IMMUNOLOGY: No


OTHER:
ESTROUS STAGE DETERMINATION: Yes
- Time schedule for examinations: at the end of treatment (i.e. day of necropsy)
- How many animals were examined: all female animals, that survived until necropsy
- examination of vaginal cytology of samples obtained by vaginal smear procedure

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.
- Organs weighed:
Brain
Epididymisa
Gland, adrenala
Gland, pituitary
Gland, prostate
Gland, thyroid
Gland, seminal vesicle
Heart
Kidney
Liver
Lung
Ovarya
Spleen
Testisa
Thymus
Uterus

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 Group 1 animals (Control group), Group 4 animasl (high dose group) and Group 3 females (mid dose group). Mid-dose group females were evaluated in complete, since the number of remaining high dose group females was too low for statistical evaluation. Remaining animals (Group 2 animals and Group 3 males) were evaluated for gross lesions and target tissues ( see below) only. 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 below:
Artery, aorta
Body cavity, nasopharynx
Bone marrow smear
Bone marrow
Bone, femur
Bone, sternum
Brain [cerebellum, mid-brain, cortex] (8 levels.)
Cervix
Epididymis
Esophagus
Eye (a)
Gland, adrenal (b)
Gland, clitoral
Gland, harderian
Gland, lacrimal (exorbital)
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, preputial
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum (b)
Large intestine, colon
Large intestine, rectum
Larynx
Liver (b)
Lung
Lymph node, mandibular
Lymph node, mesenteric
Muscle, skeletal
Nerve, optic (a)
Nerve, sciatic
Ovary
Oviduct
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testis
Thymus (b)
Tongue
Trachea
Ureter
Urinary bladder
Uterus
Vagina

(a) Optic nerve remained attached to the eye and fixed in Modified Davidson’s fixative. The remaining part of the optic nerve was placed in formalin.
(b) Target tissues evaluated in all animals

- 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 as listed above (except for bone marrow smear) from animals of each dose group


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 3"
"Tissue Weighing, Collection, Processing and Evaluation Table 2 of 3"
"Tissue Weighing, Collection, Processing and Evaluation Table 3 of 3"

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, treatment-related

Description (incidence and severity):

Group 4 (high dose) – 8/10 females found dead or killed in extremis:
- liquid to soft feces (including fecal smearing) (8/8)
- abdominal distension (7/8)
- erected fur (7/8)
- hunched posture (8/8)
- closed eyes (1/8)
- thin appearance (1/8)
- ungroomed fur (1/8)
- decreased activity (1/8)


Group 4 (high dose):
- fecal smearing and soft to liquid feces in all males and the 2 females (the females that survived until end of treatment)
- slight abdominal distension in 6 males and 2 females (the females that survived until end of treatment)
- erected fur in 8 males and 2 females (the females that survived until end of treatment)
- hunched posture in 6 males and 2 females (the females that survived until end of treatment)

Group 3 (mid dose):
- soft or liquid feces was observed in all animals from Day 35 onwards
- slight abdominal distension between Days 68-71 for 2-4 days in 2 females
- erected fur between Days 37-79 for 1-6 days in 1 male and 4 females
- hunched posture on 2-4 days between Days 43-44, Days 45-49 and Days 70-71 for 2-4 days in 2 females
- Some of these findings are suggestive of a test material-related effect on the digestive tract. In absence of an effect on body weight and food consumption, these findings were considered not adverse.

Group 2 (low dose):
- no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

Group 4 (high dose): 8/10 females (on days 30, 32, 36, 41, 57, 79)
Group 2 (low dose): 1/10 males, 1/10 females, both due to a gavage accident

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose) - females found dead or killed in extremis (8/10):
- body weight loss (-8 to -21 %) during the last few days before euthanization

Group 4 (high dose):
- terminal body weight in males decreased compared to control (-12 %, statistically significant)
- body weight in females decreased compared to control until Day 57 (-9.6 %, statistically significant), thereafter body weight decreased further compared to control (-7.3 %), but could not be assessed for statistics since only 2 females left in the group; terminal body weight in the surviving females decreased compared to control (-9 %, statistical analysis not applicable due to the low number of animals)

Group 3 (mid dose):
- no effects observed

Group 2 (low dose):
- no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- lower food consumption (9 %) in males between Days 29-36
- lower food consumption (35 %) in females between Days 29-36 which was likely a result of low food consumption in one out of two cages
- slightly higher food consumption (2-8 %) in males and females between Days 37-92

Group 3 (mid dose):
- no effects observed

Group 2 (low dose):
- no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was recorded from Day 50 onwards as higher water consumption was observed in males and females of the high dose group (Group 4) via visual inspection.

Group 4 (high dose):
- higher water consumption in males and females until end of treatment (dose-dependent)

Group 3 (mid dose):
- higher water consumption in males and females until end of treatment (dose-dependent)


Group 2 (low dose):
- higher water consumption in males and females until end of treatment (dose-dependent)


The effects in all dose groups are considered secondary to the effects on the digestive tract and subsequent fecal changes and therefore in itself not considered adverse.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- statistically significant higher neutrophil count in males
- statistically significant lower lymphocyte count in males
- statistically significant higher red blood cell count in males
- statistically significant higher reticulocyte cell count in males
- statistically significant higher hemoglobin concentration in males


- hematology parameters of the two remaining females could not be assessed, due to the low number of animals and the limited statistical power of the results. However, the two surviving females showed similar hematological changes, where the decreased leukocytes showed similar histopathological correlations, which was considered adverse in these females due the high grade of severity (up to marked degree) of the microscopic findings.
- The observed effects on neutrophil and red blood cell count and the hemoglobin concentration are considered non-adverse due to the absence of a microscopic correlated of these findings.
In addition, males showed a lower lymphocyte count with correlating macroscopic, organ weight and microscopic changes in the thymus. In absence of adversity of the histopathological changes, this effect was considered not adverse.


Group 3 (mid dose):
- statistically significant lower lymphocyte count in males and females
- statistically significant higher neutrophil count in females
- statistically significant higher red blood cell count in females
- statistically significant higher hemoglobin concentration in females
- The observed effects on neutrophil and red blood cell count and the hemoglobin concentration are considered non-adverse due to the absence of a microscopic correlated of these findings.
In addition, males and females showed a lower lymphocyte count with correlating macroscopic, organ weight and microscopic changes in the thymus. In absence of adversity of the histopathological changes, this effect was considered not adverse.



Group 2 (low dose):
- statistically significant lower lymphocyte count in females
- In the absence of correlating macroscopic, organ weight and microscopic changes in the thymus, this effect was considered not adverse.

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- statistically significant higher urea concentration in males
- statistically significant higher creatinine concentration in males
- statistically significant higher HDL cholesterol concentration in males
- statistically significant lower triglyceride concentration in males
- statistically significant lower sodium concentration in males
- statistically significant lower potassium concentration in males
- statistically significant lower inorganic phosphate concentration in males

- clinical chemistry parameters of the two remaining females could not be assessed, due to the low number of animals and the limited statistical power of the results. However, similar changes were observed in the two surviving females

Group 3 (mid dose):
- statistically significant higher urea concentration in males and females
- statistically significant higher creatinine concentration in males
- statistically significant higher cholesterol concentration in females
- statistically significant higher HDL cholesterol concentration in females
- statistically significant lower triglyceride concentration in males
- statistically significant lower sodium concentration in males and females


Group 2 (low dose):
- no effects observed

In absence of a histopathological correlate, these findings were considered non-adverse in all dose groups.

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

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- statistically significant decreased T4 levels (23.53 ng/mL; 0.53 x compared to control) and mean values below historical control in males (T3 and TSH levels were unaffected)

- Historical control data for Wistar Han rats: males (T4) (period 2019-2022): T4 (ng/mL): mean = 50.1; Central 90% = 32.5 – 66.8 (n = 65).

- thyroid parameters of the two remaining females could not be assessed, due to the low number of animals and the limited statistical power of the results


Group 3 (mid dose):
- statistically significant decreased T4 levels (30.56 ng/mL; 0.69 x compared to control) and mean values below historical control in males
- Historical control data for Wistar Han rats: males (T4) (period 2019-2022): T4 (ng/mL): mean = 50.1; Central 90% = 32.5 – 66.8 (n = 65).


Group 2 (low dose):
- no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- statistically significantly higher value for hindleg grip strength recorded for males. As no dose-dependent effect was observed on an individual animal level between the different dose groups, this effect was considered not related to treatment with the test material.
- decreased total movements (statistically significant) and decreased ambulations (not statistically significant) in males. In absence of corroborating findings, this change was considered not adverse.

Group 3 (mid dose):
- statistically significantly higher value for foreleg grip strength recorded for females. In the absence of a dose-related response, this variation was considered not related to treatment with the test material.


Group 2 (low dose):
- no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose):
- lower thymus weight (absolute and relative to bw) in males (statistically significant) and females
- lower spleen weight (absolute and relative to bw) in males (statistically significant) and females
- higher adrenal gland weight (relative to bw) in males (statistically significant) and females
- higher liver weight (relative to bw) in males (statistically significant) and females
- statistically significant lower absolute thyroid/parathyroid weight in males (considered not to be test material-related due to test material-related effect on final body weight particularly noted in males (150/300/200 mg/kg/day: -12%)
- statistically significant lower absolute heart weight in males (considered not to be test material-related due to the lack of dose-related pattern, and test material-related effect on final body weight particularly noted in males (150/300/200 mg/kg/day: -12%)



- statistical analysis of the organ weights of the two remaining females could not be conducted, due to the low number of animals and the limited statistical power of the results


Group 3 (mid dose):
- statistically significant lower thymus weight (absolute and relative to bw) in males and females. In absence of adversity of the histopathological changes related to the thymus, these effects were considered not adverse.

- statistically significant lower spleen weight (absolute and relative to bw) in males. Considered non-adverse in absence of a microscopic correlate
- statistically significant lower spleen weight (relative to bw) in females. Considered non-adverse in absence of a microscopic correlate
- statistically significant higher adrenal gland weight (relative to bw) in males. In absence of adversity of the histopathological changes, these effects were considered not adverse.
- statistically significant higher liver weight (relative to bw) in females. In absence of adversity of the histopathological changes, these effects were considered not adverse.
- statistically significant lower absolute prostate weight in males (considered not to be test material-related due to test material-related effect on final body weight particularly noted in males (40/80 mg/kg/day: -8%)
- statistically significant lower absolute thyroid/parathyroid weight in males (considered not to be test material-related due to the lack of dose-related pattern, and test material-related effect on final body weight particularly noted in males (40/80 mg/kg/day: -8%)


Group 2 (low dose):
- no effects observed

Please refer to the table in the Attachements named "Extract of organ weights"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose) - females found dead or killed in extremis (8/10):

- abnormal watery (yellow) content in the cecum (7/8), ileum (4/8), and/or jejunum (4/8)
- cecum (1/8) and jejunum (1/8) distended with gas
- enlargement of the cecum (1/8)
- decreased adipose tissue (8/8)
- small spleen (7/8)
- small thymus (7/8)

Group 4 (high dose):
- abnormal watery content in the jejunum in 1/10 males and 2/2 females
- abnormal watery content in the ileum in 1/10 males
- abnormal watery content in the cecum in 8/10 males and 2/2 females
- The findings related to the digestive tract were considered adverse in Group 4 (high dose), since they likely attributed to the poor general health of these animals
- small thymus in 3/10 males. In absence of adversity of the histopathological changes related to the thymus, these effects were considered not adverse.


Group 3 (mid dose):
- abnormal watery content in the jejunum in 2/10 males and 3/10 females
- abnormal watery content in the ileum in 3/10 males and 2/10 females
- abnormal watery content in the cecum in 4/10 males and 8/10 females
- As no adverse clinical signs, or effect on body weight and/or food consumption were observed the macroscopic findings related to the digestive tract were considered non-adverse.
- small thymus in 1/10 males. In absence of adversity of the histopathological changes related to the thymus, these effects were considered not adverse.



Group 2 (low dose):
- abnormal watery content in the jejunum in 2/9 males and 1/9 females
- abnormal watery content in the ileum in 2/9 males
- abnormal watery content in the cecum in 2/9 males
- As no adverse clinical signs, or effect on body weight and/or food consumption were observed the macroscopic findings were considered non-adverse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Group 4 (high dose) - females found dead or killed in extremis (8/10):
- minimal to marked decreased lymphoid cellularity
- mild increased apoptosis in the thymus
- mild to marked decreased lymphoid cellularity in the spleen
- minimal to mild decreased hematopoietic cellularity in the bone marrow of the sternum
- minimal attenuation in the cecum
- minimal to mild vacuolation of the zona glomerulosa in the adrenal gland
- minimal to mild centrilobular hepatocellular hypertrophy in the liver
- minimal tubular vacuolation in the kidney


Group 4 (high dose):
- minimal to mild attenuation in cecum observed in 4/10 males including minimal mononuclear cell infiltrate in 2/10 males
- moderate attenuation in cecum observed in 1/2 females
- minimal to mild increased apoptosis in thymus in 9/10 males and 1/2 females
- minimal decreased lymphoid cellularity in thymus in 2/10 males and 1/2 females
- minimal to mild vacuolation of the zona glomerulosa in the adrenal gland in 8/10 males and 2/2 females
- minimal to mild centrilobular hepatocellular hypertrophy in the liver in 7/10 males and 1/2 females
- minimal tubular degeneration in the kidney in 1/10 males associated with hyaline droplet accumulation. The minor increase in severity of hyaline droplet accumulation (a background finding) was considered non-adverse. Tubular degeneration is an adverse finding but only seen in one male

The findings related to the digestive tract were considered adverse, since they likely attributed to the poor general health of these animals


Group 3 (mid dose):
- minimal attenuation in cecum observed in 3/10 males including minimal mononuclear cell infiltrate in 3/10 males
- mild attenuation in the cecal epithelium in cecum observed in 1/10 females
- minimal increased apoptosis in thymus in 4/10 males and 5/10 females
- minimal to mild decreased lymphoid cellularity in thymus in 2/10 males and 3/10 females.
- minimal vacuolation of the zona glomerulosa in the adrenal gland in 5/10 males and 5/10 females
- minimal centrilobular hepatocellular hypertrophy in the liver in 4/10 males and 2/10 females. Considered non-adverse due to the low grade of severity.
- mild tubular vacuolation in the kidney in 1/10 females (no dose-dependency observed, considered as an incidental finding and non-adverse
- As no adverse clinical signs, or effect on body weight and/or food consumption were observed the microscopic findings related to the digestive tract were considered non-adverse.
- In absence of adversity of the histopathological changes related to the thymus, these effects were considered not adverse.
- The findings in the liver and spleen were considered non-adverse due to the low grade of severity.


Group 2 (low dose):
- minimal attenuation in the cecal epithelium in cecum observed in 3/9 males. Considered non-adverse, since no adverse clinical signs, or effect on body weight and food consumption were observed
- minimal vacuolation of the zona glomerulosa in the adrenal gland in 4/9 males and 2/9 females. Considered non-adverse due to the low grade of severity

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:

One female of Group 3 (40/80 mg/kg/day) showed reflux on Day 85 and therefore the actual dose volume could not be confirmed.
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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

ileum

jejunum

other: cecum

Treatment related:

yes

Conclusions:

In conclusion, administration of 2,2',6,6'-Tetrabromo-4,4'-isopropylidenediphenol, oligomeric reaction products with Propylene oxide and n-butyl glycidyl ether for at least 90 consecutive days by once daily oral gavage in Wistar Han rats at dose levels of 10/20, 40/80 and 150/300/200 mg/kg/day showed the following adverse effects at 150/300/200 mg/kg/day: mortality in females; clinical signs, body weight and food consumption effects consistent with effects on the digestive tract and a poor general health in males and females; histopathological changes consisting of abnormal watery content in the jejunum, ileum, and cecum of males and females, an attenuation in the cecum of males and females as well as mononuclear cell infiltrate in the cecum of males, lower lymphocyte count with correlating increased apoptosis and decreased lymphoid cellularity in the thymus of females with correlating lower thymus weight. As well as tubular degeneration in the kidney of one male. No adverse effects were observed at the other dose levels. Based on these results, the NOAEL was established to be 40 mg/kg/day in males and females.

Executive summary:

The objective of this study was to determine the potential toxicity of 2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with Propylene oxide and n-butyl glycidyl ether, when given orally by gavage for at least 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows:

Text Table1

Experimental Design

Group No.	Test Material Id.	Dose Level (mg/kg/day)	Dose Volume^a(mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	Control	0 (Vehicle)	5	0	10	10
2	2,2’,6,6’-Tetrabromo-4,4’-isopropylidenediphenol, oligomeric reaction products with Propylene oxide and n-butyl glycidyl ether	10/20^b	5	2/4^b	10	10
3		40/80^b	5	8/16^b	10	10
4		150/300^b/ 200^c	5	30/60^b/ 40^c	10	10

Id. = Identification

^aBased on the most recent body weight measurement.

^bfrom Day 28 onwards dose levels were increased by 2-fold, as the Group 4 dose level did not produce definite signs of toxicity.

^cFrom Day 37 onwards dose levels of Group 4 were decreased to 200 mg/kg/day, due to deterioration of the observed clinical signs, body weight loss in males and mortality, clinical signs, and body weight loss in females at 300 mg/kg/day. All Group 4 males and females were not dosed on Day 36.

Chemical analyses of formulations were conducted three times during the study and confirmed that formulations of the test material in corn oil were prepared accurately and homogenously.

The following parameters and endpoints were evaluated in this study: mortality, clinical signs, body weights, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, and clinical chemistry), macroscopic examination, organ weights and microscopic examination.

Mortality occurred in one male and one female at 10/20 mg/kg/day, which were considered related to complications during the gavage procedure. In addition, eight females were euthanized in moribund condition at 150/300/200 mg/kg/day, which was considered to be test material-related and adverse.

At 10/20 mg/kg/day, non-adverse erected fur in one male, higher neutrophil in males and females, and lower lymphocyte count in females, and higher cholesterol and HDL cholesterol in females were noted. In addition, a non-adverse higher water consumption was observed in males and females. In addition, a non-adverse abnormal watery content was observed in the digestive tract (jejunum, ileum, cecum) of males with correlating attenuation in the cecal epithelium, as well as a non-adverse vacuolation of the zona glomerulosa in the adrenal gland of males and females.

At 40/80 mg/kg/day, non-adverse clinical signs consisted of liquid to soft feces, abdominal distention, erected fur, and hunched posture in males and/or females. In addition, a non-adverse higher water consumption was observed in males and females. Hematology changes consisted of a non-adverse increase red blood cell count and hemoglobulin concentration in females, and a higher neutrophil and lower lymphocyte count in males and females, which correlated with a non-adverse small thymus in males, lower thymus weight in males and females and increased apoptosis and decreased lymphoid cellularity in males and females. Clinical chemistry changes consisted of non-adverse higher urea, creatinine, cholesterol, HDL cholesterol and lower sodium concentrations in males and females. In addition, higher inorganic phosphate concentrations in males and lower triglyceride concentration in males were noted. Other histopathological changes consisted of a non-adverse abnormal watery content in the digestive tract (jejunum, ileum, and cecum) in males and females, with correlating attenuation in the cecal epithelial. In addition, lower spleen weight was observed in males and females, as well as vacuolation of the zona glomerulosa in the adrenal gland of males and females with correlating higher adrenal weight in males; centrilobular hepatocellular hypertrophy in the liver of males and females with correlating higher liver weight in females; and tubular vacuolation in the kidney one female. These findings were also considered non-adverse.

At 150/300/200 mg/kg/day, eight females were euthanized in moribund condition showing clinical signs suggestive of effects on the digestive tract (liquid/soft feces, abdominal distention, body weight loss) and histopathological changes consistent with signs of a deteriorating condition. These deaths were considered test material-related and adverse. The surviving animals showed similar clinical signs consisting of liquid/soft feces, fecal smearing, abdominal distension, erected fur and/or hunched posture in males and females. In addition, at 300/200 mg/kg/day, lower body weight and body weight gain were observed in males and body weight loss in females, which was accompanied by a lower food consumption in males and females at 300 mg/kg/day. Histopathological changes consisted of an abnormal watery content in the digestive tract (jejunum, ileum, cecum) in males and females with correlating attenuation in the cecal epithelium. The combination of these findings was considered indicative of an adverse effect on the digestive tract of the animals and of a poor general health due to treatment with the test material and were therefore considered adverse. A lower lymphocyte count was observed with correlating increased apoptosis and decreased lymphoid cellularity in males and females, a small thymus in males and lower thymus weight in males and females. In the two surviving females this finding was considered adverse, whereas in males it was non-adverse. In addition, one male showed adverse tubular degeneration in the kidney.
Non-adverse effects in males and/or females at 150/300/200 mg/kg/day consisted of a dose-dependent higher water consumption, lower total movement and ambulations in males, higher neutrophil, red blood cell, reticulocyte count, higher hemoglobulin and hematocrit concentration, lower lymphocyte count, higher urea, creatinine, (HDL) cholesterol, inorganic phosphate concentration and lower sodium and potassium concentration. Furthermore, a non-adverse lower spleen weight, vacuolation of the zona glomerulosa in the adrenal gland with higher adrenal gland weight, centrilobular hepatocellular hypertrophy in the liver with higher liver weight, tubular vacuolation in the kidney and an increased incidence and severity of hyaline droplets accumulation with minimal tubular degeneration in the kidney were observed in males and/or females.

No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., ophthalmoscopy and coagulation parameters).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

gastrointestinal tract

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 administered via gavage to 5 rats/sex/dose at 0, 10, 30, 90 mg/kg bw (male) or 0, 5, 15, 45 mg/kg bw (female) in corn oil for 4 weeks. Up to and including 90 or 45 mg/kg bw no mortality occured. The behavior of the rats were not influenced by the treatment up to and including 90 mg/kg (males) or 45 mg/kg (females). There were no clinical findings induced by treatment with the test item in rats dosed at low and mid dose. In all high dose rats (both sexes) treatment with the test item induced changed feces consistency (soft, pulpish). Related to body weight mid and high dose males consumed more water. High dose females had a slightly higher water intake in the last 2 weeks.The body weight gain, the food, clinical chemistry and organ weights were not affected up to and including 90 mg/kg in males and 45 mg/kg in females. The results from Functional Observational Battery (FOB) measurements in males and females receiving up to and including 90 mg/kg bw or 45 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 90 mg/kg or 45 mg/kg.

Histopathological examinations revealed minor inflammatory changes in the intestine (mainly cecum, mid and high dose, both sexes, one low dosed male). Possibly related to this a tendency towards minor unspecific histopathological changes were noted in mesenteric lymph nodes (all treated animals, no dose relation).

Therefore, under the condition of the present study, the NOAEL (no-observed-adverse-effect-level) for the test item after 4-week daily oral treatment by gavage is 10 mg/kg bw and 5 mg/kg bw for male and female rats.

In a repeated dose oral toxicty study (OECD TG 408), administration of the substance for at least 90 consecutive days by once daily oral gavage in Wistar Han rats at dose levels of 10/20, 40/80 and 150/300/200 mg/kg/day showed the following adverse effects at 150/300/200 mg/kg/day: mortality in females; clinical signs, body weight and food consumption effects consistent with effects on the digestive tract and a poor general health in males and females; histopathological changes consisting of abnormal watery content in the jejunum, ileum, and cecum of males and females, an attenuation in the cecum of males and females as well as mononuclear cell infiltrate in the cecum of males, lower lymphocyte count with correlating increased apoptosis and decreased lymphoid cellularity in the thymus of females with correlating lower thymus weight. As well as tubular degeneration in the kidney of one male. No adverse effects were observed at the other dose levels. Based on these results, the NOAEL was established to be 40 mg/kg/day in males and females.

Justification for classification or non-classification

Although the effective dose levels (≥ 30 mg/kg bw (males); ≥ 15 mg/kg bw (females)) are below the guidance values that assist in STOT RE classification, no toxicologically significant signs of systemic toxicity were evident following oral exposure up to and including 90 mg/kg in male and 45 mg/kg in female rats in a subacute study. Treatment-related findings, indicating reduced local tolerance of the test item, are restricted to the intestine (mainly cecum). In view of the clinical observations made (changed feces consistency), the intestine findings were considered indicative of acute to subacute enteritis and to be directly test item-related. Such morphological changes are potentially reversible and provide no evidence of marked organ dysfunction. Moreover the degree of functional impairment of the intestine through local effect of the test substance seems to be rather concentration- than dose-dependent. The onset of diarrhea one day after administration followed by rapid deterioration of the general condition which results rapidly in mortality is linked to a single dose around 500 mg/kg bw for the female rat, which is the most sensitive sex. This health hazard is already addressed by allocation to acute hazard category 4. The clinical signs (changed feces consistency) and onset of lethality were comparable between the acute oral toxicity study and the dose range finding study. The comparison indicates that responses appear to be predominated by local overdosing of the intestinal tissue. The degree of initial disturbance of the intestine determines whether and how the test substance is tolerated after repeated exposure. By reducing the daily dose significantly below 100 mg/kg bw and day the local concentration in the intestine reaches a level which has an impact on the intestinal tissue as shown in terminal pathology but the animals are largely unimpaired.

In the 90 -day study, similar effects of overdosing the intestinal tissue were observed at and above 150 mg/kg bw/day i.e. soft to liquid feces, abnormal watery content in the jejunum, ileum and cecum,

an attenuation in the cecum of males and females as well as mononuclear cell infiltrate in the cecum of males

The effects in thymus and spleen are considered adaptive responses as a consequence of overdosing the intestinal tissue and subsequent immunological reactions and do not result in adverse effects in these organs or organ function. Oberseved effects associated with adrenals and liver at 40 mg/kg bw/day are not considered adverse. Consequently, these are considered toxicollogically not relevant to assist in STOT-RE classification, and the effective dose levels ( ≥ 150 mg/kg bw) are above the guidance value of 100, as well.

Hence, from an expert perspective, the findings in the intestine which is considered to be a local response, do not reflect a true repeated exposure toxicity and classification for repeated dose toxicity according to Regulation (EC) No 1272/2008, Annex I is not required.