Benzene, 4-bromo-1-chloro-2-[(4-ethoxyphenyl)(phenylmethoxy)methyl]-

Endpoint summary

Description of key information

Oral:
One 7-day study on rats is available which was conducted according to OECD 407 using rats (Beerens-Heijnen, 2015). The no-observed-adverse-effect-level (NOAEL) for this study is 1000 mg/kg.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 to 15 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 9 weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing: Group housing of 3 animals per sex in Makrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

VEHICLE
- Concentration in vehicle: 30, 60, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 consecutive days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
150, 300, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

3 animals per sex per dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals.

Body weights: On Days 1, 4 and 7.

Food consumption: Over Days 1-4 and 4-7.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical laboratory investigations: Blood samples were collected as part of the necropsy procedure under anaesthesia using isoflurane. Immediately thereafter, the animals were examined post mortem, between 7.00 and 10.30 a.m. Animals were deprived of food overnight (for a maximum of 24
hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids. The following parameters were determined:
Haematology: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets;
Clotting Potential: Prothrombin time, Activated Partial thromboplastin time;
Clinical Biochemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals surviving to the end of the observation period were deeply anaesthetized using isoflurane.The animals were subsequently exsanguinated and subjected to a full post mortem examination. One animal treated at 150 mg/kg died at blood sampling and could not be exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- liver of all males and females of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4,
- all gross lesions.

Other examinations:

Organ weights: The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Ovaries, Brain Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver.

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

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

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

Details on results:

Mortality
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One male (no.5, receiving 150 mg/kg) died during blood sampling, which was considered to be an accidental death. At microscopic examination this animal showed moderate endocardial myxomatous changes and heart hypertrophy (correlating to the enlarged heart recorded at necropsy) which may have attributed to a higher sensitivity to the procedural handling and/or anesthesia during the necropsy. Based on the incidental nature and the absence of a dose response the mortality and the morphological changes were considered not toxicologically relevant.

Clinical signs
No clinical signs were noted during the observation period.

Body weights
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption
Food consumption before or after allowance for body weight was similar between treated and control animals.

Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Slight changes in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) occurred in males in a dose related distribution. However the changes were very slight and well within the range considered normal rats of this age and strain and therefore considered not toxicologically relevant.

Clinical biochemistry
Higher alanine transferase (ALAT) and aspartate transferase (ASAT) levels were noted in one male at 1000 mg/kg (1/3).
Other values in treated females and males showing a slight dose response distribution were considered to have arisen as a result of slightly high or low control values and/or were well within the range considered normal for rats of this age and strain and therefore considered not toxicologically significant.

Macroscopic examination
Necropsy did not reveal any toxicologically relevant alterations.
Incidental findings were noted among the dose groups. These findings are occasionally seen among rats used in these types of studies. Based on the low incidence and the absence of corresponding these changes were considered not toxicologically relevant.

Organ weights
Males at 1000 mg/kg showed higher absolute and relative liver weights (17 and 18% increase compared to mean control values, respectively).
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Microscopic examination
Minimal centrilobular hepatocellular hypertrophy in the liver was recorded in 1/3 males at 150 mg/kg, in 2/3 males at 300 mg/kg and in 2/3 males at 1000 mg/kg.
There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

Wistar rats were treated with the test substance for 7 consecutive days by daily oral gavage at dose levels of 150, 300 and 1000 mg/kg.
No treatment related changes were noted in clinical appearance, body weight, food consumption and macroscopic examination.
At microscopic examination centrilobular hepatocellular hypertrophy in the liver was recorded in males at all treatment levels at a minimal degree with dose related incidence. At the highest dose level this occurred along with a higher liver weight and higher liver enzymes such as alanine aminotransferase and aspartate aminotransferase in one male.
Considering the slight morphologic alterations, slight organ weight changes and the low incidence of changes in liver enzymes, these findings were considered treatment related however not to be adverse.
From the results presented in this report it was concluded that the test substance has potential for nonadverse liver toxicity when administered to rats by daily oral gavage for a period of up to 7 consecutive days up to 1000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Additional information

Oral:

One 7-day study on rats is available which was conducted according to OECD 407 using rats (Beerens-Heijnen, 2015).

No treatment related changes were noted in clinical appearance, body weight, food consumption and macroscopic examination.

At microscopic examination centrilobular hepatocellular hypertrophy in the liver was recorded in males at all treatment levels at a minimal degree with dose related incidence. At the highest dose level this occurred along with a higher liver weight and higher liver enzymes such as alanine aminotransferase and aspartate aminotransferase in one male.

Considering the slight morphologic alterations, slight organ weight changes and the low incidence of changes in liver enzymes, these findings were considered treatment related however not to be adverse.

From the results presented in this report it was concluded that the test substance has potential for nonadverse liver toxicity when administered to rats by daily oral gavage for a period of up to 7 consecutive days up to 1000 mg/kg.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study run to a method comparable with current guidelines and to GLP, with rats

Justification for classification or non-classification

Oral: No significant toxic effects in 7-day animal study.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.9.1 the substance is not classified for the specific target organ toxicity-repeated exposure endpoint.