Dichloromethylbenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP; investigating in addition a2y-nephropathy

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Principles of method if other than guideline:

In addition the guideline and based on ECHA's request for this study kidney blocks were immunohistochemically stained for alpha 2u globulin. Kidney blocks were sectioned at 3 micrometers, mounted on charged glass microscope slides, and immunohistochemically stained for alpha 2u globulin.

Immunohistochemically stained sections were examined by light microscopy and observations were graded as follows:
Severity Grades (CDISC, 2013)

Unremarkable:
Tissue considered to be within normal limits, under the conditions of the study and considering the age, sex, and strain of the animal concerned.

Present:
A non-gradable histologic change for which no additional information would be gained by assigning a severity grade (e.g., cyst).
Grade 1 (Minimal):
The first (lowest) level of severity in an ordered list based on a five-level scale of minimal, mild, moderate, marked, and severe.
Grade 2 (Mild):
The second level of severity in an ordered list based on a five-level scale of minimal, mild, moderate, marked, and severe.
Grade 3 (Moderate):
The third level of severity in an ordered list based on a five-level scale of minimal, mild, moderate, marked, and severe.
Grade 4 (Marked):
The fourth level of severity in an ordered list based on a five-level scale of minimal, mild, moderate, marked, and severe.
Grade 5 (Severe):
The fifth (highest) level of severity in an ordered list based on a five-level scale of minimal, mild, moderate, marked, and severe.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dichloromethylbenzene

EC Number:

249-854-8

EC Name:

Dichloromethylbenzene

Cas Number:

29797-40-8

Molecular formula:

C7H6Cl2

IUPAC Name:

(dichloromethyl)benzene

Test material form:

liquid

Details on test material:

Identification: Dichloromethylbenzene
Appearance: Colourless – light yellow liquid
Test substance storage: In refrigerator (2-8°C) protected from light, container flushed with nitrogen
Molecular formula: C7H6Cl2
Molecular weight: 161.0
Volatile: vapour pressure: 65.3 Pa at 298 K
Specific gravity/density: 1.256 kg/L (20˚C)
Batch: CHT 301014

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rat: Crl:WI(Han) (outbred, SPF-Quality).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400, specific gravity 1.125.

Details on oral exposure:

Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for density specific gravity of the test item and Polyethylene glycol 400 vehicle. No correction was made for purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall a GC-FID method was developed and is experimentally demonstrated that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature protected from light. In addition, the lowest concentration used for males was considered stable in the freezer up to 61 days.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions, or 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

90 days. Animals were dosed up to the day prior to necropsy.

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Males: 0, 20, 100, 400
Basis:
other: mg/kg/day; nominal

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Females: 0, 100, 400, 800
Basis:
other: mg/kg/day; nominal

No. of animals per sex per dose:

10 males and 10 females per group (females were nulliparous and non-pregnant).

Control animals:

yes, concurrent vehicle

Details on study design:

Guidelines
The study was based on the following guidelines:
- EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008.
- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.
- OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

All phases of this study performed by the test facility were conducted in compliance with the following GLP regulations:
- OECD Principles of Good Laboratory Practice concerning Mutual Acceptance of Data in the Assessment of Chemicals, 26 November 1997 (C(97) 186 Final);
- EC Council Directive 2004 (2004/10/EC, February 11, 2004, Official Journal of February 20, 2004).

Positive control:

No applicable

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase this were also performed outside the home cage in a standard arena (collected under Project 509113 for logistic reasons and reported under Project 508051).
The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

Functional Observations: During Week 12-13 of treatment, the following tests were performed on the first 5 animals/sex/group of all dose groups:
- hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent)
- fore- and hind-limb grip strength were recorded as the mean of three measurements
- motor activity test (recording period: 1 hour under normal laboratory light conditions for individual animals, using a computerized monitoring system)
- Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Ophthalmoscopic
Examination (direct): Following instillation of tropicamide solution (5 mg/mL, Minims® Tropicamide 0.5% w/v) both eyes were examined by means of an ophthalmoscope:
at pretest : All animals (including spare animals)
at Week 13 : Groups 1 and 4

Body weights: Weekly.

Food consumption: Weekly.

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

Sacrifice and pathology:

Clinical Laboratory Investigations: Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. at the end of the treatment. 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 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 serum 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.

Urinalysis: Volume, Specific gravity (SPEC. GRAV), Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose
Nitrite, Sodium, Potassium, Calcium, Sediment (White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other).

Necropsy
Animals surviving to the scheduled day of necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex] (7 levels ) , Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Female mammary gland area (Femur including joint), Heart, Ileum, Jejunum, Kidneys, Larynx (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles, (Skeletal muscle), Skin, Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions; Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
Fixed in modified Davidson's solution: Epididymides, Eyes with optic nerve [if detectable] and Harderian gland, Testes.

Organ Weights: The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:

Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

Other examinations:

Kidneys slides of all males and 3 control females and 3 high dose females (first three of the dose group) needed for the imunohistopathology were prepared and stained or alpha 2u globulin.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortality occurred in females at 0 and 100 mg/kg/day and in males at 0, 20, 100 and 400 mg/kg/day during the study period. Two females at 400 mg/kg/day (an no 62 and 69, Day 28 and 84) and one female at 800 mg/kg/day (an no 77, Day 43) were sacrificed for ethical reasons.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality occurred in females at 0 and 100 mg/kg/day and in males at 0, 20, 100 and 400 mg/kg/day during the study period. Two females at 400 mg/kg/day (an no 62 and 69, Day 28 and 84) and one female at 800 mg/kg/day (an no 77, Day 43) were sacrificed for ethical reasons.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals considered unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight remained similar to the control level over the study period.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Subjective appraisal was maintained during the study, but no quantitative investigation introduced.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters were considered unaffected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Very slight changes were noted in clinical biochemistry parameters, such as higher alkaline phosphatase activity, higher bile acid level, lower bilirubine level and/or lower glucose level.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Slight observations on: lower pH in males at 400 mg, higher sodium and lower calcium concentrations and /or excretion.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver and kidney weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Major gross findings of the three sacrificed females consisted of emaciation (microscopically correlated to several atrophic organs/reduced size of organs) and (parts) of the gastro-intestinal tract distended with gas.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During histopathological examination morphological changes were noted in liver, forestomach and thyroid. alpha 2u globulin nephropathy was also observed in male rats.

Histopathological findings: neoplastic:

no effects observed

Details on results:

In life phase terminated. Data processing and evaluation ongoing.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Mortality

Two females at 400 mg/kg/day (an no 62 and 69, Day 28 and 84) and one female at 800 mg/kg/day (an no 77, Day 43) were sacrificed for ethical reasons. Prior to sacrifice, reduced body weights were noted and clinical signs such as hunched posture, piloerection, laboured respiration, rales, abdominal swelling, diarrhoea, dehydration and/or lean appearance. Major gross findings consisted of emaciation (microscopically correlated to several atrophic organs/reduced size of organs) and (parts) of the gastro-intestinal tract distended with gas.

Microscopic findings of note were present in the duodenum (erosion/ulceration and/or vacuolation of the enterocytes and/or granulocytic inflammation and/or hemorrhage, at minimal to slight degree), bone marrow (atrophy, up to moderate degree), thymus (atrophy, up to marked degree) and reproductive organs. These findings are most likely the consequence of emaciation but cannot explain the cause of morbidity. No cause of death could be determined histopathologically.

No mortality occurred in females at 0 and 100 mg/kg/day and in males at 0, 20, 100 and 400 mg/kg/day during the study period.

Clinical Signs

Among the surviving animals of all test item treated dose groups hunched posture and/or rales were noted. Furthermore, one female at 100 mg/kg/day showed (next to hunched posture) abdominal swelling, piloerection and lean appearance at the end of the observation period.

No findings were noted during the weekly arena observations in this study.

Salivation, seen after dosing among all test item treated animals and some control animals, was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Functional Observations

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.

Although the motor activity is slightly higher in males at 20 mg/kg/day, the motor activity was considered similar between treated and control groups, based on the absence of a clear dose response. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Body Weights

Body weights and body weight gain of treated animals considered unaffected by treatment.

Food Consumption

Food consumption before or after correction for body weight remained similar to the control level over the study period.

Ophthalmoscopic Examination

No ophthalmology findings were noted that were considered to be related to treatment.

The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Clinical Laboratory Investigations

Haematology

Haematological parameters were considered unaffected by treatment.

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these were considered to be small in nature and occurred in the absence of a dose-related trend.

Clinical Biochemistry

The following statistically significant changes in clinical biochemistry parameters were noted:

- Higher alkaline phosphatase (ALP) activity in males at 400 mg/kg/day and females at 800 mg/kg/day

- Lower bilirubin level in males at 100 and 400 mg/kg/day and females at 100, 400 and 800 mg/kg/day

- Lower glucose level in males at 400 mg/kg/day

- Higher bile acid level in females at 800 mg/kg/day (single females)

Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis

The following statistically significant changes were noted in urinalysis parameters:

- Lower pH in males at 400 mg/kg/day (only slightly affected in females at 800 mg/kg/day)

- Higher sodium concentration (mmol/L) and excretion (mmol/TPV) in males at 400 mg/kg/day and females at 400 and 800 mg/kg/day (not stat sign in females at 400 mg/kg/day)

- Lower calcium concentration (mmol/L) and/or excretion (mmol/TPV) in females at 400 and 800 mg/kg/day.

Any other statistically significant changes in urinalysis were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Pathology

Macroscopic Examination

The following test item-related macroscopic findings were present in males at 400 mg/kg/day:

- Greenish discoloration of the kidneys was observed in 3/10 males. The microscopic correlate was hyaline droplet accumulation.

- Enlargement of the liver was observed in 2/10 males. The microscopic correlate was hepatocellular hypertrophy.

No further test item related macroscopic findings were noted in males at 20 and 100 mg/kg/day and all females.

Any other statistically significant changes in macroscopic examination were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Organ Weights

Statistically significant higher liver weights were noted in the 400 mg/kg/day treated males (relative to body weight, +21%) and in the 800 mg/kg/day treated females (absolute and relative to body weight, +29% and +32%, respectively).

Statistically significant higher kidney weights were noted in the 400 mg/kg/day treated males (absolute and relative to body weight, +15% and +25% respectively). The small increases in relative kidney weights of the males at 100 mg/kg/day and females at 400 and 800 mg/kg/day were in line with the decrease in body weight.

Any other differences, including those that reached statistical significance were considered not to be dichloromethylbenzene-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Microscopic Examination

The test item-related kidney lesions in males consisted of:

- A dose-related increase in severity of hyaline droplet accumulation, consisting of 10/10 males at 100 mg/kg/day (8 slight, 2 moderate) and 10/10 males at 400 mg/kg/day (4 slight, 6 moderate) compared to 9/10 males at 0 mg/kg/day (3 minimal, 4 slight, 2 moderate), 10/10 males at 20 mg/kg/day (4 minimal, 5 slight, 1 moderate).

- An increase in incidence and severity of basophilic tubules in 7/10 males at 400 mg/kg/day (3 minimal, 4 slight) compared to incidences up to 4/10 and severities of minimal degrees at 0, 20 and 100 mg/kg/day.

- The presence of granular casts in 5/10 males at 400 mg/kg/day (3 minimal, 1 slight, 1 moderate).

The test item-related forestomach lesions in both sexes consisted of:

- Squamous cell hyperplasia in 5/10 males (5 minimal) at 400 mg/kg/day and in 8/9 females (6 minimal, 2 slight) at 800 mg/kg/day.

The test item-related liver lesions in both sexes consisted of:

- Hepatocellular hypertrophy in 1/10 males at 100 mg/kg/day (minimal) and 7/10 males at 400 mg/kg/day (6 minimal, 1 slight) and in 9/9 females at 800 mg/kg/day (7 minimal, 2 slight).

The test item-related thyroid glands lesions in both sexes consisted of:

- An increase in incidence of follicular cell hypertrophy in 8/10 males (2 minimal, 6 slight) at 100 mg/kg/day, in 8/10 males (3 minimal, 5 slight) and 4/8 females (4 minimal) at 400 mg/kg/day and in 7/9 females (7 minimal) at 800 mg/kg/day compared to 4/10 males (2 minimal, 2 slight) and 2/10 females (2 minimal) at 0 mg/kg/day, in 4/10 males (2 minimal, 2 slight) at 20 mg/kg/day and in 1/10 females (1 minimal) at 100 mg/kg/day.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Immunohistopathologic Examination

Administration of dichloromethylbenzene to male rats at dose levels of 100 and 400 mg/kg/day was associated with increased severity of globular deposits of alpha 2u globulin within the proximal tubules of the kidney, as visualized by immunohistochemistry.

Text Table 1. Incidence of Immunohistopathology Findings

Males Females

Dose Level (mg/kg/day): 0 20 100 400 0 100 400 800

Kidney (a)                      10 10 10 10 3 0 0 3

Alpha 2u Globulin IHC Positive, Globular Deposits

9 10 10 10 0 - - 0

Minimal                  3 3 1 0 - - - -

Mild                                    5               6 2 1 - - - -

Moderate                  1 1 3 4 - - - -

Marked                  0 0 4 5

Alpha 2u Globulin IHC Positive, Punctate Deposits

10 10 10 10 0 - - 0

Mild                                     4 3 3 1 - - - -

Moderate                   6 7 6 8 - - - -

Marked                   0 0 1 1 - - - -

Alpha 2u Globulin IHC Negative 0 0 0 0 3 - - 3

Increased globular staining deposits were observed in the cytoplasm of proximal tubular epithelial cells in the 100 and 400 mg/kg/day group males when compared with the control group. A slightly higher degree of punctate staining deposits was also observed in these groups. As was expected, immunohistochemical staining for alpha 2u globulin was negative in the kidney of all females examined.

Overall, administration of dichloromethylbenzene to male Crl:WI(Han) rats via oral gavage daily for at least 90 consecutive days at dose levels of 100 and 400 mg/kg/day was associated with increased severity of globular deposits of alpha 2u globulin within the proximal tubules of the kidney, as visualized by immunohistochemistry.

Applicant's summary and conclusion

Conclusions:

Based on the adverse test item-related morphologic alterations in the kidneys of males at 400 mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for Dichloromethylbenzene of 100 mg/kg/day was established for male rats.
For human risk assessment a No Observed Adverse Effect Level (NOAEL) of 400 mg/kg/day could be considered.

Executive summary:

Wistar rats were treated with Dichloromethylbenzene for at least 90 consecutive days by daily oral gavage administration at dose levels of 0, 20, 100, 400 mg/kg/day for males and 0, 100, 400 and 800 mg/kg/day for females.

During this study 3 females (2 at 400 mg/kg/day and 1 at 800 mg/kg/day) were sacrificed prematurely based on body weight loss and clinical signs, such as laboured respiration, rales, swelling of the abdomen, diarrhea, dehydration and/or lean appearance. Major gross findings consisted of emaciation and distension of (parts) of the gastro-intestinal tract with gas. No cause of death could be determined histopathologically for either of these three animals. Microscopic findings of note were present in the duodenum (erosion/ulceration and/or vacuolation of the enterocytes and/or granulocytic inflammation and/or hemorrhage), bone marrow (atrophy), thymus (atrophy) and reproductive organs (disturbed cycle pattern). These findings are most likely the consequence of emaciation but cannot explain the cause of morbidity. The histopathological findings were not observed in any of the rats who survived their scheduled study period of at least 90 days of treatment, and therefore regarded to be non-related to treatment.

However since the cause of death could not be determined, a treatment related mortality could be doubted.

Among all the surviving animals treated with dichloromethylbenzene, the clinical observations revealed a dose dependent incidence of rales and /or hunched posture during the observation period. One female at 100 mg/kg/day showed signs of poor health at the end of the observation period.

For all surviving animals in this study, no toxicologically significant changes were noted in functional observations, ophthalmoscopy, body weight, food consumption and haematology.

During histopathological examination morphological changes were noted in kidney, liver, forestomach and thyroid.

In the kidney, hyaline droplet accumulation was noted at increased severity in 100 and 400 mg/kg/day males. This microscopic finding could be correlated to higher kidney weights of males at 400 mg/kg/day (absolute and relative to body weight, +15% and +25% respectively) and the macroscopic finding in these males. In absence of hyaline droplets in females, the hyaline droplet accumulation was considered to represent alpha 2u globulin, a normal protein in male rats, not present in female rats or in higher mammals, including man (Sahota et al., 2013). This was confirmed by immunohistochemistry.

At 400 mg/kg/day, additional test item-related histopathological lesions were noted, consisting of an increased incidence of tubular basophilia and the presence of granular casts at the transition outer/inner stripe of the outer medulla. These findings are regarded to be related to hyaline droplet accumulation and this spectrum of findings is sometimes diagnosed as alpha 2u globulin nephropathy (Hard, G.C., 2008). In the male rats treated at 400 mg/kg/day, this combination of kidney findings was considered to be adverse in nature for the rat.

In the liver, hepatocellular hypertrophy was noted at increased incidences in males at 400 mg/kg/day and females at 800 mg/kg/day and could be related to higher liver weights in females (absolute and/or relative to body weight, 29% and 32% respectively). Very slight changes were noted in clinical biochemistry parameters, such as higher alkaline phosphatase activity, higher bile acid level, lower bilirubine level and/or lower glucose level. The microscopic hepatocellular hypertrophy was recorded at low severities (up to slight) and was not accompanied by other test item-related microscopic liver findings. Based on the magnitude of increase in liver weight of females at 800 mg/kg/day (both absolute and relative 29% and 31% respectively), the hepatocellular hypertrophy was regarded as adverse in rats.

The liver effects in males and females at lower dose levels are not considered adverse.

In the forestomach, squamous cell hyperplasia were observed at low degrees in the highest dose groups (400 mg/kg/day in males and 800 mg/kg/day in females), without additional test item-related morphologic changes. This was regarded to represent an adaptive protective response to the direct effect of test item by oral gavage. Therefore, this finding was not considered to be adverse.

In the thyroid, the follicular cell hypertrophy was noted at low degrees in all dose groups, including the control (males up to slight incidence and females minimal incidence). Slightly increased incidence in males 100 and 400 mg/kg and females at 400 and 800 mg/kg were without additional changes in the thyroid glands or other endocrine organs. Therefore, these thyroid changes are regarded as non-adverse.

Based on the adverse test item-related morphologic alterations in the kidneys of males at 400 mg/kg/day, a No Observed Adverse Effect Level (NOAEL) for Dichloromethylbenzene of 100 mg/kg/day was established for male rats.

For human risk assessment a No Observed Adverse Effect Level (NOAEL) of 400 mg/kg/day could be considered.

For females, adverse test item-related alterations were only noted at 800 mg/kg/day as morphological changes in the liver. Based on these results a No Observed Adverse Effect Level (NOAEL) for Dichloromethylbenzene of 800 mg/kg/day could be considered for female animals. However, based on the moribund condition of one female at 800 mg/kg/day and two females at 400 mg/kg/day, a lower No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day could be considered for female animals in this study. A treatment related mortality cannot be fully excluded, but the mortality occurred in the absence of a clear dose related distribution and the histopathological findings of these animals are most likely the consequence of emaciation and were not observed in any of the rats surviving their scheduled study period, Therefore a relation to treatment can be doubted.