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Description of key information

In a study according to OECD Guideline 407 male and female Sprague-Dawley rats received 0, 4, 20, 100 mg/kg bw/d dissolved in sesame oil by gavage for 28 days. The NOAEL was 4 mg/kg bw/d based on changes in red blood cell parameters, on hemolytic anemia with microscopic changes in the spleen and increase in relative liver weights in the next higher dose and increase in relative spleen weight at 100 mg/kg bw/d (Hoechst AG 1993).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
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
- Age at study initiation: approximately 6 week
- Housing: in groups of 5 animals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
the test compound was suspended homogenousely in the vehicle by means of a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0 - 2 % (w/v)
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
the determination of the concentration was performed by photometrical detection after HPLC seperation on a reversed phase column
Duration of treatment / exposure:
28 days
Frequency of treatment:
once per day
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
4 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary dose range finding study
Positive control:
not relevant
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly throughout the study

FOOD CONSUMPTION
twice weekly

WATER CONSUMPTION
once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
At the termination of the study, hematological examinations were performed on all animals without previous withdrawl of food. Blood samples were taken from the retrobarbital venous plexus in narcosis. In order to prevent systematic errors, blood sampling was conducted in a randomized order.
hematological parameters:
erythrocytes, hemoglobin, hematocritm mean cellular volumd (MCV), Mean cellular hemoglobin (MCH), Mean cellular hemoglobin concentration(MCHC), Leucocyte count, thrombocyte count, differential leucocyte count, and red cell morphology, reticulocyte count, Heinz bodies, coagulation time

CLINICAL CHEMISTRY: Yes
After blood sampling for hematology the animals were killed by cutting of the vena cava cranialis in deep narcosis and exanguinated. In order to prevent systematic errors, exanguination was conducted in a randomized order.
Parameters:
sodium, potassium, inorganic phosphorus, uric acid, bilirubin total, creatinene, serum-glucose, urea-nitrogen, calcium, chloride, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, total protein, albumin

URINALYSIS: Yes
urinalysis was performed on all animals a few days before termination of the study. for this purpose, the urine was collected by using metabolism cages (Overnight from day 26 to day 27)
Parameters:
appearance, colour, pH-value, hemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, specific weight, sediment, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After exanguination all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

organ weights (calculation of organ to body weight ratio): heart, lung, liver, kidneys, spleen, ovaries, testes, epididymides, adrenals, brain, thymus

HISTOPATHOLOGY: Yes
Heart, liver, kidneys, adrenals, spleen, lung, brain, thymus, ovaries, testes, epididymides, trachea, stomach, jejunum, colon, urinary bladder, uterus, prostata, seminal vesicles, skeletal muscles, N. ischiadicus, femur with bone marrow, spinal cord (cervical, thoracal and lumbal), lymph nodes (dervical, and iliacal)
Other examinations:
no further data
Statistics:
---One way analyses of variance with sequentially rejective multiple comparison
---One way analyses of variance based on ranks with equentially rejective multiple comparison
---Trend Test analyses for non-neoplastic lesions (ARMITAGE)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100 mg-group, m/f: irregular respiration, stilted gain
>= 20 mg/kg bw/day, m/f: increased salivation
Mortality:
mortality observed, treatment-related
Description (incidence):
No death occurred throughout the study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
all groups: body weight gain was not impaired
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
food consumption unaffected
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
>=20 mg/kg bw/day(m/f): slightly increased water intake (not significant, not dose dependent)
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
4 mg/kg bw/day:
male: significant decrease in erythrocyte counts (6.98 10E12/l versus 7.63 10E12/L [historical control: 6.34-8.95 10E/L])

20 mg/kg bw/day:
male: significant decrease in erythrocyte counts (6.87 10E12/L versus 7.63 10E12/L of concurrent control) and haematocrit (0.42 UNITY versus 0.46 UNITY of control)
female: significant increase in MCV (62 10E-15L versus 58 10E-15L)

100 mg/kg bw/day:
male: decrease in erythrocyte values (6.34 10E12/L versus 7.63 10E12/L of control), decrease in haematocrit (0.42/0.39 UNITY versus 0.46/0.41 UNITY of control), decrease in haemoglobin (140/131 g/L versus 149/138 g/L of control), increase in MCV values 67/66 10E-15L versus 60/58 10E-15L of control), reticulocyte counts (0.077/0.080 UNITY versus 0.011/0.008 UNITY of control)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
male: significant increase in sodium- and chlorid-ions when compared to concurrent control, which were, however, within the historical control values of this strain:
control//4/20/100 mg/kg bw/day//historical control range:
Sodium: 135//139/142//142//132-149 mmol/L, Chlorid: 98// 101/102/102//95-106 mmol/L
20 mg/kg bw:
increase in alk. phosphatase (f: 261 U/L versus 175 U/L of control)
100 mg/kg bw/day:
increase in urea values (m/f: 8.44/8.17 mmol/L versus 5.62/6.80 mmol/L of control) )m [indicative for an impaired kidney function; however, no histopathological correlates were found]
increase in ALAT(GPT) (m: 54 U/L versus 44 U/L), increase in alk. phosphatase (f: 267 U/L versus 175 U/L of control)
Endocrine findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
dark yellow discolouration of urine: male: from 20 mg/kg bw/day onwards; female: at 100 mg/kg bw/day;
pH-Value: female: at 100 mg-group significantly decreased: pH=5.4 versus pH=6.1 (control)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
absolut organ weights not affected, rel. organ weights:
20 mg/kg bw
increased relative liver weight (m: 4.545 % versus 4.036 % of control)
100 mg/kg bw/day
increase in rel. liver weight (m/f: 4.571/4815 % versus 4.036/3.926 % of control)
increased rel. spleen weight (m/f: 0.321/0.330 % versus 0.189/0.234 % of control)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw, spleen: 5/5 m and 5/5 f dark discoloration spleen
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
m(grading)-f(grading), low, mid, high dose versus control:
congestion
low: 0/5(0) - 0/5(0), mid: 3/5(slight) - 5/5(slight), high: 5/5(moderate) - 5/5(moderate) versus contr.: 0/5 - 0/5
increased extramedullary haematopoiesis:
low: 2/5(minimal) - 2/5(minimal), mid: 5/5(minimal) - 5/5(slight), high: 5/5(slight) - 5/5(slight) versus contr.: 3/5(minimal) - 0/5
increase in haemosiderosis:
low: 0/5 - 5/5(slight), mid: 5/5(minimal) - 5/5(marked), high: 5/5(moderate) -5/5(marked) versus contr.: 0/5 - 5/5(mild)
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on changes in red blood cell parameters, on hemolytic anemia with microscopic changes in the spleen and increase in relative liver weights in the next higher dose and increase in relative spleen weight at 100 mg/kg bw/d
Critical effects observed:
not specified
Conclusions:
The NOAEL was 4 mg/kg bw/d based on changes in red blood cell parameters, on hemolytic anemia with microscopic changes in the spleen.
Executive summary:

In a study according to OECD Guideline 407 male and female Sprague-Dawley rats received 0, 4, 20, 100 mg/kg bw/d dissolved in sesame oil by gavage for 28 days. The NOAEL was 4 mg/kg bw/d based on changes in red blood cell parameters, on hemolytic anemia with microscopic changes in the spleen and increase in relative liver weights in the next higher dose and increase in relative spleen weight at 100 mg/kg bw/d (Hoechst AG 1993).

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

1,2-Dichloro-4-nitrobenzene shall be registrated according to Regulation (EC) 1907/2008 (REACH) Article 18 (transported isolated intermediate) and no studies are required according to REACH Annex VII.

Justification for classification or non-classification

1,2-Dichloro-4-nitrobenzene shall be registrated according to Regulation (EC) 1907/2008 (REACH) Article 18 (transported isolated intermediate, > 1000 tpa) and no studies are required according to REACH Annex VII.