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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUCLID4 Test substance: other TS: 1,2-dichloro-4-nitrobenzene, purity 99 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 6 week
- Housing: in groups of 5 animals
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To:

Administration / exposure

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

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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 4, 20 or 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: sacrifice on day 29
Positive control:
not relevant

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / 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, coleur, 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 macrosscopically vilible abnormalities . the autopsy included macroscopic ecamination 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, testesm 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)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
RS-Freetext: OBSERVATIONS: No death occurred throughout the study unspecific signs of intoxication:
100 mg-group, m/f:
irregular respiration, stilted gain,
>= 20 mg/kg bw/day, m/f:
increased salivation; all groups:
body weight gain was not impaired, food consumption unaffected,
>=20 mg/kg bw/day(m/f):
slightly increased water intake (not significant, not dose dependant) URINALYSIS: dark yellow discolouration of urine: m, from 20 mg/kg bw/day onwards; f, at 100 mg/kg bw/day; pH-Value: f, 100 mg-group, significantly decreased: pH=5.4 versus pH=6.1 (control) HEMATOLOGY: 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 CHEMISTRY: male: significant increase in sodium- and chlorid-ions when compared to
concurrent control, which were, however, within the historicol 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) ORGAN WEIGHTS: 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); PATHOLOGY: 100 mg/kg bw, spleen: 5/5 m and 5/5 f, dark discolouration spleen: 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)

Effect levels

Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (actual dose received)
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RS-Freetext:
OBSERVATIONS:
No death occurred throughout the study
unspecific signs of intoxication: 100 mg-group, m/f: irregular respiration, stilted gain, >= 20 mg/kg bw/day, m/f: increased salivation;
all groups: body weight gain was not impaired, food consumption unaffected, >=20 mg/kg bw/day(m/f): slightly increased water intake (not significant, not dose dependant)
URINALYSIS:
dark yellow discolouration of urine:
m, from 20 mg/kg bw/day onwards; f, at 100 mg/kg bw/day;
pH-Value:
f, 100 mg-group, significantly decreased: pH=5.4 versus pH=6.1 (control)
HEMATOLOGY:
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 CHEMISTRY:
male: significant increase in sodium- and chlorid-ions when compared to concurrent control, which were, however, within the historicol 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)
ORGAN WEIGHTS:
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);
PATHOLOGY:
100 mg/kg bw, spleen: 5/5 m and 5/5 f, dark discolouration spleen:
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)

Applicant's summary and conclusion

Executive summary:

According to OECD TG 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).