Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Sep. 19, 1988 to Jan. 17, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
according to U.S. Fed.Reg., 21 CFR Part 58, GLP Regulation
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol, Chlororange
- TSIN: COS 198
- Substance type: Pure active substance
- Physical state: Orange- yellow fine grained powder
- Stability under test conditions: Not reported
- Storage condition of test material: Normal room temperature, in darkness

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wella AG ; n°Batch COS 198
- Expiration date of the lot/batch: No data
- Purity test date: No data
- Purity : 100% (HPLC at 254 nm)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, protected from light
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
strain Crl: Wi/br (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Wistar derived SPF-Albino rats of the Crl: Wi/Br strain
- Source: Charles River Wiga GmbH Sulzfeld
- Age at study initiation: about 6 weeks
- Weight at study initiation: Males: 122 g - 169 g; Females: 116 g - 146 g
- Fasting period before study: Not reported
- Housing: Animals were single-housed in Makrolon cages
- Bedding: "Altromin Laboreinstreu" produced from pure soft wood, dried, disdusted and sterilized at 180°C and renewed weekly.
- Diet: "Ssniff R" pelleted diet (standard laboratory rat diet) produced by Ssniff Spezialdiaten GmbH; ad libitum
- Water: Tap water from Makrolon drinking bottles was given with free access.Consumption was controlled visually daily.
- Acclimation period: 10 days
- Animal health: On arrival, animals were examined for health status. Only animals in good health were used for this study. Before initiating the study a second health examination was carried out to assure that all animals were in the best condition for this investigation.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 ± 1.5 °C
- Relative humidity: 55% ± 10% (recorded by thermohygrographs)
- Air changes: 16 times per hour (SPF quality grade)
- Photoperiod: 12 hrs artificial light (120 lux) daily from (7 a.m. - 7 p.m.)

EXPERIMENT INITIATION DATE: Sep. 19, 1988

EXPERIMENT COMPLETION DATE: Jan. 17, 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance was suspended in Carboxy methyl cellulose (CMC). Suspensions were prepared fresh daily using a magnetic stirrer and protected from light in aluminum foil covered beakers. Stirring was continued until all animals were treated in order to maintain homogeneity of the test article.

APPLICATION OF THE TEST SUBSTANCE: Test substance was administered by oral gavage once daily at the same time in the morning. The control animals received the vehicle (0.5 % CMC) only. The dosage was individually adapted to the weight of the animals which was determined weekly. The relation between the administered volume and body weight remained constant at 10 mL per kg.

VEHICLE
Carboxy methyl cellulose (CMC)
- Concentration in vehicle: 0.1, 0.3 and 0.9% test substance in CMC for dose levels of 10, 30 and 90 mg/kg bw, respectively.
- Lot/batch no.: Batch# 09 52 995
- Purity : contains 5% (w/w) water

Details on dosage was provided in table 1 under ‘Any other information on materials and methods incl. Tables’
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the concentration used for treatment were analyzed by photometric analysis 4 times throughout the study. Analyses were performed at the IBR-Bio analytical Centre. Concentration checks found samples of the dosing suspension to be good in accordance with the nominal values. Details on results of analyses were presented in the appendix of the study report.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily for seven days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
Dose / conc.:
90 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
Main study: 15 animals/sex/dose group
Recovery period (satellite group): 10 animals/sex in control and 90 mg/kg dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used for this study were chosen according to the results of a dose range finding test
- Rationale for animal assignment: Animals were assigned to treatment groups using computerized randomization with regard to similar group mean body weights.
- Rationale for selection of animal: The rat is the standard experimental animal of choice. Because of its large amount of physiological and historical background data it is especially suitable for toxicological investigations.
- Justification for the Route of Administration: The oral route was chosen to increase plasma concentrations.
- Rationale for selecting satellite groups: For recovery observations, satellite groups were chosen for the control and high dose group (10 animals/sex/group) These animals were deprived from treatment after 13 weeks.
- Post-exposure recovery period in satellite groups: 4 weeks (subsequent to the 13 weeks of treatment)
Positive control:
None

Examinations

Observations and examinations performed and frequency:
MORTALITIES/VIABILITY: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily;
Animals were observed for sensory and motor behavior, hair coat, body orifices, urine and fecal excretions, general health status and dose responses.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to initiation (0-value), after 6 weeks and 3 months in 10 males and 10 females of each group and in the recovery animals at termination, the following evaluations were conducted:
1. Ophthalmoscopic examinations: Cornea and episcleral vessels, anterior chamber, pupil, lens and retina (if possible) were examined using an ophthalmoscope, fitted with switchable lens systems and a slit lamp.
2. Hearing: Tested by simple noise production
3. Reflex – examinations: Testing was performed (modified IRWIN-Screen) with special regard to awareness, emotion, coordination and autonomic functions.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (individually)

FOOD CONSUMPTION AND COMPOUND INTAKE: Weekly.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: Not reported

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 6 and 13 treatment weeks in all test groups and after 17 weeks in the recovery groups
- Anaesthetic used for blood collection: Not reported
- Animals fasted: Not reported
- How many animals: 10 males and 10 females of each test group.
- Collection site: From the retrobulbar venous plexus by means of non heparinised hematocrit tubules.
- Parameters checked included: Erythrocytes (RBC), Leukocytes (WBC), Thrombocytes, Reticulocytes (inclusion bodies), Hemoglobin, Hematocrit, MCV, MCH and MCHC, Differential blood count, Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 6 and 13 treatment weeks in all test groups and after 17 weeks in the recovery groups
- Animals fasted: Not reported
- How many animals: 10 males and 10 females of each test group.
- Parameters checked included: a) Glucose, triglyceride and cholestrol; b) Protein (total) and albumin; c) Bilirubin (total), Creatinine, Urea Nitrogen (BUN) and Uric acid; d) Calcium, Chloride, Inorganic Phosphorus, Iron, Potassium, Sodium and Na/K ratio by statistical evaluation; e) Alanine Aminotransferase (ALT/GPT) (EC 2.6.1.1), Alkaline Phosphatase (EC 3.1.3.1), Aspartate Aminotransferase (AST/GOT) (EC 2.6.1.2), Creatine Kinase (CK) (EC 2.7.3.2), Glutamat Dehydrogenase (GLDH) (EC 1.4.1.3), AST/ALT ratio by statistical evaluation

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to first treatment, after 6 and 13 treatment weeks in all test groups and after 17 weeks in the recovery groups
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not reported
- Parameters checked include: Color, protein, pH-value, glucose, bilirubin, urobilinogen, blood, nitrate, ketones, sediment, spec. weight
Samples were collected from 5 males and 5 females of each test group in the frame of blood sampling.
Performance of Laboratory Examinations: The investigations of hematology, clinical chemistry parameters and urinalyses were carried out at IBR-Laboratory. Details on methods used are provided in the study report.
Sacrifice and pathology:
SACRIFICE: All animals were sacrificed at the end of their experimental phase by CO2 asphyxiation

GROSS PATHOLOGY: Yes
Macroscopic examination of the cranial, thoracic, abdominal and pelvic cavity was carried out.

ORGAN WEIGHTS: The following organ weights were determined and recorded from all male and/or female animals of all groups animals necropsied at termination of treatment: Cerebrum with cerebellum, adrenal (left and right), pituitary, spleen, heart, prostate gland, liver, testis (left and right) with Epididymis, kidney (left and right), ovary (left and right), lung (prior to perfusion), uterus, thymus. Paired organs were weighed as pairs.

HISTOPATHOLOGY: Yes
Organ Fixation and Tissue Preparation: All dissected organs were preserved in buffered 10 % formaldehyde solution except for kidneys, pancreas, testes with epididymides and an additional sample of the liver, which were fixed in Bouin's solution. Eyes were fixed in Zenker's solution.
Procedure: Samples from all tissues were removed from all males or females, respectively, and blocks and slides were prepared from 10 males and 10 females of the control group and the high dose group (90 mg/kg) and examined histopathologically by board certified veterinary pathologists: Skin (untreated), cecum, mammary gland, colon, salivary gland, rectum, trachea, lymph node (cervicalis), esophagus, lymph node (mesenterialis), thryoid (2x), ovary (2x)/testis + epididymis (2x), parathryoid (1x), seminal vesicle, thymus, prostate/uterus and vagina, heart, urinary bladder, lung with main stem bronchi, sciatic nerve, aorta, skeletal muscle, liver, bone (sternum), spleen, bone (femur), pancreas, marrow (sternum), kidney (2x), marrow (femur), adrenal (2x), eye with N. opticus (2x), stomach, cerebrum with brain stem, duodenum, cerebellum, jejunum, spinal cord, illeum, pituitary, lacrimal gland (2x), tongue.

All tissues were stained with hematoxylin and eosin. From heart, liver and adrenals additionally frozen sections were prepared and stained with Sudan III for fat content judgements. Lungs were perfused slightly with formaldehyde solution.
Statistics:
Statistical analyses of data were performed separately for male and female animals.
Methods used:
- Body weight changes and food consumption were evaluated by a one-resp. two-factorial analysis of variance. To compare the group mean values, the method of "Scheffe" was employed. Food conversion ratio: (Weight changes per week/food consumption per week) x 100 was calculated
- The organ weights were evaluated by analysis of co-variance. The comparison of the mean values was performed by the method of "Scheffe" for the analysis of co-variance.
- Clinical chemistry and hematology were analysed by ‘Analysis of variance’ with subsequent Scheffe test.
Significance levels: p<0.05 slightly significant; p<0.01 significant; p<0.001 highly significant

Staistical analyses of data were performed seperately for male and female animals. Further details are provided in the study report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All animals of the control and treatment groups showed a healthy habit and normal motor and sensoric activity throughout the entire test period. Urine of the mid and high dose animals were orange discolored (test substance induced discoloration) during the entire study and the abdominal hair coat was stained accordingly. About 10 out of 50 animals of the high dose group showed slight diarrhoea.
No test substance related findings were observed in reflex examinations, ophthalmoscopic investigations and hearing tests.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain rates of the high dose group males were significantly reduced during the second half of the study.The reduced male body weight was nearly compensated by significantly increased weight gain rates during the recovery period.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Mean food consumption was comparable in all test groups of males or females over the entire study period.
Food efficiency:
not examined
Description (incidence and severity):
Food Conversion Ratio: No significant differences were seen in the food conversion ratio during this study.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Description (incidence and severity):
No test substance related findings were observed in reflex examinations, ophthalmoscopic investigations and hearing tests.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological investigation revealed intergroup differences in prothrombin time, leukocyte and reticulocyte count, which occurred sporadically within the limits of the normal range.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical chemistry values indicated several statistical significant intergroup differences. These changes, however, were not persistent, not dose-related and randomly sex-related. They were therefore considered to be due to coincidence
Urinalysis findings:
no effects observed
Description (incidence and severity):
These parameters revealed not test substance-relatated differences between control and treatment animals, except for an orange-red (test compound) induced discoloration in mid- a,d high dosed animals.
Behaviour (functional findings):
not examined
Description (incidence and severity):
No test substance related findings were observed in reflex examinations, ophthalmoscopic investigations and hearing tests.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weight changes of the liver, lung and thymus of males and females in the high dose and recovery group were not consistent. However, increases in kidney weight were found in males and females both of the high dose and recovery .
See tables in "any other informations"
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance related findings were seen at terminal autopsy in any animal from the experimental groups. Findings were those which are commonly seen in this strain of rat.
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
The histomorphological examinations revealed no treatment related organ alterations in high-dose group animals. Therefore no investigations were made from low-dosed and recovery animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
90 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

The coincidental findings observed in a small number of control and test animals were proteinaceous content in the urinary bladder, dilatation of the colon and rectum, hydronephrosis in the kidney, hydrometra, infiltration of neutrophilic granulocytes in the uterus, impairment of spermiogenesis, intertubular edema of the testes, hyperplasia of Leydig cells, purulent prostatitis, alveolar pulmonary edema and suppurative pneumonia.Gonads were examined for possible effects

Kidney - Organ Weights

Terminal Sacrifice, week 13 - Male

 Group Not corrected   Percent  Corrected  Percent
 I - XM  3.288 100 3.212  100 
  SD  0.435   0.376   
 II - XM  3.237 98.4  3.154  98.2 
   SD  0.454   0.269   
 III - XM  3.279 99.7  3.279  102.1 
   SD  0.409   0.214   
 IV - XM  3.311 100.7  3.471  108.1 
   SD  0.310   0.281   

Terminal Sacrifice, week 13 - Female

 Group Not corrected   Percent  Corrected  Percent
 I - XM 1.871  100  1.877  100 
  SD  0.263   0.221   
 II - XM  1.928 103  1.898  101.1 
   SD  0.227   0.169   
 III - XM  2.057 110  2.021  107.7 
   SD  0.261   0.214   
 IV - XM 2.036  108.8  2.095  111.6 
   SD  0.221   0.181   

Recovery Sacrifice, week 17 - Male

 

 Group Not corrected   Percent  Corrected  Percent
 I - XM 3.203 100 3.187 100 
  SD  0.231   0.229   
 IV - XM 3.624 113.1  3.641 114.2 
   SD  0.249   0.237   

Recovery Sacrifice, week 17 - Female

 

 Group Not corrected   Percent  Corrected  Percent
 I - XM 1.904 100 1.906 100 
  SD  0.173   0.172   
 IV - XM 1.985 104.2  1.983 104.1 
   SD  0.217   0.198   

Applicant's summary and conclusion

Conclusions:
Chlororange (2-amino-6-chloro-4-nitrophenol) when administered by oral gavage daily for 13 weeks to male and female Crl: Wi/Br strain Wistar rats at dose levels of 0, 10, 30 and 90 mg/kg bw/day, revealed a no observed adverse effect level (NOAEL) at 30 mg/kg bw/day for male and female rats.
Executive summary:

The repeated dose oral toxicity study of Chlororange (2-amino-6-chloro-4-nitrophenol) was performed by following methods similar to the OECD Guideline 408 (Repeated Dose 90 -Day Oral Toxicity in Rodents).

Male and female Wistar derived SPF-Albino rats of the Crl: Wi/Br strain (source: Charles River Wiga GmbHSulzfeld) were used in this study. The body weight range of animals was 122 - 169 g (males) and 116 - 146 g (females). Animals were individually housed in Makrolon cages and maintained under controlled environmental conditions (temperature: 21.5 ± 1.5°C, humidity: 55% ± 10%, air changes: 16 times per hour and 12 hours light /12 hours dark). Ssniff R pelleted diet and tap water were provided ad libitum. Male and female rats were assigned to 4 groups (15 animals/sex/group;for control and 90 mg/kg dose groups an additional 10 animals/sex formed recovery groups) and were treated for 90 days as follows:

Group 1: Control (0.5% Carboxymethylcellulose)

Group 2: 10 mg/kg bw/day

Group 3: 30 mg/kg bw/day

Group 4: 90 mg/kg bw/day

During the in-life phase, rats were observed for mortality, clinical signs body weight and food consumption. Ophthalmoscopic examinations, hearing tests and reflex-examinations were carried out priorto initiation (0 -value), after 6 weeks, after 3 months and in the recovery animals.

After 6 and 13 treatment weeks in all test groups and after 17 weeks in the recovery groups, blood and urine samples were collected for hematology, clinical chemistry, and urinalysis. All animals were sacrificed at the end of their experimental phase by CO2 asphyxiation.

No animals died during the study. No adverse clinical signs, except for an orange-red discoloration of urines in the mid and high dose animals, were observed. Body weight gain rates were significantly reduced in high-dose males during the second half of the study. The reduced male body weight was nearly compensated by significantly increased weight gain rates during the recovery period. No significant differences were seen in food consumption andfood conversion ratios.

Hematological investigation revealed intergroup differences in prothrombin time, leukocyte and reticulocyte count, which occurred sporadically within the limits of the normal range. Clinical chemistry values indicated several statistical significant intergroup differences. These changes, however, were not persistent, not dose-related and randomly sex-related and were therefore considered to be coincidental.

No test substance-related findings were seen at terminal autopsy. Organ weight changes of the liver, lung and thymus of males and females in the high dose and recovery groups were not consistent. However, increases in kidney weight were found in males and females both of the high dose

and recovery groups.The histomorphological examinations revealed no treatment related organ alterations in high-dose group animals.

Based on above, treatment related adverse effects were observed at the highest tested dose level i.e.90 mg/kg bw/day (reduced body weight and increased kidney weights).

In conclusion, Chlororange (2-amino-6-chloro-4-nitrophenol) when administered by oral gavage daily for 13 weeks to male and female Crl: Wi/Br strain Wistar rats at dose levels of 0, 10, 30 and 90 mg/kg bw/day, revealed a no observed adverse effect level (NOAEL) at 30 mg/kg bw/day for male and female rats.

This repeated dose oral (3 month) toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 408 method.