4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 407, EEC Directive 84/449/EEC, Part B, B.7 and in accordance with the Principles of Good Laboratory Practice (GLP).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

MORTRACE SB CONC.

IUPAC Name:

MORTRACE SB CONC.

Constituent 2

Reference substance name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

IUPAC Name:

4-(4-Nitrophenylazo)-2,6-di- sec.butylphenol

Constituent 3

Reference substance name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

EC Number:

410-610-2

EC Name:

4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Cas Number:

111850-24-9

Molecular formula:

C20 H25 N3 O3

IUPAC Name:

2,6-bis(butan-2-yl)-4-[2-(4-nitrophenyl)diazen-1-yl]phenol

Test material form:

other: dark red-brown liquid

Details on test material:

- Name of test material (as cited in study report): Mortrace SB Conc.
- Physical state: dark red-brown liquid
- Analytical purity: concentrate
- Lot/batch No.: MR 26592 SBC
- Expiry date: June 03, 1993
- Stability under storage conditions: stable
- Stability in vehicle: stabile for at least 4 hours in corn oil
- Storage condition of test material: at room temperature protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 6 weeks
- Housing: group housed 5 rats/sex/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C
- Humidity (%): 55%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: In order to melt the test substance completely, it was placed for 3 days in a water bath at 60°C. Chemical analysis (HPLC) of’ the test substance after melting revealed that the test substance is stable for 3 days at 60°C.
The test substance was heated to maximally 60°C and subsequently weighed into a glass flask on an analytical balance and the vehicle (w/w) added. Adjustment was made for specific gravity of vehicle. Dosing solutions were prepared daily immediately prior to dosing. The homogeneity of the solution was maintained by heating the test substance formulations to maximally 60°C, and by the use of an electric and magnetic stirrer. Formulations were allowed to cool down to approximately 25°C before dosing. Homogeneity during treatment was maintained using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): recommended by various regulatory agencies

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations in corn oil were noted as being stable for at least 4 hours at all concentrations tested. In addition, these test substance preparations formed a homogeneous suspension. Analysis of the accuracy of dose preparations revealed values within the range of -3% to +1% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

28 days

Frequency of treatment:

single administration daily 7 days/week for 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg body weight
Basis:
other: 10 mg/l = 10.9 mg/g (nominal concentration)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg body weight
Basis:
other: 40 mg/l = 43.3 mg/g (nominal concentration)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
800 mg/kg body weight
Basis:
other: 160 mg/l = 172 mg/g (nominal concentration)

No. of animals per sex per dose:

5 males + 5 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration.
Animals receiving 1000 mg/kg/day showed low body weight gain and food consumption over the 5-day treatment period. Changes in clinical appearance no ed in these animals during the 5 days of treatment were rough coat and orange appearance of the skin and urine. On day 5, macroscopic changes among animals receiving 1000 mg/kg/day consisted of enlarged stomach, irregular surface of the forestomach and yellow discolouration of various tissues. In males and females of this group liver weights were noted as increased. Increased liver weights were also noted in females of the 200 mg/kg dose group and orange staining of various tissues was also noted in animals of the 200 and 50 mg/kg dose groups.
Based on these observations, dose levels for a study of 28 days duration were selected to be 0, 50, 200 and 800 mg/kg/day.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily which included time of onset, degree and duration of the clinical sign

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding termination, prior to overnight fasting

FOOD CONSUMPTION: Yes (weekly)

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined by means of an ophthalmoscope following instillation of tropicamide solution (5 mg/rnl) during the last week of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: standard hematological parameters

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: standard clinical chemistry parameters

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (including organ weights - adrenals, brain, heart, kidneys, liver, spleen, testes)
HISTOPATHOLOGY: Yes

Other examinations:

not applicable

Statistics:

Standard statistical methods were employed

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Changes in clinical appearance or behaviour that were considered to be toxicologically significant were swelling of the abdomen, lethargy and rough coat. In the 800 mg/kg group 2 females died during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Changes in clinical appearance or behaviour that were considered to be toxicologically significant were swelling of the abdomen, lethargy and rough coat. In the 800 mg/kg group 2 females died during the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain values of males receiving 800 mg/kg/day were low during the 4-week treatment period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment food consumption and relative food consumption were lower than controls in animals receiving 800 mg/kg/day

Food efficiency:

not examined

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

not examined

Description (incidence and severity):

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

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Pallor of the retina was noted in 1 out of 5 males and 1 out of 3 females receiving 800 mg/kg/day during week 4 of treatment. No other changes were observed in this group or in animals receiving 200 or 50 mg/kg/day.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes in red blood cell parameters were indicative of’ an anaemic response. There were no changes observed in red blood cell parameters of animals receiving 50 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALAT and ASAT - 800 mg/kg (increase in males, only); T.bil. - 800 mg/kg (marked increase in males); Creatinine - 800 mg/kg (decreased in males and females); ALP - 800 mg/kg (increase in females, only) and other effects (refer below).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver, kidney, adrenal and spleen weights were noted to be affected by treatment with MORTRACE SB CONC.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY - Changes in clinical appearance or behaviour that were considered to be toxicologically significant were swelling of the abdomen, lethargy and rough coat. Abdominal swelling was observed in all animals treated with 800 mg/kg/day during the last 3 weeks of treatment. In 2 males treated with 800 mg/kg/day, lethargy or rough coat were noted on days 27 and 28 of treatment. No such changes were observed among animals treated at 200 or 50 mg/kg/day.
Other marked changes noted in this study were excessive salivation and yellow or orange appearance of the skin and fur. Excessive salivation was noted periodically In animals receiving 50 or 200 mg/kg/day and during weeks 1 to 4 of treatment in animals receiving 800 mg/kg/day. The degree of salivation was also noted as being increased at higher dose levels. However, excessive salivation is commonly noted among rats of this age and strain following oral gavage administration and considered to be related to a bad taste or irritant effect of the test substance. Although a dose related increase was apparent in this study, this finding was considered not to represent a sign of systemic toxicity. The yellow or orange discolouration of the skin and fur was attributed to the staining properties of the test substance, which was a red-brown liquid. Therefore no toxicological significance was attached to this finding.
In the 800 mg/kg Group, 2 females died during the treatment period. One female (animal 39) was killed in extremis on day 3. The animal was noted with a poor physical condition, dragging its hind body around the cage. Animal 40 was found dead on day 9 without any associated clinical signs being observed.

BODY WEIGHT AND WEIGHT GAIN - Body weight and body weight gain values of males receiving 800 mg/kg/day were low during the 4-week treatment period. With the exception of’ day 15 all differences were statistically significant when compared to controls.
In contrast with males, females of the 800 mg/kg dose group showed increased body weights and body weight gain over the 4-week study period. In the case of body weight gain the difference between control and treated females was statistically significant at all weeks.
Body weight values for animals receiving 200 or 50 mg/kg/day were similar to those of controls, as were body weight gain values of these animals.

FOOD CONSUMPTION - During the first week of treatment food consumption and relative food consumption were lower than controls in animals receiving 800 mg/kg/day. However, animals of this group showed an Increased food consumption before and after allowance for body weight (during weeks 2 to 4) when compared to control. In females, these differences were more marked than in males. rood consumption and relative food consumption values by animals receiving 200 or 50 mg/kg/day were considered to remain in the same range as control animals.

OPHTHALMOSCOPIC EXAMINATION - Pallor of the retina was noted in 1 out of 5 males and 1 out of 3 females receiving 800 mg/kg/day during week 4 of treatment. No other changes were observed in this group or in animals receiving 200 or 50 mg/kg/day.

HAEMATOLOGY - Treatment related changes in red blood cell parameters were indicative of’ an anaemic response.
Low red blood cell counts were statistically significant in comparison with control animals in males and females receiving 200 of 800 mg/kg/day. Haemoglobin values measured in animals receiving 800 mg/kg/day were decreased with statistical significance when compared to control animals. A statistically significant increase, when compared to controls, of the mean corpuscular volume (MCV) and the mean corpuscular haemcglobin (MCH) was observed among animals receiving 200 or 800 mg/kg/day. In females receiving BOO mg/kg/day, a statistically significant decrease in mean corpuscular haemoglobin concentration (MCHC) and increase in red cell distribution width (ROW) and in males receiving 800 mg/kg/day a statistically significant decrease in mean corpuscular haemoglobin concentration was noted when comparing these values with those of corresponding controls. There were no changes observed in red blood cell parameters of animals receiving 50 mg/kg/day.

CLINICAL CHEMISTRY - The following changes in biochemistry parameters were considered to be of toxicological significance:
Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) were increased males receiving 800 mg/kg/day only. In males receiving 200 or 50 mg/kg/day or in treated females these values were comparable to control values.
A marked increase in total bilirubin was noted in males and females treated with 800 mg/kg/day. Total bilirubin in animals receiving 200 or 50 mg/kg/day was similar to control animals.
Creatinine values were noted as decreased in males and females receiving 800 mg/kg/day. Animals receiving 200 or 50 mg/kg/day showed creatinlne values in the same range as controls.
Alkaline phosphatase values in females receiving 800 mg/kg/day were Increased with statistical significance when compared to control females. There was no such change in males receiving 800 mg/kg/day or animals receiving 200 or 50 mg/kg/day.
A statistically significant increase In sodium, potassium and inorganic phosphate was noted in females receiving 800 mg/kg/day and a statistically significant increase in inorganic phosphate was noted in males receiving 800 mg/kg/day. Serum electrolytes in males and females receiving 200 or 50 mg/kg/day were comparable to control animals.
Small statistically significant differences in total protein and albumin remained in the range normally expected for rats of this age and strain and were therefore considered to have occurred by chance. Urea values in treated females showed a statistically significant decrease in comparison with controls. In males this change was not observed. Since the female control value is high for rats of this age and strain and there is no clear biological explanation for a decrease in serum urea, these changes were considered not to represent a toxic effect of the test substance.

ORGAN WEIGHTS - Liver, kidney, adrenal and spleen weights were noted to be affected by treatment with MORTRACE SB CONC.
Absolute and relative liver weights were increased with statistical significance, when compared to control animals, in males receiving 800 or .200 mg/kg/day and females receiving 800 mg/kg/day but not in animals receiving 50 mg/kg/day.
A statistically significant increase in kidney weight was noted, between animals receiving 800 mg/kg/day and control animals, before and after allowance for body weight. No such change was observed between control animals and animals treated at 200 or 50 mg/kg/day.
Adrenals, which are principally not dependant on body weight, were increased In males and females receiving 800 mg/kg/day when compared to controls. In animals of the 200 and 50 mg/kg dose group, adrenal weights remained in the range of control animals.
Increased spleen weights and relative spleen weights from animals receiving 800 mg/kg/day were statistically significant when compared to controls. Among animals receiving 200 mg/kg/day, this value was also considered to be increased although this difference did not attain a level of statistical significance when compared to controls. The spleens of animals receiving 50 mg/kg/day showed similar weights as control animals.
Statistically significant differences between relative brain or testes weights of males receiving 800 mg/kg/day and those of controls were considered to be of no toxicological significance, as these organs are principally not related to the body weight.

GROSS PATHOLOGY - The following macroscopic changes, noted at necropsy were considered to have arisen as a result of’ treatment with MORTRACE SB CONC.
A number of tissues, i.e. stomach, liver, spleen, adrenals, kidneys and caecum, were noted to be enlarged in aninals receiving 800 mg/kg/day. An enlarged spleen was also noted in males receiving 200 mg/kg/day. These changes were most frequently noted in the stomach, liver and spleen.
In 2 males of the 800 mg/kg dose group, the kidneys were noted with a granulated appearance of the surface, which in 1 male was accompanied with pale appearance of the cortex.
In the present study, various tissues were noted with a yellow-like appearance. All animals of all treatment groups showed yellow-like fat tissue. In animals receiving 200 or 800 mg/kg/day the stomach also had a yellow appearance. In addition, animals receiving 800 mg/kg/day showed yellow discolouration of the skin and in some cases of the genital region and pancreas. These changes were however, attributed to the staining properties of the test substance and considered not to be of toxicological significance. Other observed changes, such as small thymus, dark red appearance of the kidney, hernia diaphragmatica and watery cyst in the ovary occurred incidentally in animals receiving 800 mg/kg/day and were therefore considered to have arisen fortuitously.

HISTOPATHOLOGY: NON-NEOPLASTIC - Treatment-related changes were detected in the liver, spleen, kidneys and the adrenals.
In the liver, diffuse or centrilabular hepatocellular hypertrophy was observed in all male rats from the 200 and 800 mg/kg/day body weight groups and in most females from all treated groups. The severity of this finding increased with increasing dose levels.
In the spleen a dose related increased haemopoiesis was noted in all treatment groups. In addition, congestion of the spleen was observed In the 800 mg/kg dose group.
In the kidneys, several signs of tubular degeneration were noted in the 800 mg/kg Group. The degeneration consisted of cystic tubular dilation, proteinaceous cast formation and pigment accumulation in the tubular cytoplasm and increased tubular basophilia in males.
In the adrenals, increased cortical vacuolatlon was noted in both males and females in the 800 mg/kg dose group.
The enlargement and yellowish discolouration noted in the stomach were not accompanied by histopathological changes.

Effect levels

Dose descriptor:

NOEL

Remarks:

not identified

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on overall effects

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

From the results presented in this report a No Observed Effect Level (NOEL) could not be established. Repeated oral treatment with MORTRACE SB CONC. caused a slight anaernic response in animals treated with 200 or 800 mg/kg/day. In animals of the 50 mg/kg dose group an increased haemopoietic activity was apparent in the spleen. In addition, hepatocellular hypertrophy and associated lesions were noted in the liver of males and females receiving 200 or 800 mg/kg and females receiving a dose of 50 mg/kg of MORTRACE SB CONC. These liver changes may represent an increased activity in metabolising a xenobiotic agent. However, given the changes in serum enzyme levels, a slight hepatotoxic effect can not be ruled out.

Executive summary:

In this subacute 28 -day toxicity study, MORTRACE SB CONC. was administered once daily by gavage to SPF-bred Wistar rats. The study comprised of four groups, each group comprising of 5 male and 5 female rats/dose. All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and on the day before necropsy. During week 4 of treatment, both eyes of all animals were examined. On the day of termination blood was collected from each animal for clinical laboratory investigations. Subsequently, macroscopic observations and organ weights were recorded. A histopathological examination was performed on adrenals, heart, kidneys, liver, spleen, stomach and testes.

At 50 mg/kg/day, haemopoiesis was noted to be increased upon microscopic examination of the spleen of males as well as females. and microscopically observed liver cell hypertrophy was noted in females only.

At 200 mg/kg/day, a decrease of red blood cell numbers and increase of mean corpuscular volume and mean corpuscular haemoglobln values were noted in males and females, macroscopically enlarged spleen was observed in males only, increased liver weights were noted in males and increased spleen weights were noted in males and females and microscopically observed liver cell hypertrophy was noted in males and females.

At 800 mg/kg/day, changes in clinical appearance Included, swelling of the abdomen, lethargy and rough coat, one female was killed for humane reasons on day 3 and 1 female was found dead on day 9. During the 4 week treatment period, low body weights and body weight gain was noted in males, whereas females were noted with high body weights and body weight gain and food consumption (both absolute and relative) were low over the first week of treatment and high over the weeks thereafter. Pallor of the retina was seen at ophthalmoscopic examination in 1/5 males and 1/3 females. Slight anaemic changes, including, decreased red blood cell numbers and haemoglobin and increased mean corpuscular volume and mean corpuscular haemoglobin were noted in males and females. In females, a decrease in mean corpuscular haemoglobin concentration and increase in red cell distribution width and in males a decrease in mean corpuscular haemoglobin concentration was observed. Changes in clinical biochemistry parameters included an increase of total bilirubin and inorganic phosphate in males and females, an increase of alanine aminotransf’erase and aspartate eminotransferase in males only and an increase of alkaline phosphatase, sodium, potassium and inorganic phosphate in females only. Macroscopically enlarged appearance or the stomach, liver, spleen, adrenals, kidneys and caecum was noted in both males and females. Furthermore, a granulated appearance of the kidney surface and pale renal cortex was noted in 2/5 males. Increased liver, kidney, adrenal and spleen weights were noted in males as well as females. Microscopically observed changes were noted in males and females and consisted of increased hepatocellular hypertrophy in the liver, increased extrarnedullary haemopoiesis in the spleen, tubular degeneration in the kidneys and cortical vacuolatiori in the adrenals.

From the results presented in this report a No Observed Effect Level (NOEL) could not be established. Repeated oral treatment with MORTRACE SB CONC. caused a slight anaernic response in animals treated with 200 or 800 mg/kg/day. In animals of the 50 mg/kg dose group an increased haemopoietic activity was apparent in the spleen. In addition, hepatocellular hypertrophy and associated lesions were noted in the liver of males and females receiving 200 or 800 mg/kg and females receiving a dose of 50 mg/kg of MORTRACE SB CONC. These liver changes may represent an increased activity in metabolising a xenobiotic agent. However, given the changes in serum enzyme levels, a slight hepatotoxic effect can not be ruled out.