6-(2-chloro-6-cyano-4-nitrophenylazo)-4-methoxy-3-[N-(methoxycarbonylmethyl)-N-(1-methoxycarbonylethyl)amino]acetanilide

Endpoint summary

Description of key information

50 mg/kg body weight/day of FAT 41'024/B was established as the no-observed-effect-level (NOEL) and, in view of the reversibility of minor kidney findings observed only in males, 200 mg/kg body weight/day is the no-observed-adverse-effect level (NOAEL).

Key value for chemical safety assessment

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:

key study

Study period:

11 February 1999 to 01 April 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP methodology followed and OECD Guideline 407 used to performed the experiment.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: HanIbm: WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf / Switzerland.
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 124 - 136 grams (mean 129 grams)
Females: 116 - 125 grams (mean 120 grams)
- Diet : ad libitum Pelleted standard Provimi Kliba 3433 (batch no. 33/98) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland)
- Water : Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): relative humidity between 40-70 %.
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/ 12 hours dark (light period between 06.00 and 18.00), music during the light period.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article formulations were prepared weekly.
FAT 41'024/B was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a glass rod and a magnetic stirrer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. Homogeneity of the test article in the vehicle
was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Identity: PEG 300
- Batch number: 395304/1 20199
- Expiry date December: 29, 2003
- Storage conditions: at room temperature (20°C) in original container.
- Safety precautions: Routine hygienic procedures (gloves, goggles, face mask):

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

RCC Project 692627: Determination of concentration, homogeneity and Stability of FAT 41024/A in PEG 400.
ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken during acclimati
zation. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The
analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a HPLC method supplied by the Sponsor.

Acclimatization / Pretest:
The mean concentrations of the homogeneity samples were found to be 95.4%, 104.7%, and 97.4% of the nominal concentrations of dose group 2 (10 mg/ml), dose group 3 (40 mg/ml), and dose group 4 (200 mg/ml), respectively. The individual concentrations varied in the range from -2% to +2% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Administration / Test:
The mean concentrations of the homogeneity samples were found to be 86.7%, 90.2%, and 90.1% of the nominal concentrations of dose group 2 (10 mg/ml), dose group 3 (40 mg/ml), and dose group 4 (200 mg/ml), respectively. The individual concentrations varied in the range from -3% to +6% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.
FAT 41'024/B was found to be stable in the vehicle at room temperature for two hours and for seven days.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test article as supplied.

Remarks:

Doses / Concentrations:
50 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test article as supplied.

Remarks:

Doses / Concentrations:
200 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test article as supplied.

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test article as supplied.

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females

Group 1: 0 mg/kg body weight
Group 2: 50 mg/kg body weight
Group 3: 200 mg/kg body weight
Group 4: 1000 mg/kg body weight

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

CAGE SIDE OBSERVATIONS: Yes
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3 (ca 1 and 3 hours after administration); as well as once daily on days 4- 28 (ca. 1 hour after administration), and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed
in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
The determination of the terminal body weight was performed immediately prior to necropsy.

FOOD CONSUMPTION :
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
The test was performed approximately 1h 30 hours after application.


CLINICAL LABORATORY INVESTIGATIONS
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.


HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Parameter examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, reticulocyte count, nucleated erythocyte, heinz body, methemoglobin, total leukocyte count, red blood cell morphology, thromboplastine time, ect..

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
Anticoagulant used during blood collection: Lithium heparin 15I.U./ml)
- Parameters examined: Glucose, urea, creatinine, uric acid, bilirubin total, cholesterol total, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total, globulin, albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-hour fasting period into a specimen vial.
Sampling dates: after 4 weeks: 18 March 1999 after 6 weeks: 01 April 1999 (post recovery)
- Animals fasted: Yes
- Parameters examined: Specific gravity, osmolality, color, appearance, pH, protein, glucise, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment, white blood cells, crystals (triple phosphate).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: grip strength / motor activity.

GRIP STRENGTH:
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY:
Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were randomized monitored during the fourth treatment week for a 60-minute period and the total activity of this
time period was recorded. Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.

Sacrifice and pathology:

NECROPSY
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
after 4 weeks: 18 March 1999
after 6 weeks: 01 April 1999 (post recovery)
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
Adrenal glands, Ovaries, Aorta, Pancreas, Bone (sternum, femur including joint), Pituitary gland, Bone marrow (femur), Brain (cerebrum,cerebellum, pons), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution),
Harderian gland (fixed in Davidson's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Prostate
gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data :
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied to macroscopical findings.
References :
- C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

VIABILITY/MORTALITY:
All animals survived until scheduled necropsy.

GENERAL CAGESIDE OBSERVATIONS (DAILY):
Dark discoloration of feces was observed in all animals treated at 200 and 1000 mg/kg/day from study day 2 onwards. This finding persisted for one day of the recovery period in the high dose group. This finding is considered to be an effect of treatment with a dye stuff as test article rather than a toxic effect caused by the treatment.

DETAILED CLINICAL OBSERVATIONS (WEEKLY):
Reduced activity was observed in male animals throughout all dose groups in week 2 and 3 of treatment. Because this finding was observed only in males and in all groups, it is considered to be incidental. Dark discoloration of feces in one male of the high dose group is considered to be an effect of treatment with a dye stuff rather than a toxic effect of treatment with test article.

FUNCTIONAL OBSERVATIONAL BATTERY (WEEK 4):
No clinical signs of toxicity were observed in any animal of all dose groups.

*Grip Strength:
Fore- and hindlimb grip strength was increased in males treated with 1000 mg/kg/day. The differences attained statistical significance. In the absence of similar findings in the females treated with 1000 mg/kg/day, this finding was considered to be incidental.

*Locomotor Activity:
No differences in overall locomotor activity were noted in test article-treated animals whencompared to the controls.

FOOD CONSUMPTION:
The mean daily food consumption of the test article-treated animals compared favorably with those of the control animals. Evaluation of the relative food consumption did not indicate test article-related differences between groups.

BODY WEIGHTS:
The mean body weight development of the test article-treated animals was considered to be unaffected when compared with those of the control animals.

HEMATOLOGY:
No test article-related differences in hematology parameters were noted when compared with the control values.

CLINICAL BIOCHEMISTRY:
Creatinine levels were higher in males and females treated with 1000 mg/kg/day. The differences attained statistical significance (p<0.01) when compared with control values. Although higher levels of creatinine were noted after the 14-day recovery period, the differences attained statistical significance (p<0.05) only in males. In view of the microscopical changes noted in the kidney, this finding was considered to be related to the treatment with the test article. All remaining differences to the control values were considered to be incidental.

URINALYSIS:
No test article-related differences in urinalysis parameters were noted when compared with the control values.

ORGAN WEIGHTS:
No test article-related differences in absolute or relative organ weights were noted when compared with the control values. The kidney to brain weight ratio of the females treated with 1000 mg/kg/day was increased with statistical significance (p<0.05) when compared with the control females. This finding was not noted in the males of this group and was therefore considered to be incidental.

MACROSCOPICAL FINDINGS:
A number of scattered macroscopic findings were recorded. These findings did not distinguish test article-treated rats from those of the control group and are commonly seen in animals of this age and strain.

MICROSCOPICAL FINDINGS
A number of findings were noted after 28 days' treatment. The following distinguished rats treated with 1000 mg/kg/day from those of the controls:
Kidneys
• Tubular degeneration/regeneration with cellular atypia (nuclear pleomorphism) was noted in three males and five females at 1000 mg/kg/day. In one of these males, this change was associated with a minimal papillary mineralization. The severity of the tubular degeneration/regeneration in rats
treated at 1000 mg/kg/day was slight to massive in males and slight to marked in females.
The minimal tubular degeneration/regeneration noted in one female treated at 200 mg/kg/day was similar to the type of tubular changes occurring rarely but spontaneously in control rats of this strain and age. Therefore, the change noted in this female was considered to be incidental.
A tubular single cell necrosis was noted in four males and five females at 1000 mg/kg/day. The severity of this findings was minimal to moderate
A minimally increased incidence and moderately increased severity of hyaline droplets were noted in male rats treated at 200 mg/kg/day or 1000
mg/kg/day.

Thymus
• At 1000 mg/kg/day, a minimally increased incidence of cortical atrophy was noted in males and a minimally increased incidence and severity were noted in females.
A number of findings were diagnosed in other organs and tissues examined at the termination of the treatment period. Their incidence, severity and morphologic appearance did not distinguish test article-treated rats from control rats.
Microscopic findings noted in rats treated with 1000 mg/kg/day after recovery included:
Kidneys
• Post necrotic tubular regeneration was noted in two males and four female rats. The severity of this finding was minimal slight in males and minimal to moderate in females.
• Interstitial fibrosis was noted in one male and two female rats. The severity of this finding was minimal to slight.
• Slightly increased incidence and severity of hyaline droplets was noted in males

Dose descriptor:

NOEL

Effect level:

50 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Dose descriptor:

NOAEL

Effect level:

200 other: mg/kg/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: In view of the reversibility of minor kidney findings observed only in males

Critical effects observed:

not specified

Conclusions:

Based on the results of this study, 50 mg/kg body weight/day of FAT 41'024/B was established as the no-observed-effect-level (NOEL) and, in view of the reversibility of minor kidney findings observed only in males, 200 mg/kg body weight/day is the no-observed-adverse-effectlevel (NOAEL).

Executive summary:

This experiment was performed according to the OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) and follows GLP methodology.

GENERAL

In this subacute toxicity study, FAT 41'024/B was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Detailed outside cage observations were performed during pretest and weeks 1-3. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

From the animals of the low and middle dose groups, kidneys and thymus sections were examined to establish a no-effect level.

*MORTALITY / VIABILITY

All rats survived until scheduled necropsy.

*CLINICAL SIGNS

No test article-related signs of toxicity were evident in any group.

*FUNCTIONAL OBSERVATIONAL BATTERY

No clinical signs were evident in any group.

*GRIP STRENGTH AND LOCOMOTOR ACTIVITY

No test article-related differences between groups were ascertained.

*FOOD CONSUMPTION AND BODY WEIGHT

The mean daily food consumption and the mean body weight development were unaffected by the test article, when compared with the control values.

CLINICAL LABORATORY INVESTIGATIONS

Hematology: No test article-related changes were noted when compared with the control data.

Clinical Biochemistry: Higher creatinine levels noted in the males and females treated with 1000 mg/kg/day were considered to be a result of the treatment with the test article.

Urinalysis: No test article-related changes were noted when compared with the control data.

ORGAN WEIGHTS:

No test article-related differences in absolute organ weights or relative organ weights were ascertained when compared with the control data.

MACROSCOPIC / MICROSCOPIC FINDINGS

No test article-related gross macroscopic changes were noted at any dose level. A number of microscopic findings were considered to distinguish test article-treated rats from control rats at the termination of the treatment period:

• Kidneys: At 1000 mg/kg/day, tubular degeneration/regeneration in three males and all females; tubular single cell necrosis in four males and all females, increased hyaline droplets in all males. At 200 mg/kg/day, increased hyaline droplets in all males.

• Thymus: Increased cortical atrophy in all males and all females treated with 1000 mg/kg/day.

All other microscopic findings noted at the end of treatment were considered to be incidental findings commonly occurring in rats of this strain and age.

After recovery, the following findings were noted in rats treated with 1000 mg/kg/day:

Kidneys: post-necrotic tubular regeneration in two males and four females; interstitial fibrosis in one male and two females; increased hyaline droplets in males.

All other microscopic findings noted at the end of recovery were considered to be incidental findings commonly occurring in rats of this strain and age.

Based on the results of this study, 50 mg/kg body weight/day of FAT 41'024/B was established as the no-observed-effect-level (NOEL) and, in view of the reversibility of minor kidney findings observed only in males, 200 mg/kg body weight/day is the no-observed-adverse-effectlevel (NOAEL).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is a Klimisch 1.

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

In this subacute toxicity study, FAT 41024/B was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 0, 50, 200 and 1000 mg/kg body weight/day for a period of 28 days.

In the macroscopic and microscopic observations this following effects were observed:

• Kidneys: At 1000 mg/kg/day, tubular degeneration/regeneration in three males and all females; tubular single cell necrosis in four males and all females, increased hyaline droplets in all males. At 200 mg/kg/day, increased hyaline droplets in all males.

• Thymus: Increased cortical atrophy in all males and all females treated with 1000 mg/kg/day.

All others microscopic findings noted at the end of treatment were considered to be incidental findings commonly occurring in rats of this strain and age.

After recovery, the following findings were noted in rats treated with 1000 mg/kg/day:

Kidneys:post-necrotic tubular regeneration in two males and four females; interstitial fibrosis in one male and two females; increased hyaline droplets in males.

All other microscopic findings noted at the end of recovery were considered to be incidental findings commonly occurring in rats of this strain and age.

In the clinical biochemistry investigations, higher creatinine levels were noted in the males and females treated with 1000 mg/kg/day and were considered to be a result of the treatment with the test article.

Based on the results of this study, 50 mg/kg body weight/day of FAT 41024/B was established as the no-observed-effect-level (NOEL) and, in view of the reversibility of minor kidney findings observed only in males, 200 mg/kg body weight/day is the no-observed-adverse-effect level (NOAEL).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only this study is available.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).