Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

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:

07 February 2012 to 06 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20 % of the sex mean.
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 – 23.3 ºC
- Humidity (%): 41 - 61 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

On one day during acclimatization period the lights were turned off for 20 minutes, due to a technical error. Deviation was of a slight and incidental nature, and was therefore considered not to have adversely affected the study outcome.

IN-LIFE DATES: From: 07 February 2012 to 06 March 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. No correction wasmade for the purity of the test substance.

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

On Day 3 and 11 the use of magnetic stirrer was not recorded in the study daybook. The use of the magnetic stirrer is standard in these kinds of studies. Furthermore, the formulation was considered to be homogeneous to visually acceptable levels by the bio-technicians prior to dosing. Based on the low incidence of this deviation and the accepted homogeneity to visually acceptable levels, not recording the use of magnetic stirrer is considered to have no adverse influence on the study outcome.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the pretreatment and/or treatment phase, according to a validated method (NOTOX project 497818). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110 % of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10 %. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels are based on results of a10-day dose range finding study with FAT 40854/A (NOTOX project 497845).
- Selection of animals for selected measurements: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

On Days 8, 15 and 21 the weekly clinical observations (outside the home cage in a standard arena) were performed prior to dosing instead of after dosing. Since no peak period of clinical signs occurred in this study and no clinical signs of toxicity occurred. The time of this weekly observation has no adverse influence on the outcome of the study.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes / No / No data
- All animals were fasated overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines (+ deviations)

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group. All tests were two-sided and in all cases p <0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

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:

no effects observed

Urinalysis findings:

not examined

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period. Red faeces was noted in the highest dose group of males and females. This is considered to have arisen as a result of treatment (red test substance). Incidental findings in single animals that were noted included salivation, scabs and/or a wound. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN:
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION:
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY:
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes at 100 and 300 were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These findings included: Slightly lower mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in males at 300 mg/kg and higher neutrophils (%) and lower lymphocytes (%) in females at 300 mg/kg and lower basophils (%) in females at 100 mg/kg. The increases of neutrophil counts with concurrently reduced lymphocyte counts, which were noted in females without a treatment related distribution, could be considered to be a secondary non-specific response to stress and to be of no toxicological significance.

CLINICAL CHEMISTRY:
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Lower bilirubin levels were noted in males and females at 1000 mg/kg, which remained within the range considered normal for rats of this age and strain. A lower level bilirubin is considered to be not relevant in toxicological terms. Furthermore, any statistically significant changes in females at 100 and 300 mg/kg were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These findings included higher level of aspartate aminotransferase (ASAT) and chloride and lower level of inorganic phosphate.

NEUROBEHAVIOUR:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. A lower total movement count was noted in females at 1000 mg/kg and a higher total movement count was noted in males at 100 mg/kg. These changes occurred in the absence of a clear dose related distribution, supportive clinical signs and similar results in the opposite sex. Therefore the variation in motor activity is considered to be of no toxicological significance. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS:
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. Statistically significant changes were noted in seminal vesicles weights (males 300 mg/kg), relative adrenal weight (males 100 mg/kg) and relative spleen weight (females 300 mg/kg) between treated and control animals. These findings were considered not to be a sign of toxicity as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

GROSS PATHOLOGY:
Necropsy did not reveal any toxicologically relevant alterations. Reddish discolouration of the glandular mucosa of the stomach was noted in one male at 1000 mg/kg. This finding is considered to be caused by pigment of the test substance, corresponding with the microscopic finding in the stomach. The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included scab formation, foci in the stomach, discolouration of the thymus and fluid in the uterus.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the kidney a dose-dependent increased incidence and severity was noted in hyaline droplets (up to moderate) in males at 300 and 1000 mg/kg/day. In this study the occurrence of cortical hyaline droplets was accompanied by a slightly increased incidence and severity of corticomedullary tubular basophilia in males at 300 and 1000 mg/kg/day compared to control. (Group 1: 3/5 grade 1, Group 2: 1/5 grade 1, Group 3: 1/5 grade 1 and 1/5 grade 2, and Group 4: 1/5 grade 1 and 2/5 grade 2). Red/brown pigment was noted in the mucosa of the stomach (minimal to slight) in half of the animals of Group 4. In addition red/brown pigment was noted in the Peyer’s patches (minimal) in half of the animals of Group 4. The nature and severity of these microscopic findings were not considered to be toxicologically relevant and not adverse. All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for FAT 41043/A of at least 1000 mg/kg was established.