Reaction mass of [(2-substituted-4-nitroaryl)diazenyl]methyl(arylamino)-[(2-arylethyl)amino]pyridinecarbonitrile and [(2-substituted-4-nitroaryl)diazenyl]methyl(arylamino)-[(2-arylethyl)amino]pyridine-3-carbonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 04 January 2002 to 05 July 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed according to test guideline in compliance with GLP with FAT 41030 a structural analogue of FAT 41045. Both substances are very similar in their chemical structure and, as demonstrated, in a number of physicochemical properties. Therefore, this study is used for read-across. Information from this study provides further support for non-classification of FAT 41045 regarding acute toxicity based on read-across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Directive 96/54/ EC, B. 7. "Repeated dose (28 days) toxicity (oral)", September 30, 1996.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

FAT 41'030/A

IUPAC Name:

FAT 41'030/A

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

FAT 41'030/A

Test animals

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Test system: Rat, HanBrl:WIST (SPF)
Source: RCC Ltd Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
Number of animals: 30 males and 30 females
Age at delivery: 6 weeks
Body weight range at acclimatization: Males: 133 - 161 grams (mean 147 grams); Females: 112 - 131 grams (mean 122 grams)
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Conditions
Standard Laboratory Conditions:
Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a range for room temperature of 22±3 °C, and for relative humidity between 30-70 %. The animals were provided with a 12-hour artificial fluorescent light, 12-hour dark cycle. Music was played during the light period.
Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz / Switzerland).
Diet: Pelleted standard Provimi Kliba 3433 (batch no. 77/01 and 119/01) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) was available ad libitum.
Water: Community tap-water from Itingen was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

Daily dose levels: 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
Dose volume: 10 ml/kg body weight
Duration of recovery: 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg body weight
Basis:
other:

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

TEST ITEM PREPARATION
FAT 41'030/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

MORTALITY/VIABILITY: Observations for mortality/viability were recorded twice daily.
GENERAL CAGESIDE OBSERVATIONS (DAILY): The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28, and once daily during days 29-42 (recovery).
DETAILED CLINICAL OBSERVATIONS (WEEKLY): The animals were observed in their horne cages, outside their horne 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.
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.
BODY WEIGHTS: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, 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.

Sacrifice and pathology:

NECROPSY
Sacrifice: after 4 and 6 weeks
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

Other examinations:

CLINICAL LABORATORY INVESTIGATIONS
Blood and urine sampling: after 4 and 6 weeks
Blood samples for hematology and clinical chemistry were collected from all surviving animals under light isoflurane anesthesia. The animals were fasted 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 micro-hematocrit glass capillary tubes. Urine was collected into specimen vials during the 18-hour fasting period while animals were housed in metabolism cages.

Statistics:

The Dunnett-test (many to one t-test) based on a pooled variance estimate was used to analyze body weight, food consumption, organ weights and ratios if the data can be assumed to follow a normal distribution.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
Student's t-test was applied to grip strength and locomotor activity.
Fisher's exact-test was applied to the macroscopic findings.
Analyses of clinical laboratory data were carried out using the statistical routines contained in the NOVATOX System. Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to the Bartlett test.
Alternatively, if the variances were considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were then compared to the control group using Dunnett's test if the ANOVA was significant at the 5 % level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<=0.05).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals survived until scheduled sacrifice.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals survived until scheduled sacrifice.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by treatment with the test item.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

GENERAL CAGESIDE OBSERVATIONS (DAILY): Test item-related red-brown discoloration of feces was noted in males and females treated with 50, 200 and 1000 mg/kg/day, starting at day 5 at 50 mg/kg/day, at day 3 at 200 mg/kg/day and at day 2 at 1000 mg/kg/day. The finding of red-brown feces disappeared within the first four days of the recovery period in all males and females treated with 1000 mg/kg/day. This discoloration was considered to be a passive effect of the dyestuff rather than a sign of systemic toxicity. Additionally, red-brown bedding was noted in cages of males treated with 200 or 1000 mg/kg/day. This was due to the red-brown feces. All remaining findings (soft feces, salivation, tail bent, and a bleeding wound, which developed into scab on the shoulder, localized alopecia and bleeding wound at the end of the recovery period) were considered to be incidental.
DETAILED CLINICAL OBSERVATIONS (WEEKLY): Red and/or red-brown discoloration of feces occurred in some males treated with 50, 200 and 1000 mg/kg/day and in few females treated with 1000 mg/kg/day. As indicated above, this finding is of no toxicological relevance. A dose-related increase of miosis was noted during weeks 2 and/or 3 in males and females treated with 200 and 1000 mg/kg/day. This finding was transient in nature and does not represent an adverse effect. All other clinical signs (soft feces, tail bent, and a bleeding wound) were considered to be incidental.
FUNCTIONAL OBSERVATIONAL BATTERY: All findings (tail bent, salivation, and a wound) were considered to be incidental findings.
Body weight development was not affected by the test item at any dose level in males and females. The slight retardation in body weight gain in males treated with 50 mg/kg/day during weeks 3 and 4 of the treatment period was not seen at higher dose levels and therefore considered to be incidental. There was no difference in weight development between control and high dose group animals during the recovery period.
The mean food consumption was comparable at all dose levels during the treatment and recovery periods, except for a slightly lower food intake during the treatment period in males treated with 50 mg/kg/day. However, this difference was not reflected at higher dose levels and therefore considered to be incidental.
Hematology parameters were not affected by treatment with the test item. Minor effects seen in male groups were restricted to significantly lower counts (p<0.01) of white blood cells at 50 mg/kg/day and 1000 mg/kg/day, a significant decrease (p<0.05) in absolute neutrophils at 200 mg/kg/day and 1000 mg/kg/day, a significant decrease (p<0.05) in lymphocytes at 50 mg/kg/day, and a significant decrease (p<0.05) in the amount of relative methemoglobin at 200 mg/kg/day after 4 weeks treatment. All values remained within (or were only marginally outside) the normal range of the historical control data, and these changes were considered to be unrelated to the test item. Minor changes seen in female groups were limited to a significant increase (p<0.01) in relative reticulocytes at 200 mg/kg/day, significant effects (p<0.05) on the reticulocyte matrix index (a decrease at low f1uorescence and an increase at high f1uorescence) at 200 mg/kg/day after 4 weeks' treatment, and a significant reduction (p<0.05) in large unstained cells (Luc) in the at 1000 mg/kg/day after two weeks' recovery. All values remained within the normal range of the historical control data, and together with the absence of a dose response-relationship, the differences were considered to be unrelated to the test item.
Clinical chemistry parameters were not affected by treatment with the test item. Some minor changes seen in males were confined to significant changes (p<0.05) on electrolyte concentrations (sodium, potassium and chloride): The amount of potassium was increased at 200 mg/kg/day, and chloride- was increased at 50 mg/kg/day and 1000 mg/kg/day at treatment end, whereas sodium was decreased at 1000 mg/kg/day at the end of the recovery period. All values remained within the range of the historical control data, and together with the absence of a dose response-relationship, the differences were considered to be unrelated to the test item. The clinical chemistry parameters of the test item-treated females were similar to those of the controls.
After 4 weeks, absolute heart weights were elevated in females treated with 200 mg/kg/day and 1000 mg/kg/day (p<0.05), and the heart-to-brain weight ratio was increased (p<0.05) in the latter group. The absolute liver weight of females and the liver-to-brain weight ratio treated with 1000 mg/kg/day were also increased (p<0.05). Significantly increased absolute thymus weights (p<0.01), thymus-to-body weight (p<0.05) and thymus-to-brain weights (p<0.05) were also noted in females treated with 1000 mg/kg/day. In the absence of any correlating histopathological changes, the organ weight changes seen in females were considered to be incidental findings. Organ weights of all other females and all males were unaffected by treatment. After 6 weeks, organ weights of all animals sacrificed after the recovery period revealed no difference between control animals and those previously treated with the test item at 1000 mg/kg/day.
The macroscopic findings noted in this study were restricted to discoloration of the jejunum of one male treated with 1000 mg/kg/day and in the ileum and cecum of four males treated with 1000 mg/kg/day. Discoloration of the cecum was noted in three females treated with 1000 mg/kg/day. At recovery sacrifice, there were no test item-related gross findings.
The terminal sacrifice revealed no test item-related microscopic findings. No histopathological correlation was found to explain the discoloration of the intestinal mucosa observed at terminal sacrifice in several animals given 1000 mg/kg/day. At recovery sacrifice, there were no test item-related histopathological findings.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: due to discoloration of feces, ileum and caecum at higher doses

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

50 mg/kg body weight/day of FAT 41'030/A was established as the no-observed-effect-level (NOEL) due to discoloration of feces, ileum and caecum at higher doses, not considered adverse, and 1000 mg/kg body weight/day of FAT41'030/A as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

In the subacute toxicity study, FAT 41'030/A was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only. 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.

Oral administration of FAT 41'030/A to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days was generally well tolerated and did neither produce early mortality or treatment-related signs of toxicological relevance (daily, weekly or functional observational battery) nor effects upon fore- and hindlimb grip strength or locomotor activity. Furthermore, this test revealed no effects on food consumption and body weight development, and there were no treatment-related changes in hematology parameters and no macroscopical and microscopical findings related to the administration of the test item. Test item-related findings were generally restricted to dark-red discoloration of feces and bedding in males and females treated with 50, 200, and 1000 mg/kg/day, to discolorations present in the jejunum of one male and the ileum and caecum of four males and the caecum of three females treated with 1000 mg/kg/day. These discolorations were considered to be a passive effect of the dyestuff rather than a sign of systemic toxicity and in the absence of physiological or histopathological findings considered to be of no toxicological relevance.

Based on the results of this study, 50 mg/kg body weight/day of FAT 41'030/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg body weight/day of FAT41'030/A as the no-observed-adverse-effect-level (NOAEL).