5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile

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:

1996-02-22 to 1996-05-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-300-1

EC Name:

-

Cas Number:

138564-59-7

Molecular formula:

C12H9N3O2S

IUPAC Name:

5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

97.7%
Item code QA405K
Lilly lot no: 356MO1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 356M01
- Purity test date: 1995-11-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at ambient room temperature (<30 degrees Centigrade)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

(Cr1:CD BR) was the noted strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4 weeks old
- Weight at study initiation: males: 78-83g females: 55-60g
- Fasting period before study: None, the only fasting occured during urine sampling.
- Housing: One or two per cage in suspended polypropy Jene cages ( overall dimensions ca 42 x 27 x 20 cm) with stainless steel wire grid tops and bottoms. Beneath each cage was suspended a polypropylene tray containing absorbent paper. Tray paper was changed once each week during the study. Cages were changed at least once every 2 weeks during the study. Water bottles were changed once each week during the course of the study.
- Diet (e.g. ad libitum):Rat and Mouse (Modified) No. 1 SQC Expanded (Pelleted) Maintenance Diet supplied by Special Diets Services Limited, Stepfield, Witham, Essex was available to the animals ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:
None of the contaminants revealed by the analysis of diet and waterwere considered to have affected the outcome of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 35-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered once daily for 4 weeks orally via gavage using a steel dosing cannula at a constant dose volume of 10 ml dosing formulation/kg body weight

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance dissolved completely to the necessary concentration in this vehicle.
- Concentration in vehicle: 99.5% test material / 0.5% vehicle
- Amount of vehicle (if gavage): 10 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples were taken from each formulation containing test material and from Control formulation (containing vehicle only). Samples were taken from the formulations used on the first and twenty third day of treatment and analysed for concentration and homogeneity in the Inveresk Product Chemistry Laboratory

Duration of treatment / exposure:

28 d

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of an earlier range finding study that found an increase in liver weight in borth male and females treated with the 1000 mg test ingredient/kg bodyweight/day level of treatment.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: During each day at least once in the morning and once as late as practical.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

BODY WEIGHT: Yes
- Time schedule for examinations: Twice in the week prior to the start of treatment. Then, they were recorded daily to determine the dose volume to be administered.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The quantity of food consumed by each cage of animals was recorded twice weekly commencing one week before the start of treatment up until the end of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 and at the day of terminal nercropsies
- Anaesthetic used for blood collection: Yes, under light isofluorane anaesthesisa into plastic tubes
- Animals fasted: No
- How many animals: All
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 of treatment
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes
- The tissues detailed were processed and examined histologically from all animals in the Control and High dose groups, including premature decedents. In addition following initial examination from the animals in the Control and High dose groups, the kidneys from males in the Low and Intermediate groups were also examined.

Statistics:

Body weight, food consumption, water consumption, haematology, clinical chemistry, urinalysis and organ weight data were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt stabilise the variances. If the variances remained heterogeneous then a non-parametric test such as Kruskal-Wallis ANOVA was used. The statistical to analysis of water consumption was additional to that required by the protocol.

Organ weights were also analysed conditional on body weight by using analysis ofcovariance (ANCOVA).

Histological data were analysed using Fisher's Exact Probability test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations noted included orange staining of the cage and tray paper. This staining was noted for all animals treated with the test substance from generally the first day of treatment throughout the treatment period. Several animals, throughout all groups treated with the test substance, showed orange staining of the tail. Two animals treated at 1000 mg/kg/day (Animals 19 and 39) also showed orange staining on the thoracic and abdominal regions. The staining observed was considered to be related to the orange colour of the test material. Three animals treated at 1000 mg/kg/day (Animals 17, 20 and 38) showed signs of irregular breathing on Day 6 of treatment. In addition, Animal 20 also showed piloerection at this time.

Other occasional clinical observations were considered to be incidental and therefore not related to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

Ther were 3 premature deaths during the study. Of these one was considered to be related to treatment (Animal 19, a male treated at 1000 mg/kg/day), This animal showed substantial weight loss (38 g) from Day 21 to Day 23 of treatment. In addition, dark faeces, animal cold to touch, piloerection, eyes, ears and body pale were also noted . during this period. This animal was killed on Day 23 of treatment. The remaining premature decedents were killed due to conditions unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Although males treated at 1000 mg the test substance kg/day appeared to have a reduced body weight gain, compared to Controls, from Day 21 of treatment, this did not achieve statistical significance.
There were no differences in group mean body weight at any level of the test substance when compared to Control in the females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences among groups in food consumption. From Day 18-28 of treatment, an apparent reduction in group mean total food consumed in 1000 mg test substance/kg/day groups, compared to Control, but was not significant. During this period food consumed values at 100 and 10 mg of the test substance kg/day were similar to Control. For females, the group mean consumption during the treatment period was considered to be all similar at levels of the test substance.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Visual inspection of water consumption during the first week of treatment revealed an increase in water consumed for animals treated at 1000 mg the test substance kg/day. Thereafter, a gravimetric increase in water consumption was observed for both sexes at 1000 mg test substance/ kg/day when compared to Control. At 100 and 10 mg/kg test substance kg/day water consumption during the treatment period was essentially similar to Control.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in the haematology or coagulation parameters analysed were noted for males at any level of test substance, when compared
to Control.

A reduction in group mean Prothrombin time, (P< 0.01), was observed for females treated at 10 mg/kg/day. However, the lack of similar effects for females treated at 100 and 1000 mg/kg/day and the lack of significane of a minor decrease in this parameter indicated that this reduction may have been a result of the variation associated with small group sizes as opposed to a treatment related effect.

There were no to other significant differences in the haematology and coagulation poarameters for females when compared to Control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For Males. at 1000 mg test substance/kg/day the group mean Total bilirubin was increased compared to Control, attaining statistical significance of P<0.05. Although all of the total bilirubin individual values at 1000 mg test substance/kg/day were greater than Control individual values, indicating a treatment related effect, the increase in the group mean Total bilirubin at 1000 mg test substance/kg/day was considered to be exaggerated owing to the influence of Animal 17.

Animal 16, treated at 1000 mg test substance/kg/dayshowed a pattern of increase in both Urea and Creatinine, when compared to Control. A similar relationship however was not observed for other animals at this level.

No effects on the other clinical chemistry parameters were noted.

Females
An increase in the group mean Total protein at 1000 mg test substance/kg/day (P<0.001),was noted when compared to Control. Although a relationship to treatment was indicated, the extent of the effect could be considered be to biased by Animal 39. Therefore the significance of this effect is equivocal. No effects on the other clinical chemistry parameters were noted.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences among groups in urine specific gravity, volume or pH. Urinary volume was very slightly increased in both sexes treated at 1000 mg of the test substance /kg/day when compared to Control. A large number of organisms was noted in the small amount of urine from Animal 18, treated at 1000 mg/kg/day. The biological significance of this finding was unclear.

The urine of animals treated at 1000 mg/kg/day was generally generally considered to be darker/more concentrated colour when compared to Control. This finding was considered to be related to the formulated concentration of the coloured test material.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male
An increase in group mean liver weights (P<0.05), was noted at 1000 and 100 mg test substance/kg/day after covariance analysis when compared to Control.

An increase in group mean kidney weights was noted after absolute and covariance analysis at 1000 and 100 mg test substance/kg/day when compared to Control. These increases did not attain statistical significance.

A slight reduction in group mean adrenals weight was noted at 1000 mg test substance/kg/day after both absolute and covariance analysis when compared to Control. Due to the magnitude of this reduction being slight, the variability of this finding and the lack of any associated effects, it is unlikely to be attributable to administration of the test substance.

There were no notable intergroup differences in the remaining organ weights.

Females
An increase in group mean liver weights was noted after both absolute and covariance analysis at 1000 mg test substance/kg/day(P<0.01 andP<0.001 after absolute and covariance analysis respectively). The visible increase in group mean liver weights at 10 mg test substance/kg/day was considered to be within background variation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings for premature decedent Animal 19 included dark foci on all lung lobes, pituitary pale in colour, generally all organs pale and stomach distended with dark contents.

Two female animals, treated at 10 mg test substace/kg/day, showed dilated kidney(s}. Due to the lack of an effect at higher dose levels, these findings were considered to be coincidental.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histological findings for premature decedent Animal 19, treated at 1000 mg test substance/kg/day included centrilobular necrosis of the liver and increased haemopoiesis in the spleen. The dark faeces noted during the study and the dark stomach contents noted at necropsy were suggestive of haemorrhage in the upper gastrointestinal tract, however this was not determined histologically.

For animals surviving to the end of the treatment period, the following histological findings were made:
For males at 1000mg test substance/kg/day, mild papillary oedema in the kidneys (defined as loss of structural definition of the interstitum at the papillary tip with a myxomatous appearance) was noted in 2 males. Both of these cases were accompanied by interstitial papillary mineralisation. The finding was bilateral in one case and unilateral in the other. This finding was considered to be related to treatment.

There was no evidence of papillary oedema in the kidney of males treated at 100 or 100mg test substance/kg/day or in females treated at any level of the test substance.

Centrilobular hepatocellular hypertrophy of the liver was noted in on Control male, and 2 males treated at 1000mg test substance/kg/day. This finding was also observed in one female treated at 1000mf test substance/kg/day.

All other histological or necropsy findings were considered to be usual for animals of this strain and age and therefore not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

2

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The observed effects did not satisfy the criteria for classification in accordance with (EC) 1272/2008