S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-660-1

EC Name:

-

Cas Number:

104797-47-9

Molecular formula:

C14H10N4O3S3 (Hill formula) C14H10N4O3S3 (CAS Formula)

IUPAC Name:

(Z)-[1-(2-amino-1,3-thiazol-4-yl)-2-(1,3-benzothiazol-2-ylsulfanyl)-2-oxoethylidene]amino acetate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: T15-AE
Chemical name: syn-2-(2-Aminothiazol-4-yl)-2-(methylcarbonyloximino) aceticacid-mercaptobenzthiazolylester.
Supplier: Sponsor.
Batch No.: 36429032.
Molecular formula: C14 H10 N4 O3 S3.
CAS No.: 104797-47-9
Appearance: Yellow powder.
Purity: 92.9 % (HPLC)
Conditions of storage: In the deep freezer, in the dark; may be used under light.
Stability at conditions of storage: 12 months.
Stability at room temperature: Ca. 2 weeks.
Expiry date: 8 April 2005 according to the Certificate of Analysis, but extended to October 2005 based on investigations by the sponsor.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman GmbH. 33176 Borchen. Germany.
- Age at first dosing: Ca. 7 w.
- Weight at first dosing: mean of ca. 140 g for males; mean of ca. 110 g for females.
- Housing: Single caging in Makrolon cages
- Bedding material: Aspen wood chips
- Diet: Altromin 1324 forte, gamma-irradiated, ad libitum
- Water: Tap water, acidified to pH 3 with HCl, ad libitum
- Acclimation period: 6 d.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21.9
- Humidity (%): ca. 53.7
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.1 %

Details on oral exposure:

The test substance was administered as a suspension in 0.1 % Na-CMC. Dose volume: 10 mL/kg bw.
Preparations of the test substance were made freshly every day shortly before the administration to the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test substance solutions
Determinations of the test substance in some selected preparations for stability, concentration and homogeneity were performed by HPLC using the following conditions:

An HPLC-method was developed and validated to determine the test substance in solutions.
HPLC-System: Thermo Separation Instruments Thermoquest; SpectraSystem UV6000LP Photodiode Array detector with 50 mm flowcell
Software:ChromQuest Chromatography Data System version 2.51
Column: Hy Purity C18 3 µm 2.1 *100 mm, SN 0244049Y; Lot No. 6375 Thermo Electron Co
Column temperature: 35 °C
Autosampler temperature: 15 °C
Flow: 0.5 mL/min
Injection volume: 10 µL
Detection: UV-absorption at 218 nm
Mobile phase: 60 % buffer pH 7; 40 % acetonitrile LiChrosolv
Calibration was performed with standard solutions in the appropriate concentration range. A regression line was calculated from these results.
The concentrations of the test substance were determined by relating the areas under the curve to standards.

The following analyses were performed:
Stability: The stability of the test substance in suspension was investigated on Day 7 for the low and the high dose. Analyses were performed immediately after the preparation and 4 hours later. The stability was considered to be sufficient, if the loss in concentration was less than 5 % within 4 hours.
Conditions for the solutions before analysis: Ambient temperature, ambient light.

Concentration and homogeneity: Investigations were performed on Day 7. Three samples of 2 mL each were taken from preparations of low, mid and high dose. Concentration was considered as sufficient, if the mean of the test substance concentrations of the 3 samples taken was within ± 10 % of the target concentration. Homogeneity was considered to be sufficient, if the test substance concentrations of the 3 samples taken did not exceed ± 10 % of their mean.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week.
Day 1 was the first day of dosing the test substance.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 - 100 - 316 - 1000 mg/kg bw (groups K-A-B-C).
Basis:
other:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 - 1000 mg/kg bw (groups KS - CS)
Basis:
other: nominal, for the satellite recovery groups.

No. of animals per sex per dose:

5 males and 5 females in each treated groups and in the control group.
Additional 5 males and 5 females in each of the recovery group (1000 mg/kg bw) and the recovery control group.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based on a dose range finding study.
Control and high dosed recovery groups were observed for further 14 days without test substance administration.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

Daily observations
All animals were carefully observed for general signs and the health status once a day and additionally checked once a day for viability.

Detailed clinical observations
More detailed clinical observations were made while the animals were either in the standard arena or in the hand of the animal technician on:
• Days -1, 6, 13, 20, 27: all animals of all groups.
• Days 34, 41: all animals of the recovery groups.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

Functional observations
Assessment of the behaviour, of the motor activities, and of the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) was performed outside the home cage in a standard arena. The eyelid and auricular reflexes (tactile and proprioceptive) were tested by slightly touching of cornea and the interior of a pinna with a nylon cord of approximately 0.6 mm diameter.
Acoustic reactivity was tested by reaction to a sudden moderate sound (clapping of the hands).
Visual reactivity was derived from the reaction to a white sheet, brought near to the animal without any physical contact.
For the testing of the righting reflex the animals were held between the front- and hindlegs, turned on their backs and dropped from a height of 30 cm into the bedding material of the standard arena. A landing on all four legs is a normal reaction. This and a measurement of the forelimb grip strength were conducted in all animals on Day 27, and on Day41 for animals of the recovery groups.

Body weight
Body weight was determined of all animals once a week, and on Days 35 and 43 in all animals of the recovery groups.

Feed consumption
Determined per cage in weekly intervals in all animals.

Sacrifice and pathology:

Necropsy
Animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death on
• Day 29: groups K, A, B and C.
• Day 43: groups KS and CS.

The following organs / tissues were taken from all animals and fixed in Bouin's solution:
• gross lesions, tissue masses or tumours • ovaries • adrenal glands • pancreas* • aorta* • pituitary * • brain • prostate • caecum* • rectum* • coagulating glands * • salivary glands * • colon • seminal vesicles * • duodenum • sciatic nerve • epididymes * • skeletal muscle (thigh) • eyes* • skin and mammary gland * • femur bone with joint * • spinal cord (cervical, thoracal, lumbar) • heart • spleen • ileum • sternum • jejunum • stomach • kidneys • testes • lacrimal glands (extraorbital) * • thymus • liver • thyroid and parathyroid glands • lungs • trachea • lymph nodes (mandibular, mesenteric) • urinary bladder • oesophagus • uterus
*:not included in the routine histopathology of groups K and C.

Organ weights
Weights of the following organs were determined of all animals:
• adrenal glands • kidneys (both together) • brain • spleen • epididymis (both together) • testes (both together) • heart • thymus • liver
Relative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.

Histopathology
Groups K and C: Histopathological examination was performed in all animals of all fixed organs or tissues listed above, except for those labelled with a "*".
Groups KS, A, B and CS: No histopathological examination was performed, as there was no indication for test substance related effects found in group C.

Appropriate parts of organs or tissues were embedded in paraffin. Sections of about 5 µm were stained with haematoxylin and eosin. Evaluation of slides was performed using a light microscope Leica-DMRB. To describe the severity of lesions, the following grades were applied, if appropriate: minimal (1), mild (2), moderate (3), marked (4), severe (5). The term "focal" together with a higher degree of severity also stands for "multifocal ".

Other examinations:

Haematology
Blood samples were taken from the retrobulbar vein plexus of the left eye each in slight ether anaesthesia. Feed was withdrawn in the evening of the day before and offered again immediately after the end of the blood sampling. Blood was taken on Day 29 from all animals of groups K, A, B and C , and on Day 43 of groups KS and CS.
Parameters determined:
• Red blood cell count (RBC)
• Haemoglobin concentration (Hb)
• Haematocrit (Hct)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• White blood cell count (WBC)
• Mean cell volume (MCV)
• Platelet count (PL T)
• Differential white blood cell count (Leucocyte count, % of the different cell species)
• Prothrombin time (Quick) as indicator of blood clotting capacity.

Clinical biochemistry
Blood samples were taken from the same animals and at the same time as for haematology. Plasma was obtained by centrifugation of the blood samples after incubation at 37 °C for about 1 hour.
Parameters determined:
• alanine aminotransferase (AL T, GPT)
• albumin (ALB)
• alkaline phosphatase {AP)
• aspartate aminotransferase (AST, GOT)
• calcium (Ca)
• cholesterol (CHOL)
• creatinine (CREA)
• gamma glutamyl transferase (GGT)
• glucose (GLU)
• potassium (K)
• sodium (Na)
• total protein (TP)
• urea (UREA)

Statistics:

Statistical method:
Analysis of variance followed by the Scheffe-test: all data with means and standard deviations determined, comparison of more than two groups
t-test: all data with means and standard deviations determined, for comparison of two groups only
H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled
Chi2 -test: counted events
Fisher's exact test: counted events, if the Chi2 -Test was not applicable

Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used. Groups K and KS and C and CS were treated separately for statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance related effects were observed in the detailed clinical observation and the functional observations.

Mortality:

no mortality observed

Description (incidence):

All animals survived until the scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See also the attachment.
The high dosed satellite group males had significantly lower body weights than the corresponding control group from Day 15 onwards until the end of the recovery period. A parallel trend in the high dosed males failed to gain significance.
The body weight gain of the high dosed satellite males was significantly lower than the control values in the first two weeks of dosing, with a similar !trend, though without significance, in both high dosed and high dosed satellite males later on.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See also the attachment.
The feed consumption of the high dosed and high dosed satellite males was repeatedly lower, when compared with the controls, gaining several times significance. In the high dosed and high dosed satellite females, feed consumption was significantly lower in the first week of dosing.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See also the attachment.
The group differences in red blood cell count, haemoglobin concentration, haematocrit, white blood cell count, lymphocyte count are attributed to the test substance. In spite of their significant differences, changes in MCH, monocyte count and neutrophil count are not attributed to the test substance, as they all lack a dose relationship. They are interpreted as incidental significances.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See also the attachment.
Changes in cholesterol, glucose and total protein are attributed to the test substance. Group differences in albumin, K+ and Ca++ are not related to the test substance as to the lack of a dose relationship. Group differences in ALT, alkaline phosphatase and AST are not considered to be a toxic effect, considering the trend of the values. An increase in values may serve as an indicator for toxicity, while here a decrease was present.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

See also above under clinical signs.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The changes in liver and heart weights are interpreted as effects of the test substance. The relative lower brain weight may be due to the more rapid recovery of the body weights vs. the brain weights after cessation of dosing.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no significant differences between the high dosed group and the control group. Therefore in the other groups only some isolated individual lesions were examined histopathologically. All alterations found are interpreted as background lesions, inconspicuous in type and incidence.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on body weights, feed consumption, haematology, clinical biochemistry and organ weight. No specific mode of action was detected.

2

Dose descriptor:

NOEL

Effect level:

316 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on body weights, feed consumption, haematology, clinical biochemistry and organ weight. No specific mode of action was detected.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

316 mg/kg bw/day (nominal)

System:

other: unspecific: body weight, feed consumption, haematology, clinical chemistry, organ weight

Organ:

not specified

Any other information on results incl. tables

Stability of the test substance preparations: The test substance was sufficiently stable in the preparations for 4 h.

Concentration and homogeneity of the test substance preparations: The concentrations and the homogeneity of the test substance in the preparations were within the chosen limits.

Applicant's summary and conclusion

Conclusions:

The NOAEL is 100 mg/kg bw.

Executive summary:

The toxicity of the substance was investigated after a repeated oral administration to rats, according to the EC-method B.7. and to the OECD Guideline 407. Methods:

Test substance administration:

The test substance was administered as a suspension in 0.1 % aqueous Na-CMC orally by gavage to 3 groups of 5 male and 5 female F344 rats each, once a day on 7 days per week for 28 consecutive days (Day 1 to 28). Doses used:

• Group A (low dose): 100 mg/kg bw and day

• Group B (mid dose): 316 mg/kg bw and day

• Group C (high dose): 1000 mg/kg bw and day

An equally sized negative control group (group K) received the vehicle of the test substance.

In addition, two groups of 5 males and 5 females each, i.e. one high dose recovery group (group CS) and one control recovery group (group KS), were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration.

Investigations:

• Animal observations: all animals, once a day.

• Detailed clinical observations: all animals, once a week.

• Functional observations: all animals, once, at the end of the dosing and of the recovery period.

• Body weights: all animals, once a week.

• Feed consumption: all animals, for each week.

• Haematology: all animals of groups K, A, B and C, on Day 29 and all animals of groups KS and CS on Day 43.

• Clinical biochemistry: all animals of groups K, A, B and C on Day 29 and all animals of groups KS and CS on Day 43.

• Necropsy with gross pathological examination: all animals of groups K, A, B and C on Day 29 and all animals of groups KS and CS on Day 43.

• Organ weight determination: all animals at necropsy.

• Histopathological examination: all animals of groups K and C.

• Stability, homogeneity and actual concentration of the test substance preparation.

Results

The test substance was sufficiently stable in the preparations for 4 h. The concentrations and the homogeneity of the test substance in the preparations were within the chosen limits.

The administration of the substance caused several effects on body weights and feed consumption, in haematology and clinical biochemistry and on organ weights. The effects observed did not give an indication for a specific mode of action of the test substance. None of the effects is considered as severe or became life threatening. The males were found to be somewhat higher susceptible to the test substance than the females. The alterations persisted partly until the end of the recovery period.

The NOAEL of T15 -AE is 100 mg per kg body weight and day for males and 316 mg per kg body weight and day for females.