Benzoic acid, 2-amino-5-fluoro-, methyl ester

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: GLP- and Guideline-Study with no or minor deficiencies.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

other: U.S. EPA: OPPTS 870.3050, Health Effects Test Guidelines: Repeated dose 28-day oral toxicity study in rodents, July 2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Directive 96/54/EC Annex IV D Method B.7."Subacute Oral Toxicity"

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-amino-5-fluorobenzoate

EC Number:

608-681-1

Cas Number:

319-24-4

Molecular formula:

C8 H8 F N O2

IUPAC Name:

methyl 2-amino-5-fluorobenzoate

Test material form:

other: brown liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Room temperature: 22±3°C
Relative humidity: 50±20 %
Lightning hours: 12 hours light/dark cycle
Diet: Ssniff (ad libitum)
Water: tap water in plastic bottles (ad libitum)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

sesame oil

Details on oral exposure:

Application volume: 0,5 ml/kg bw

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw per day (control)
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
62.5 mg/kg bw per day
Basis:

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg bw per day
Basis:

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg bw per day
Basis:

No. of animals per sex per dose:

5

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

General health conditions: twice daily (weekend, public holiday once daily)
clinical symptoms of toxicity: once daily
functional observation battery: neurotoxicological examinations: weekly
motor activity, sensory reactivity, grip strength: week 4
body weight development: twice weekly
food consumption: twice weekly
hematology: once, end of treatment
clinical chemistry: once, end of treatment
urinalysis: once (week 4)

Sacrifice and pathology:

Organs examined (macroscopic and microscopic): Heart, kidneys, liver, lungs and trachea, spleen, thymus, iliac and mandibular lymph node, stomach, small (ileum) and large intestine (colon), brain with medulla oblongata, spinal cord (cervical), sciatic nerve, adrenals, thyroid gland with epithelial body, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, and bone marrow.

Statistics:

two-sided ordinal step-down test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

in high dose group

Mortality:

mortality observed, treatment-related

Description (incidence):

in high dose group

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

cholesterin and triglycerid levels increased in the high dose group

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

decreased pH in the high dose (both sexes) as well as an increased urine volume

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Female animals of the intermediate dose showed a significant reduction in the number of movements

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

absolut liver weight was increased

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

the female animal found dead had an enlarged spleen

Effect levels

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

No deaths occurred throughout the study. Only one female animal of the 1000 mg/kg body weight group was found dead on day 27 of the study showing immunodeficiency. In the high dose group (1000 mg/kg) several animals showed clinical symptoms such as stupor, prone position, hypoactivity, stilted gait, squatting posture, irregular respiration, bristled coat, drawn in flanks as well as straddled hind limbs. Clinical signs started between day 14 and 26.

Behavior, body weight development, food consumption and neurotoxicological parameters remained unaffected by the administration of the test compound in all dose groups.

Evaluation of hematological data indicated reduction in red blood cell counts in females of the high dose group correlated with an increase in reticulocytes.

Clinical chemistry revealed findings in the high dose groups such as increases in total cholesterin and triglycerid levels exceeding the normal range. A slight but statistically significant increase in total cholesterin was also observed in male animals of the low and intermediate dose group. This slight increase represents no adverse effect, because values did not exceed the normal range and showed no histopathological correlation (e. g. in liver). As the range in low dose and control animals showed distinct overlapping, the slight increase in mean values is not considered as substantial substance related effect in this dose group. Moreover, total bilirubin levels were increased in male animals of the high dose group and calcium levels were slightly increased in the intermediate and high dose group in both sexes, but not in a toxicologically relevant manner.

Urine in females of the high dose group showed red discoloration, as well as urine of two male animals of the intermediate dose group and of four males in the high dose group. No blood was detected in urine. Bilirubine was detected in urine of all animals of the high dose group. Moreover, urinalysis revealed decreased pH values (below the historical control data range) in the high dose group with both sexes as well as increased urine volume. Alterations in urine volume and/or pH in animals dosed with 62.5 or 250 mg/kg test item were slight compared with controls in indi-vidual sexes, and are therefore not considered as toxicologically relevant effects.

Organ weight analysis revealed increased absolute liver and spleen weights in female animals of the high dose group. Both, relative and absolute values in this group, were above the historical control data range. Male animals showed also significantly increased relative liver weights, which exceeded the normal range only slightly. No histopathological correlates were observed in liver.

Histological assessment revealed erythroid hyperplasia in the spleens of nearly all female animals in the high dose group with an average grading of 2.3, compared to an average grading of 1.0 to 1.3 in the other groups including the control. Additionally, atrophy/hypoplasia/hypocellularity in the spleen (in combination with an haemorrhage), the thymus, iliac and mandibular lymph nodes (partly with haemorrhage) and the bone marrow were observed in one female (animal 36, which died on day 27) and one male animal (animal 19, showing clinical signs) of the high dose group reflecting immunodeficiency.

Applicant's summary and conclusion

Conclusions:

In conclusion, repeated administration of 2-Amino-5-fluorbenzoesaeuremethylester at a dose level of 62.5 mg/kg body weight and day did not cause any substantial substance related alterations. Slight alterations were observed after administration of 250 mg/kg, which did not represent ad-verse findings. Repeated administration of 1000 mg/kg caused alterations such as clinical signs of toxicity in several animals, deviations in cholesterine and triglycerides, alterations in hematology in females correlated with erythoid hyperplasia in spleen, as well as death of one female animal showing immunodeficiency.