2-Butenoic acid, 4,4,4-trifluoro-3-(methylamino)-, ethyl ester, (2Z)-

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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)

Deviations:

yes

Remarks:

deviations are considered to have no impact on the validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

deviations are considered to have no impact on the validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

EC Number:

801-277-8

Cas Number:

507448-65-9

Molecular formula:

C7H10F3NO2

IUPAC Name:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division. CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: Approximately 4 weeks (at delivery)
- Weight at study initiation: Males: 143 - 176g. Females: 117 - 142 g.
- Housing: Individual
- Diet: ad libitum (except for overnight fasting prior to blood collection)
- Water: ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance was administered once daily by gavage (rubber catheter).

Preparation of suspension:
Suspensions of the test item in the standard vehicle (distilled water, 0.5% CMC, 0.1% Tween 80) yield low recovery upon content and homogeneity determinations at dose levels of 1 and 5 mg/ml. Therefore corn oil was selected as an alternative vehicle.

Suspensions of the test item in the vehicle (corn oil) at the appropriate concentrations were freshly prepared every day immediately prior to dosing.

Volume applied: 5 ml/kg body weight
Vehicle: corn oil (Fluka Biochemika, no 63156), batch no. 360327/1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The received solutions were quantitatively poured into a 100 ml volumetric flask. The sample tubes were washed with acetone, afterwards diluted to 100 ml with acetone. The solutions are diluted acetone to nominal concentrations of 40 µg.ml. 1 µl was analysed by GC with FID-detection, using the following conditions:

Column: DB-5, 30m x 0.53 mm ID
Film: 1.0 µm
Carrier gas: Helium
Head pressure: 55 kPa (bei 55 °C)
Flow: 10 ml/min
Temperatures
Injector: Cold on Column
Detector: 280 °C
Column: 55 °C, isothermal for 2 min., program 55 – 170 °C with 10 °C min., isothermal for 1.5 min.
Injection volume: 1 µl
Run Time: 15 min.
Retention Time: approx. 6.3 min

As reference sample CA 2455 A was dissolved in acetone and diluted with acetone to a final concentration in the range of the test samples. The concentration of the test product in the samples was calculated from the peak area in comparison with the peak area obtained with the reference solutions.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 300 and 1000 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale:
The dose level selection was based on the results of the following study:
Project No. 973054
Acute Oral Toxicity in the Rat (preliminary results)
Toxicology, Novartis Crop Protection AG, 4332 Stein / Switzerland.

A total of 80 animals used:
5 males and 5 females per dose group, additionally, 5 males and 5 females in dose groups 1, 4 and 5 for recovery evaluation.

Summary – Table 1

The test item was administered orally by gavage, over a period of 4 weeks to animals of both Experimental Groups I and II. Subsequently, surviving animals of Experimental Group II were kept for an additional 4 recovery weeks in order to determine reversibility, persistence of, or delayed occurrence of possible adverse effects.

Examinations

Observations and examinations performed and frequency:

Schedule of investigations:

Parameter Time Point
Clinical signs and mortality Twice daily
Body weight Once weekly
Food consumption Once weekly
Water consumption Once weekly
Detailed clinical observations During pre-test and once weekly
FOB and motor activity During pre-test, week 4 and week 8 (recovery)
Laboratory investigations Week 5, and week 9 (recovery)
Necropsy Week 5, and week 9 (recovery)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table) / No / No data
HISTOPATHOLOGY: Yes (see table) / No / No data

Results and discussion

Results of examinations

Details on results:

Mortality:
At study day 3 two females (No. 61, 63) of group 4 (300/200 mg/kg) were found dead and all females of the high dose group (1000 mg/kg) and 2 females (no. 68, 69) of group 4 were sacrificed in moribund.
At day 4 two males (no. 36, 36) of the high dose group were found dead and the remaining males were sacrificed in moribund condition.
In addition, one female (no. 46) of the control group died accidentally at day 29.

Clinical signs:
In the high dose group (1000 mg/kg), piloerection, hypoactivity, hypotonia and prostration were apparent in the majority of males and females. Lacrimation, dyspnea, ataxia, micturition and soft feces were noted in some animals before early sacrifice.
In group 4 (300/200 mg/kg), hunched posture and/or piloerection were observed in most males and females, and hypoactivity, hypotonia, prostration, dyspnea, and soft feces in few animals only.

Body weight and body weight changes:
High dose males and females (1000 mg/kg) showed negative body weight gain between day 1 and 3, i.e. they lost 7 % and 8 % of their weight, respectively. Also in group 4 (300/200 mg/kg) males (-6 %) and females (-8 %) lost weight during the first treatment days before regaining weight from day 6 and 5 onwards, respectively. At treatment end, the mean body weight in group 4 males was still 14 % below the control mean, whereas females had recovered to a similar mean weight as female controls. During the treatment-free period group 4 males showed complete recovery.
Male and female groups 2 and 3 (10 and 100 mg/kg) were not considered affected.

Food consumption:
Significantly depressed intake was recorded for high dose males (-77 %) and females (-79 %) during week 1 and for group 4 males during weeks 1 (-42 %) and 2 (-16 %) and females during week 1 (-48 %).
Food intake values similar to the respective control groups were noted during the recovery period.

Water consumption:
Increased water intake was recorded during week 1 for high dose males (+ 37 %) and females (+ 41 %) and for group 4 males during weeks 2 to 4 (+28 to +40 %). Group 4 females had lower water consumption (-44 %) during the first week.
The mean water consumption of males and females of groups 2 and 3 (10 and 100 mg/kg) were not considered affected.

Neurotoxicological examinations:
Due to compound-related toxicity group 5 animals died or had to be killed before the week 1 detailed clinical examinations.
Treatment-related signs were observed in group 4 animals only and included reduced activity, reduced muscle tone, piloerection and abnormal gait (females only). In the absence of a dose relationship, reduced sensorimotor responsiveness and few other signs observed in single animals were all considered as incidental. Group comparisons using multiple t-tests showed a statistically significant decrease in forepaw grip strength in group 4 males as compared to controls. This effect was mild, limited to forepaws and seen only in males at 4 weeks. As chemical-induced neuromotor deficits usually are more prominent and first seen in hind limbs, this finding most likely is not compound-related. This is further supported by the absence of any other functional deficits in animals of this dose group. Therefore, this effect was considered as an incidental finding not related to exposure to the test item.

Motor activity measurements conducted at the end of the treatment and recovery periods did not reveal any compound-related effects. Group comparisons using multiple t-test that were corrected for multiple testing using bootstrap resampling did not reveal any difference compared to controls.

Hematology:
At the end of the treatment period, males of group 4 (300/200 mg/kg) had a slight, hypochromic, microcytic anemia associated with anisocytosis of red blood cells. Aniocytosis was also detected in males of group 3 (100 mg/kg). A tendency to hypochromia and micrcytosis of red blood cells, substantiated by lower mean values for MCH and MCV, was also seen in females of groups 3 and 4. In addition, males and females of group 4 (300/200 mg/kg) had a thrombocytopenia. Leukocyte parameters showed some disturbance with males of group 4 (300/200 mg/kg) having higher values for neutrophils, monocytes and large unstained cells (LUC), and lower values for eosinophils. Males of group 3 (100 mg/kg also had higher monocyte counts. In females, lower values of eosinophil counts were recorded for group 3 and 4 (100 mg/kg and 300/200 mg/kg) and higher monocyte and LUC counts were recorded for group 4.

No toxicological relevance was attached to the lower mean value for relative lymphocyte counts in males of group 4 as absolute values did not differ appreciably.

Following the 4-week recovery period, values for the above parameters, were essentially similar to those of the respective controls. However, anisocytosis (males) and the tendency to hypochromasia and microcytosis of red blood cells (females) persisted.

Blood chemistry:
At the end of the treatment period, lower values for protein and globulin were recorded for males and females of group 4 (300/200 mg/kg) and for females of group 3 (100 mg/kg), resulting in a higher albumin to globulin ratio in males of group 4. Males and females of groups 3 and 4 (100 and 300/200 mg/kg) had higher plasma urea levels. In addition, males and females of group 4 had elevated alkaline phosphatase activities and group 4 males had lower calcium and higher phosphate levels.

After the recovery period, the above parameters had returned to values within expected range.

Although some other inter-group differences attained a level of statistical significance, they were not treated to treatment as they did not form a dose-response relationship and/or the magnitude of the change was too small to be of toxicological relevance. In males, these differences included lower mean values for bilirubin in group 3 at week 5 and in group 4 at week 9, and a higher mean potassium value in group 4 and week 9. In females, these differences included a higher mean glucose value for group3, lower sodium values for groups 3and 4 at week 5, a lower calcium value for group 4 at week 9, and a lower aspartate aminotransferase activity for group 4 at week 5.

Urine analysis:
At the end of the treatment period, females of group 3 and 4 (100 and 300/200 mg/kg) excreted slightly lower volumes of more concentrated urine. Following the recovery period values for volume and density were similar to those of the control group.

The quantity and quality of the urine excreted by all males and females of group 2 were not disturbed by treatment. No toxicological relevance was attributed to the higher urine volume recorded for males of group 4 at the end of the recovery period.

Organ weights and ratios:
Due to early sacrifice no organ weights were taken from group 5 animals (1000 mg/kg). At treatment end significantly lower absolute mean weights were recorded in group 4 males (300/200 mg/kg for carcass (-21 %, compared to control mean), thymus (-68 %), testes (-11 %), and spleen (-16 %). The changes to mean organ to body weiht ratios included brain (+25 %), heart (+16 %), kidney (+23 %), thymus (-61%), and adrenals (+31 %) in males and liver (+18 %) in females.

In group 3 females (100 mg/kg) the relative mean liver weight was increased.

At recovery end depressed absolute and relative mean weights for epididymides of group 4 males were recorded.

Pathology:
In this study all 10 males and 10 females of group 5 (1000 mg/kg) and 4/10 females of group 4 (300/200 mg/kg died or were terminated prematurely on days 3 to 4. Although some of these decedents were scheduled for the recovery period, their pathology is restricted to the early changes induced at the beginning of treatment, and is not comparable to that of animals surviving until the scheduled termination.

Macroscopic findings:
Treatment-related early changes:
2/10 males and 3/10 females of group 5 (1000 mg/kg), as well as 1/4 early examined females of group 4 (300/200 mg/kg) had black-brown foci on the inner surface of their stomach. On histopathyology these changes mostly correlated with defects of the gastric mucose membrane.

Treatment-related changes at the end of the treatment period:
4/5 males of group 4 (300/200 mg/kg) had the thymus diminished in size. This observation reflected cortical thymic atrophy on histology.

Changes at the end of the recovery period:
No gross lesions related to treatment were observed after the recovery period.

Incidental findings:
All other necropsy findings were incidentally distributed among the group and corresponded to the findings occasionally occurring in this rat colony. They did not represent an effect of treatment. This includes also female animal no. 45 (group 1), presenting a cyst of the left kidney corresponding microscopically with hydronephrosis.

Microscopic findings:
Treatment-related early changes – Lymphoreticular organs:
Hypocellularity of the bone marrow was observed in all 10 males and 10 females of group 5 and in 3 of 4 examined females of group 4. The average grading of fatty atrophy of the bone marrow occurring in these animals was slightly increased. The spleen showed atropy of PALS in 1/10 male of group 5 and hyperplasia of white pulp in 5/10 females of group 5. Cortical atrophy occurred in the thymus of 8/10 males and 7/10 females of group 5. Phagocytic cells were found in the thyimc cortex of 1/10 males and 4/10 females of group 5 and 4/4 examined females of group 4.

Treatment-related early changes – Stomach:
The forestomach had inflammatory edema in 9/10 males and 5/10 females of group 5 and 1/4 examined females of group 4; there was ulceration in 6/10 males and 2/10 females of group 5, and inflammatory cell infiltration in 1/10 females of group 5 and ¼ examined females of group 4. Erosion of the glandular stomach occurred in 1/10 males and females each of group 5 and in 2/4 examined females of group 4.

Treatment-related early changes – Epididymides:
Cellular debris was found in the epididymis of all 10 males of group 5.

Treatment-related changes at the end of the treatment period – Lymphoreticular organs:
Fatty atrophy of the bone marrow had slightly increased incidence and average grading in male group 4 and average grading in female group 4 (the incidence in females was 2/2 examined, that means the maximum possible value was reached). The spleen had a minimal hemosiderosis in 2/5 males of group 4. In contrast, the incidence of splenic hemosiderosis was slightly decreased in female groups 4 and 3. Cortical atrophy occurred in the thymus of 4/5 males and 2/2 females of group 4 and 1/5 females of group 3. Phagocyclic cells were found in the thymic cortex of 3/5 males of group 4 and 1/5 males of group 3.

Treatment-related changes at the end of the treatment period – Epididymides:
Cellular debris was observed in 2/5 males of group 4.

Treatment-related changes at the end of the treatment period – Liver:
Centriboular hepatocellular hypertrophy occurred with an increased incidence and grading in male groups 4, 3 and 2. The incidences were, in the descending order of groups, 5/5, 4/5 and 2/5, and the average grading 1.4, 1.5 and 1.0. In female groups 4 and 3 2/5 animals each showed a minimal hepatocellular hypertrophy as well.

Treatment-related changes at the end of the treatment period – Thyroid gland:
Hypertrophy of the thyroid follicular cells occurred with a slightly increased grading in male groups 4, 3 and 2.

Changes at the end of the recovery period – Lymphoreticular organs:
Fatty atrophy of the bone marrow occurred with a higher incidence in female group 4 and higher average grading in male and female group 4.

Changes at the end of the recovery period – Abdominal cavity:
Lymphoytic infiltration of the abdominal cavity was observed in 2/5 males of group 4. This finding could reflect gastric lesions occurring early during treatment but inapparent after the recovery period.

Changes at the end of the recovery period – Thyroid gland:
Hypertrophy of thyroid follicular cells had higher incidences and average grading in male group 4 than in male control group.

Changes at the end of the recovery period – Epidiymides:
No pathological changes were observed in the epididymides of group 4 males at the end of the recovery period, although weights of epididymides were still decreased in this group.

Incidental findings:
Additionally, a variety of other changes was found in this study. They commonly occur in laboratory rats of this colony, and, neither their incidences nor their morphologic features gave any indication of a treatment-related association. In particular, two females of group 5, sacrificed moribund on day 3, had degeneration of nerve fibres in the sciatic nerve. This finding is attributed no toxicological relevance because of its low incidence and short duration of exposure of the affected animals of this study. This change occurs spontaneously with variable incidences in rats of this strain and age and is therefore considered devoid of toxicological relevance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results obtained during this study, a “no-Observable-Adverse-Effect level” (NOAEL) for CA 2455 A when administered by daily gavage is defined at 10 mg/kg body weight per day for males and females.

Executive summary:

Under the conditions of this test the treatment (gavage of albino Wistar rats with CA 2455 A (Intermediate of CGA 276854) at dose levels of 1000 and 300 mg/kg exceeded the maximum tolerated dose (MTD) and produced early mortality and moribund condition in all high dose animals and some females at 300 mg/kg (group 4). Consequently, the surviving high dose animals and some females from group 4 were taken to early sacrifice and the daily dose for surviving group 4 animals was adjusted to 200 mg/kg. 

 

Slight changes to hematologic and blood chemistry parameters were associated with histological changes and lymphoreticular tissues including bone marrow, spleen and thymus. Fully reversible haematological and hepatotropic effects were noted at all dose levels tested. However, the effects obtained at the 10 mg/kg dose level were considered adaptive and, consequently, of a not adverse nature. In addition, signs of local irritation, inflammation, ulceration, and erosion of the stomach were noted. 

 

Based on the results obtained during this study, a “no-Observable-Adverse-Effect level” (NOAEL) for CA 2455 A when administered by daily gavage is defined at 10 mg/kg body weight per day for males and females.