Isobutyl (S)-2-chloropropionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-11-15 to 1989-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

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)

Version / remarks:

adopted May 12, 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA-TSCA Test Guideline "Functional Observational Battery" (Federal Register 50 (188): 39458 - 39461, 1985-09-27; amended 1987-05-20, Vol 52 (97): 19081 - 19082.

Deviations:

no

Principles of method if other than guideline:

The study protocol, based upon OECD-Guideline 407 was amended by adding investigations based on EPA's "functional observational battery". According to the authors, special attention was paid to neurotoxicity, because neurotoxicity had been reported in the literature for alpha-chloropropionate or alpha-chloropropionic acid, the latter possibly being generated from L-2-chloropropionic acid isobutyl ester by hydrolysis.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl (S)-2-chloropropionate

EC Number:

280-349-5

EC Name:

Isobutyl (S)-2-chloropropionate

Cas Number:

83261-15-8

Molecular formula:

C7H13ClO2

IUPAC Name:

isobutyl (S)-2-chloropropanoate

Details on test material:

- Name of test material (as cited in study report): L-2-chloropropionic acid isobutyl ester
- Physical state: liquid, colourless
- Analytical purity: 99.7%
- Impurities (identity and concentrations): no data
- Lot/batch No.: B 219 A, date of production: 1988-05-11
- Expiration date of the lot/batch: no data
- Stability under test conditions: confirmed
- Storage condition of test material: 4°C, dark (refrigerator)

The analytical results are included in Volume IV of IV of the complete study report.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Male and female Wistar rats (chbb = THOM (SPF)
- Source: Dr. Karl Thomae GmbH, D-W7950 Biberach/Riss, Germany
- Age at study initiation: 36 days (at the day of delivery), 42 day (first day of dosing)
- Weight at study initiation: males: 165 (156-173)g; females: 147 (138-155) g (first day of dosing)
- Fasting period before study: no
- Housing: single housing in type DK III stainless steel wire cages (floor area about 800 sqcm)
- Diet (ad libitum): Kliba rat/mouse/ hamster maintenance diet, "A" 343 meal, Klingentalmühle
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1988-11-02 (arrival) To 1989-03-07 (first necropsies) until 1989-03-16 (last necropsies)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once per week
- Mixing appropriate amounts with (Type of food): plain diet
- Storage temperature of food: 4°C, dark (refrigerator)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Substance-feed mixtures were taken for analytical investigations according to the following schedule:
1. directly after mixing
2. after storage of 4 days and
3. after storage of 7 days in the refrigerator (+4°C) in closed bowls .

Duration of treatment / exposure:

4 months

Frequency of treatment:

continuously in the diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
low dose group: 70 mg/kg bw/d; mid dose group: 210 mg/kg bw/ increased to 420 and 560 mg/kg bw/d; high dose group: 700 mg/kg/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
low dose group: 100 mg/kg bw/d; mid dose group: 300 mg/kg bw/ increased to 600 and 800 mg/kg bw/d; high dose group: 1000 mg/kg/d
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 females per dose group and per control group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The selection of the doses for this study was based on the results of the following studies:
(1) Testing of the acute oral toxicity of L-2-chloropropionic acid isobutyl ester in rats (BASF AG, 1984)
(2) Range-finding study with L-2-chloropropionic acid isobutyl ester: Oral application in rats over 3 weeks, prolonged to 6 weeks (BASF AG, 1988, Project No . 10C0740/87108)
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: no recovery groups included
- Section schedule rationale (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

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: Yes

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 28 days after the beginning of the administration period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 28 days after the beginning of the administration period
- Animals fasted: No
- How many animals: all
- Parameters examined: thromboplastin time (Hepato Quick's test), alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The neurofunctional observations were carried out in all animals one day before the start of the study and thereafter on days 1, 7, 14, 29, 62, 73, 80, 87, 94, and 118 in male and female animals . An additional neurofunctional test was performed in the female rats 125 days after the beginning of the administration period.
- Dose groups that were examined: all animals
- Battery of functions tested: neurofunctional observations
parameters examined: general state, tremors, convulsions, piloerection, lacrimation/secretion of pigmented tears, salivation, pupil size, diarrhea, vocalization, paresis, ataxia, body tone, posture, animal body/appearance, locomotor activity, respiration, skin color, righting reflex, behaviour, grip strength, pupillary reflex, winking reflex, vision, audition, olfaction, sensitivity of the body surface, pain perception, tail pinch, toe pinch, visual placing response.

Sacrifice and pathology:

At the end of the administration period non-fasted 9 male and 9 female animals were sacrificed by decapitation under carbon dioxide anesthesia and 3 animals per sex and dose, each from control and dose group 2 were deeply anesthetised and sacrificed by perfusion fixation.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

For the description of methods, see Volume III, PATHOLOGY REPORT.

Statistics:

The statistical evaluation of the data was carried out on the computer systems of the Department of Toxicology of BASF Aktiengesellschaft.
1. Clinical examinations
For the statistical evaluation of the study, means and standard deviations were calculated for the variables feed consumption, body weight and body weight change for the animals in each test group, and printed out in the form of tables.
For the clinical data (feed consumption, body weight, body weight change and special neurofunctional tests), a statistic one-way analysis of variance (ANOVA) was done via the Kruskal-Wallis-h-test. If the resulting p value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was carried out. This comparison was done using the Mann-Whitney-U-test for the hypotheses of equal medians.
2. Clinical chemistry and hematology
Mean and standard deviation were calculated for each test group and tabulated together with the individual values. Except for the differential blood count, a statistic one-way analysis of variance is done via the Kruskal-Wallis-h-test. If the resulting p value is equal or less than 0 05, a pairwise-comparison of each dose group with the control group was carried out. This comparison is done using the Mann-Whitney-U-test for the hypotheses of equal medians.

Results and discussion

Results of examinations

Details on results:

No substance-related findings were noted for the low dose group. The following, statistically significant findings were obtained in the mid and/or high dose group and assessed as being related to the test substance administered:

CLINICAL SIGNS AND MORTALITY
1) high dose group:
- poor general state (both sexes)
- clinical signs of neurological symptoms (e.g. ataxia, waddling gait, tremor, hyperesthesia, oblique body posture, spasms, lateral position) (both sexes)
- all animals died or were killed in a moribund state in the first three weeks (females) or six weeks (males) of the study
2) mid dose group:
- reduced body weight change (both sexes)
- clinical signs of neurological symptoms (e.g. slight ataxia, vocalization, staggering gait, spasms, tremor, or waddling gait) (both sexes)

BODY WEIGHT AND WEIGHT GAIN
1) high dose group:
- reduced body weight and body weight change (both sexes)
2) mid dose group:
- reduced body weight change (both sexes)

FOOD CONSUMPTION
1) high dose group:
- reduced feed consumption
2) mid dose group:
- reduced feed consumption (both sexes)

HAEMATOLOGY and CLINICAL CHEMISTRY
1) high dose group:
- no substance-related effects
2) mid dose group:
- no substance-related effects

NEUROTOXICITY
1) high dose group:
- clinical signs of neurological symptoms (e.g. ataxia, waddling gait, tremor, hyperesthesia, oblique body posture, spasms, lateral position) (both sexes)
2) mid dose group:
- clinical signs of neurological symptoms (e.g. slight ataxia, vocalization, staggering gait, spasms, tremor, or waddling gait) (both sexes)

PATHOLOGY
1) high dose group:
- cachexia (both sexes)
- ulcer in glandular stomach (both sexes)
- multifocal necrosis in the stratum granulosum of the cerebellum (both sexes)
- focal degeneration of testes
2) mid dose group:
- reduced body weight (both sexes)
- multifocal necrosis in the stratum granulosum of the cerebellum (both sexes)
Detailed results of the pathological examination are included in volume III of IV Pathology report.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

70 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

LOAEL

Effect level:

ca. 210 - ca. 560 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Severe neurological symptoms, morphological changes in the cerebellum and changed body weight gain

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

Report summary

L-2-chloropropionic acid isobutyl ester was administered to 5 male and 5 female Wistar rats.(Chbb = THOM SPF) per group in nominal dose levels of 100 (test group 1), 300 (test group 2), and 1000 mg/kg body weight (test group 3) over a period of 4 months. As a control, 5 male and 5 female animals were used (test group 0).

The main objective of the present study was to investigate the neurotoxicologic potential of the test substance. However, during the first 4 weeks of the study most of the animals of test group 3 died or were killed in a moribund state, and in test group 2 only slight neurological symptoms were observed. Thus, the study was prolonged up to 4 months and the dose of test group 2 (300 mg/kg body weight) were elevated twice: after 70 days of treatment to 600 mg/kg body weight and after 105 days of treatment to 800 mg/kg body weight.

However, based upon extensive analytical investigations only ingestion of about 70% of the nominal dose levels can be assumed, resulting in an actual substance intake of about 700; 560; 420; 210; and 70 mg/kg body weight instead of 1000; 800; 600; 300; and 100 mg/kg body weight, respectively.

Feed consumption and body weight were determined once a week.

The general state of health of the rats was checked at least daily and the animals were additionally thoroughly examined and palpated once a week.

Twenty-eight days after the start of the study blood samples were taken from all survival animals for clinicochemical and hematological examinations.

In addition to the clinical examinations, neurofunctional observations were carried out in all survival animals one day before the start of the study and thereafter on days 1, 7, 14, 29, 62, 73, 80, 87, 94, and 118 in male and female animals. An additional neurofunctional test was performed in the female rats 125 days after the beginning of the administration period.

At the end of the study 9 male and 9 female animals were sacrificed by decapitation under carbon dioxide anesthesia and assessed by gross pathology. This was followed by a histopathological examination.

Three animals per sex and dose, each from control and dose group 2, were deeply anaesthetised and sacrificed by perfusion fixation. The sacrificed animals were necropsied and the visible organs were assessed by gross pathology.

The following findings were obtained and assessed as being related to the test substance administered:

Test group 3 (nominal dose 1000, actual dose 700 mg/kg body weight):

Clinical examinations:

- poor general state (both sexes)

- reduced feed consumption, body weight, body weight change (both sexes)

- clinical signs of neurological symptoms (e.g. ataxia, waddling gait, tremor, hyperesthesia, oblique body posture, spasms, lateral position) (both sexes)

- all animals died or were killed in a moribund state in the first three weeks (females) or six weeks (males) of the study

Clinical chemistry and hematology:

- no substance-related effects

Pathology:

- cachexia (both sexes)

- ulcer in glandular stomach (both sexes)

- multifocal necrosis in the stratum granulosum of the cerebellum (both sexes)

- focal degeneration of testes

Test group 2 (nominal doses 300, 600, and 800; actual doses 210, 420, and 560 mg/kg body weight):

Clinical examinations:

- reduced feed consumption (both sexes)

- reduced body weight change (both sexes)

- clinical signs of neurological symptoms (e.g. slight ataxia, vocalization, staggering gait, spasms, tremor, or waddling gait) (both sexes)

Clinical chemistry and hematology:

- no substance-related effects

Pathology:

- reduced body weight (both sexes)

- multifocal necrosis in the stratum granulosum of the cerebellum (both sexes)

Test group 1 (nominal dose 100, actual dose 70 mg/kg body weight):

Clinical examinations:

- no substance-related effects

Clinical chemistry and hematology:

- no substance-related effects

Pathology :

- no substance-related effects

In summary it can be stated that the oral administration of L-2-chloropropionic acid isobutyl ester to Wistar rats via the diet in concentrations of about 700 mg/kg body weight led to severe neurological symptoms (e.g. ataxia) in male and female animals in the presence of general toxic symptoms (e.g. body weight reduction) accompanied by morphological changes in the cerebellum and testes; after a few weeks of treatment the animals of this dose group died or were killed in a moribund state.

The administration of about 210 to 560 mg/kg body weight also led to severe neurological symptoms (e.g. ataxia) and morphological changes in the cerebellum of male and female animals; additional general toxic symptoms (e.g. body weight reduction) were observed at doses of 1 420 mg/kg body weight in males and of 700 mg/kg body weight in females.

At a dose of about 70 mg/kg body weight no substance-related effects were detected.

Therefore the "no adverse effect level" for the repeated oral administration of 'L-2-chloropropionic acid isobutyl ester can be fixed under the chosen test conditions at 70 mg/kg body weight for male and female rats.