Butyl (S)-2-hydroxypropionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-08-19 to 1986-02-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Reference 2

Reference Type:

publication

Title:

n-Butanol (CAS No. 71-36-3)

Author:

ECETOC Joint Assessment of Commodity Chemicals (JACC)

Year:

2003

Bibliographic source:

JACC No. 41, ISSN-0773-6339-41

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butan-1-ol

EC Number:

200-751-6

EC Name:

Butan-1-ol

Cas Number:

71-36-3

Molecular formula:

C4H10O

IUPAC Name:

butan-1-ol

Specific details on test material used for the study:

- Name: normal butanol
- Source: American Scientific Products, Romulus, Michigan, USA
- Lot/Batch: 3597 KVEE

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat was chosen as a test sytem because of its established usefulness in toxicological studies and as a pharmacological model.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: 22-23 days at arrival
- Housing: individual in wire-bottom cages
- Diet: Purina Certified Rodent Laboratory Chow were available ad libitum
- Water: Filtered muncipal water, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Water used at TRL was analyzed periodically for the presence of contaminants as defined by the Environmental Protection Agency "National Interim Primary Drinking Water Regulations" Code of Federal Regulation. Title 40-Protection of Environment Part 141.11 and 141.12. Records retained at TRL.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.2 ± 2.2 °F
- Humidity (%): 47.6 ± 9.2 %
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

A plastic syringe and an 18 gauge ball-tipped metal dosing cannula ensheathed in a number 8 French catheters were used to administer the solution.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The amounts of the test material administered were based on individual weekly body weight values. Fresh solutions of normal butanol in deionized water were prepared weekly and dosed orally at a volume of 10 mL/kg bw. Controls received deionized water at the same volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each dose concentration were saved during weeks 1, 4, 6, 10 and 13 and taken to the Muskegon County Wastewater Treatment Facility for chemical analysis.

Duration of treatment / exposure:

The rats scheduled for the interim sacrifice were dosed daily for 42 or 43 days and the rats scheduled for the final sacrifice were dosed daily for 91 or 92 days.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

- Interim sacrifice: 10
- Final sacrifice: 20

Control animals:

yes, concurrent vehicle

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were observed at least twice daily for mortality and clinical effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
All rats received an ophthalmoscopic examination during the pre-treatment period and week 13 by a veterinary ophthalmologist. Ophthalmologic examinations were conducted on all rats in a darkened room with an indirect ophhalmoscope.

Clinical pathology:
Blood and urine samples were collected from the 10 male and 10 female rats in group V prior to initiation of dosing. Blood was obtained at the time of necropsy from all surviving rats scheduled for the interim sacrifice and from the first ten rats/sex(group at the final sacrifice. Urine was collected in metabolism cages 3-5 days prioi to the scheduled sacrifices. The rats were anesthetized with CO2, the thoracic cavity was opened and blood was collected by cardiac puncture. A necropsy was then done on each of these rats (except group V) and the tissues preserved.
The following hematology parameters were examined: hemoglobin, hematocrit, erythrocyte count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, total and differential leucocyte counts, estimated platelet count
The following clinical chemistry parameters were examined: alkaline phosphatase (Alk phos), blood urea nitrogen (BUN), glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), glucose (gluc), total protein (TP), albumin (Alb), A/G ratio (calculated), globulin (calculated), total bilirubin (Tot. bili.), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphate (phos), carbon dioxide (TCO2), total serum cholesterol (chol), creatine.
The following urinalysis parameters were examined: pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, microscopy of sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy was performed on all surviving rats of the first 10 males and 10 females from each dose group on day 43 or 44 of the study. On days 92 or 93 the remaining rats were necropsied. Rats found dead were also necropsied. Interim and final sacrifice rats were anesthetized with CO2 and exsanguinated by cardiac puncture. The cranial, thoracic and peritoneal cavities were opened, and the contents examined macroscopically. The organs and tissues lwere removed from each animal and preserved. Lungs were inflated with formalin via the trachea. eyes with attached optic nerve from all rats killed were preserved in a modified Zenker's fixative. The testes with attached epididymides from all male rats were preserved in Bouin's fixative. All other tissues were preserved in 10 % neutral-buffered formalin. Feet were preserved with the tissues for positive identification of the rat. Prior to fixation at the final sacrifice only, the following organs were weighed: brain, heart, liver, spleen, kidneys, testes with epididymides and ovaries. After fixation, the adrenals and thyroids with parathyroids were weighed. For paired organs, the organ weight was the combined weight of right and left members of the pair. Organ/body weight ratios were determined for each tissue. No organ weights were taken on rats found dead.

HISTOPATHOLOGY: Yes
On the full tissue list shown in box "Any other information on materials and methods incl. tables" a microscopic examination was done in the control and high-dose rats, on one rat sacrificed in extermis, amd on those found dead. Also, livers, hearts and kidneys of low- and mid-dose rats and all gross lesions seen at necropsy were examined microscopically. As tissues were trimmed, the presence or absence of tissues and lesions was noted. The tissues were placed in Tissue Tek cassettes that were labeled with study number, rat number and the cassette number. They were then processed on a Fisher Scientific Histomatic or an AO TP/8000. After processing, the tissues were embedded in paraffin using a Tissue Tek embediing system. They were sectioned at 5-6 µm, mounted on numbered slides and stained with hematoxylin and eosin.

Statistics:

Body weight, food consumption, clinicopathologic, and organ weight data were tested for homogeneity of variance by Bartlett's method (Steel and Torrie, 1980). If the data were found to be homogeneous, differences between control and treatment means were tested for statistical significance by the method of Dunnett (Dunnett, 1964). If the data were found not to be homogeneous, the method of Gill (modified Dunnett's) was employed (Gill, 1977).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related ataxia first appeared in the high-dose group (500 mg/kg bw/day) during week 8. Ataxia and hypoactivity occurred infrequently during week 9 and 10. These signs increased to a weekly incidence of 32 and 29 % for ataxia and hypoactivity, respectively. At week 11 and continued at approximately the same frequency during weeks 12 and 13. Onset of ataxia and hypoactivity was about 2-3 minutes after dosing and duration was less than one hour. Other clinical signs observed did not appear to be directly related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three rats died during the study. Two of these deaths were the result of the rubber catheter slipping off the metal dosing cannula. And a mid-dose (125 mg/kg bw/day) male died during week 1.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effect was present on body weight or weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effect was present on food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The pathology observed was considered to be within normal limits for the age, sex and strain of the animal.

Haematological findings:

no effects observed

Description (incidence and severity):

Only one alteration in clinical pathologic parameters occurred that was suggestive if a treatment-related effect. At the interim sacrifice, RBC (p< 0.05), PVC (p< 0.01) and HGB (p< 0.01) averages in the high dose (500 mg/kg bw/day) females were 5 % less than the corresponding control average. The RBC and PCV (p< 0.05) averages for the middle-dose (125 mg/kg bw/day) females were also slightly (4 % and 3 % respectively) below those of the controls. However, RBC PVC and HGB averages were similar for control and treated groups of males at the interim evaluation and for control and treated groups of both sexes at the final evaluation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Small yet statistically significant differences between control and treated groups averages occurred in one sex and at one evaluation only, and there was no dose response relationship. Thus, they were not considered to be treatment related. They were: a lower (p< 0.05) cholesterol average in the high-dose males at the interim evaluation, a higher (p< 0.05) absolute neutrophil count in the middle-dose males at the interim evaluation, a higher (p< 0.05) relative segmented neutrophil count and a lower (p< 0.05) relative lymphocyte count in the low-dose females at the final evaluation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Small yet statistically significant differences between control and treated groups averages occurred in one sex and at one evaluation only, and there was no dose response relationship. Thus, they were not considered to be treatment related. They were: higher (p< 0.05) urine pH values in the low dose males at the interim and low-dose females at the final evaluations.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant difference between control and treated group averages was a slightly (p< 0.05) higher thyroid weight average in the high-dose males. No dose response relationship was present, as the absolute thyroid weights were similar for all three treated groups of males. Moreover, they were only 14 % above control average. Thyroid weight averages of the treated females were not above those of the controls. Thus, this difference appears to be a chance occurrence rather than a treatment-related effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related lesion was observed in gross necropsy at the interim or final sacrifices or of the rats found dead or sacrificed in extremis. The lesions present were those commonly observed in laboratory rats and they were present in control and treated groups at similar frequency or were one-time occurrences. The enlarged uterine horns are related to the stage of the oestrus cycle. Three rats died during the study no gross lesions were seen in rat no 224. Rat no 202 (which had a catheter in its stomach) had dark area on the glandular mucosa of the stomach. In rat no. 133 (middle-dose) the left lobe of the lung was red, and the cranial and middle lobes were shrivelled.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related lesion was observed at the histopathologic evaluation. The lesions that were observed were one-time occurrences or were present in the control and treated groups at a similar frequency. The diffuse subacute lymphadenitis of the mandibular lymph node was visible grossly as red or enlarged lymph nodes. This is a commonly observed lesion on laboratory rats. The cause of death of the mid-dose rat (no. 133) that died during the study was determined to be a gavaging accident since a perforated oesophagus was found at histopathologic examination.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

nervous system

Any other information on results incl. tables

Test material analysis:

The results of the analysis of the samples revealed that the stability and concentration of the samples were acceptable.

Applicant's summary and conclusion

Conclusions:

The NOAEL of n-butanol after oral administration via gavage over a period of 90 days is considered to be 125 mg/kg bw/day for both sexes.

Executive summary:

A repeated sub-chronic dose toxicity study was conducted equivalent to OECD 408. Male and female rats (20/sex/group) at doses of 0, 30, 125 or 500 mg/kg bw/day were treated orally with n-butanol. The animals received the test item daily over a period of ca. 90 days via gavage. The only unequivocal effects produced by n-butanol were ataxia and hypoactivity at the 500 mg/kg bw/day dose level. The maximum weekly incidence was 32 % and 29 %, respectively. No dose-related differences were observed between treatment or control rats in body or organ weight changes, food consumption or mortality, gross pathology and histopathological and ophthalmic evaluations. Ataxia and hypoactivity (lasting less than 1 hour) were observed 2-3 minutes after dosing in both sexes of the high dose group during the last 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. No treatment-related signs were observed in the 30 and 125 mg/kg bw/day treatment groups. Based on the results reported, the NOAEL for orally administrated n-butanol via gavage is 125 mg/kg bw/day for both sexes.