Butyl (S)-2-hydroxypropionate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997-03-13 to 2000-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Adopted 12th May, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butyl (S)-2-hydroxypropionate

EC Number:

252-036-3

EC Name:

Butyl (S)-2-hydroxypropionate

Cas Number:

34451-19-9

Molecular formula:

C7H14O3

IUPAC Name:

butyl 2-hydroxypropanoate

Specific details on test material used for the study:

- Systemic name: butyl-S-(-)-2-hydroxypropionate
- CAS Reg. No.: 34451-19-9
- Molecular formula: C7H14O3
- Molecular weight: 146 g/mol
- Purity: 98.9 %
- Batch no.: BU-6001K and BU 5001H
- Appearance: liquid/ water white
- Boiling point: 189 °C
- Flash pomt: 79 °C
- Vapour pressure: 0.4 mbar at 20 °C
- Storage conditions: ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species is used because it is considered the most suitable for this type of study, and is usually required by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Females (if applicable) Not applicable
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: mean body weight of 255 g
- Housing: housed in groups of three, in suspended, stainless steel cages, fitted with wire mesh floor and front. The cages were randomly divided over the cage racks according to a computer randomization program and each cage was provided with a coloured card showing the animal identification numbers, the cage number, the group letter and the study number
- Diet (e.g. ad libitum): ad libitum, commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RMS) obtained from SDS, Special Diets Services, Witham, England
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 35-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

other: humidified air

Remarks on MMAD:

Particle size was not measured

Details on inhalation exposure:

EXPOSURE LEVELS:
The exposure levels of the test item have been established based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other local toxic effects at levels of 400 or 600 mg/m³ and higher.

EXPOSURE UNITS:
The animals were exposed to the test atmosphere in nose-only inhalation units. Each unit consisted of a cylindrical column surronded by a transparent cylinder. The column has a volume of ca. 50 L and consisted of a top assembly with the entrance of the chamber, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animals exposure. Several empty ports were used for test atmosphere sampling, particle size analysis, and for measurement of temperature and relative humidity. The animals were secured in plastic animal holders, positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column.

GENERATION OF THE TEST ATMOSPHERE:
To generate the test atmospheres complete evaporation was used, using a rotating liquid film evaporator. The glass flask containg the test item was placed in an oil bath at a temperature of 125 °C. A small metered flow of dry clean air was passed through the test material and subsequently mixed with a secondary metered flow of dry clean air (main flow). Appropriate fractions of this main flow were taken using eductors and were diluted with metered amounts of humidified air and passed to the inhalation units. The remaining part of the main flow was passed directly to the exhaust. The test material was changed daily.

TEMPERATURE AND RELATIVE HUMIDITY:
The temeprature and the realtive hunidity of the test atmospheres were recorded at least three times per exposure day using a RH/T device (Testo 610).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total Carbon Analysis:
The amount of test item vapozr was monitored semi-continuously by means of a total carbon analyser using flame ionisation detection (FID). The settings of the total carbon analyser were as follows: oven temperature: 150 °C, H2 flow: 0.5 bar, air flow: 0.8 bar, sampling back pressure: 200 mbar, sampling temperature: 130 °C. The samples were taken sequentially from each of the units at the animals breathing zone. The samples were drawn through sampling lines and were passed via a controlled valves system to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). Each unit was monitored approximately once each half hour for about 7 min, resulting in about 13-14 measurements per concentration level per day.

Duration of treatment / exposure:

20 days

Frequency of treatment:

6 hours a day, 5 days a week for a period of 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

75 mg/m³ air

Remarks:

Low concentration

Doses / concentrations 3

Dose / conc.:

200 mg/m³ air

Remarks:

Mid concentration

Doses / concentrations 4

Dose / conc.:

600 mg/m³ air

Remarks:

High concentration

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The exposure levels have been established in consultation with the sponsor. They are based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other (local) toxic effects at levels of 400 or 600 mg/m³ and higher.

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon (shortly after exposure) for dead or moribund animals, to minimize loss of animals from the study. At weekends only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the day of the start of the study (allocation procedure), just prior to the start of the first exposure (day 0), on nominal days 7, 14, 21, 28 and on the day of scheduled necropsy (day 29). In addition, weekly body weight gain was calculated. Body weights were also recorded on day -3 to monitor adequate growth during the acclimatization period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 29, the day after the last exposure, all rats were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia. Subsequently they were examined macroscopically for pathological changes.

HISTOPATHOLOGY: Yes
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. The respiratoty tract (nose, larynx, trachea and lungs) was processed as follows: The nose (nasal cavity) was cut at 6 levels. Levels of cross sections through the nasal cavity were assigned according to international standards (Woutersen et al., 1994). The larynx was cut longitudinally at three levels. The trachea with the bifurcation was cut longitudinally/transversally at three levelsEach lung lobe was cut at one sagittal level. Histopathological examination was performed on the nose of all animals of all groups. The other respiratory tract organs were examined in the control and high concentration group.

ORGAN WEIGHTS:
The following organs were weighed and the relative organ weights (g/kg bw) were calculated based on the final body weight of the rats: adrenals, heart, kidneys, lungs with trachea and larynx, liver, spleen and testes. Samples of these organs and the nose of all animals were preserved in a neutral aqueous phosphate-buffered solution of formaldehyde.

Statistics:

Body weight data were analysed by one-way analysis of covariance (COVAR) using pre-exposure (day 0) weights as the covariate. Organ weights were analysed by one-way analysis of variance (ANOVA). When group means were significantly different (p < 0.05), individual pairwise comparisons were made using Dunnett's multiple comparison method. The incidence of histopathological changes was evaluated by Fisher's exact probability test. All pairwise comparisons were two tailed. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) may be greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

General condition and behaviour were not adversely affected by exposure to the test material. Clinical signs included a visually slightly increased superficial breathing pattern in animals exposed to the highest concentration during exposure and areas of sparsely haired skin in one control rat.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed until the scheduled autopsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in mean body weights and mean body weight gain between the treatment groups and the controls although body weight gain in animals exposed to the high concentration vapour test atmosphere appeared slightly lower than those in the other groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant difference in the mean absolute and relative organ weights between the treatment groups and the controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examination at autopsy revealed findings in the epididymides, kidneys, mediastinal lymph nodes, skin/subcutis and testes in one or two animals only. Moreover, the findings are common for rats of this strain and age. Most animals exhibited gross findings in the lungs. However, the incidences of these findings were about equally distributed between the groups, including the control group. Therefore, the changes were considered not to be related to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the nasal tissues showed very slight to slight transitional epithelium hyperplasia at level 2 in 5 of 6 animals exposed to 600 mg/m³. Slight focal transitional epithelium hyperplasia was also observed in one animal exposed to 600 mg/m³ (nasal level 1) and in one control animal (nasal level 2). One animal exposed to 600 mg/m³ additionally showed very slight focal squamous epithelial hyperplasia (nasal level 1). Very slight focal goblet cell hyperplasia (nasal level 3) was seen in two animals exposed to 600 mg/m³. No nasal tissue changes were observed in animals exposed to 75 or 200 mg/m³.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

600 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse systemic effects observed in any test group

2

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Very slight to slight transitional epithelium hyperplasia of the nasal passages.

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/L air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Analytical results on actual concentration:

The overall mean daily concentrations of the test item vapour in the test atmospheres were 73 ± 7, 199 ± 11 and 591 ± 16 mg/m³ for the low, mid and high concentration test atmosphere, respectively.

Airflow:

Mean airflow through the control unit was 33.2 L/min, mean airflow through the low concentration unit was 28.5 L/min. The total airflow through the mid and high concentration unit could not be established, but was minimally 20.5 and 28.5 L/min, respectively.

Nominal concentration:

Nominal concentrations of the mid and high concentration test atmosphere could not be calculated. The mean nominal concentration of the low BL vapour test atmosphere was 112 ± 29 mg/m³, which was of the same order of magnitude as the actual concentration.

Temperature and relative humidity:

The daily mean temperatures in the test atmospheres were between 20.8 and 21.2 °C, the daily mean relative humidities were between 38 and 43%.

Applicant's summary and conclusion

Conclusions:

In a sub-acute inhalation toxicity study, Butyl-S-lactate (98.9% purity) showed no effects on survival, body weight, organ weights and gross pathology. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 600 mg/m³. Therefore, the NOAEC (local) for male rats is considered to be 200 mg/m³ and as no systemic effects were noted, the NOAEC (systemic) for male rats is considered to exceed 600 mg/m³.

Executive summary:

In a sub-acute inhalation toxicity study, toxicity of the test item (purity 98.9 %) was examined in male Wistar rats. Groups of 6 male rats were exposed nose-only to target vapour concentrations of 0, 75, 200 or 600 mg/m³ test item for 6 hours a day, 5 days a week during a period of 4 weeks (a total of 20 exposure days). No adverse effects were observed on survival, body and organ weights, and gross pathology. The most prominent finding consisted of histopathological changes in the nasal tissues, which showed transitional epithelium hyperplasia in almost all animals exposed to 600 mg/m³ test item but not in animals exposed to lower concentrations. These nasal changes are related to treatment and point to an irritating effect of Butyl-S-lactate on the nasal epithelium. Therefore, the local NOAEC is considered to be 200 mg/m³. The systemic NOAEC is considered to exceed 600 mg/m³ as no systemic effects were observed. This sub-acute toxicity study in the rat is acceptable and satisfies the guideline requirement according to OECD 412 for a sub-acute inhalation study in the rat.