Propanoic acid, 2-hydroxy-, propyl ester, (2S)-

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:

1990-01-24 to 1995-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl (S)-2-hydroxypropionate

EC Number:

211-694-1

EC Name:

Ethyl (S)-2-hydroxypropionate

Cas Number:

687-47-8

Molecular formula:

C5H10O3

IUPAC Name:

ethyl 2-hydroxypropanoate

Specific details on test material used for the study:

- Name of test material used in the report: ethyl lactate
- Trade name: Purasolv EL
- Batch number: EA 0188F
- Appearance: liquid
- Purity: 99.8%
- Storage: at room temperature, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF-reared, Wistar derived rats were obtained from Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: mean weight 198 g (male), 149 g (female)
- Fasting period before study: half an hour before the start of the exposure and during the exposure the animals had no access to food or water
- Housing: the rats were individually housed in Hazleton 1000 inhalation chambers
- Diet (e.g. ad libitum): the rats were provided ad libitum with the Institute's stock diet. Fresh food was supplied twice weekly.
- Water (e.g. ad libitum): the rats were provided ad libitum with unfluoridated community tap water from an automatic drinking system
- Acclimation period: the animals were acclimatised for one week in a cleaned and disinfected inhalation chamber

ENVIRONMENTAL CONDITIONS
- Temperature (°C): generally between 22 and 23 °C, with minimum/maximum temperature of 21/26.5 °C
- Humidity (%): generally between 40 and 65%, with min/max of 37/82%
- Air changes (per hr): between 15 and 22
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: vapour

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed tot the test atmosphere in H 1000 multitiered inhalation chambers manufactured by Hazleton Systems Inc., USA. The chambers were constructed of stainless steel with glass doors on two sides, which allows for observation of the animals during exposure.
- Method of holding animals in test chamber: The rats were housed individually in wire mesh stainless steel cages.
- System of generating particulates/aerosols: The test atmospheres were generated by nebulizing ethyl lactate. During nebulizing the aerosol particles evaporated. For the generation of the aerosol ethyl lactate was metered by roller pumps to air driven nublizers. For the two highest concentration levels Mikro-2 nebulizers (Spuittechniek b.v., the Netherlands) and for the lowest concentration level a Lee Check TA 150 liquid dispenser (Lee Co., USA) was used. The aerosol was sprayed into the inhalation chamber at the top level. To allow all aerosol particles to mix with air and evaporate, the animals were placed in the cage unit located at the right bottom level.
- Temperature, humidity, pressure in air chamber: the temperature in the inhalation chambers varied generally between 22 and 23 °C, with a minimum/maximum of 21/26 °C. The relative humidity could generally be maintained between 40 and 65%, with a min/max of 37/82%. Each chamber was fitted with a micro manometer which showed the negative pressur inside (5-52 Pas).
- Air flow rate: Total air flow through the chambers varied between 36 and 50 m³/hour.
- Air change rate: between 15 and 22 air changes per hour


TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test material was determined approximately twice each hour by means of a total hydrocarbon analyzer. Atmosphere samples were taken continuously from each of the chambers at a location close to the cage unit in which the animals were housed. The samples were drawn through sampling lines and passed via a computer controlled valves system to the total hydrocarbon analyzer. The response of the hycrocarbon analyzer (mV's) was recorded and converted into concentration values (mg/m³). To ensure that no test material would condensate during sampling the sampling lines and the valve system were heated. Each chamber was monitored approximately once each half hour during a period of approximately 7 minutes, resulting in 11 concentration values per concentration-level per day.
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the study was started, the response time of the flame ionization detector (FID) of the total hydrocarbond analyzer (Ratfish RS 55, W. Germany) was calibrated by passing flows of 150, 600, or 2500 mg/m³ over the FID. The calibration mixtures were prepared by injecting calculated quantities of the test material in teflon bags, which were filled with 25 L of air. After evaporation of the test material and careful mixing with the air, samples were passed from the bag through the total hydrocarbon analyzer. The response of the FID (in mV's) was recorded by a HP Vectra PC. The concentration of the test material was determined approximately twice each hour by means of a total hydrocarbon analyzer. Atmosphere samples were taken continuously from each of the chambers at a location close to the cage unit in which the animals were housed. The samples were drawn through sampling lines and passed via a computer controlled valves system to the total hydrocarbon analyzer. The response of the hycrocarbon analyzer (mV's) was recorded and converted into concentration values (mg/m³). To ensure that no test material would condensate during sampling the sampling lines and the valve system were heated. Each chamber was monitored approximately once each half hour during a period of approximately 7 minutes, resulting in 11 concentration values per concentration-level per day.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

150 mg/m³ air (nominal)

Remarks:

Low concentration,
Mean daily concentration (analytical): 145 +/- 2 mg/m³

Doses / concentrations 3

Dose / conc.:

600 mg/m³ air (nominal)

Remarks:

Mid concentration,
Mean daily concentration (analytical): 603 +/- 2 mg/m³

Doses / concentrations 4

Dose / conc.:

2 500 mg/m³ air (nominal)

Remarks:

High concentration,
Mean daily concentration (analytical): 2451 +/- 21 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The concentrations were selected based on the results of an acute inhalation study with ethyl lactate (see IUCLID section 7.2.2). During that study it was found that rats could be exposed to an actual concentration level of 5.4 g/m³ for a period of 4-hours without mortalities or severe clinical signs.
- Rationale for animal assignment (if not random): The study rats were allocated - males and females separately - to the four groups by computer randomization proportional to body weight.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were visually observed for mortality, clinical symptoms and behaviour before the start of the exposure and after the exposure. During the weekends the anmials were observed only once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the weekly weighings the animals were carefully examined for health condition and clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each anmial was recorded during the acclimatization period on day -7 and -1, and just before the start of the first exposure to the test material (day 0), weekly thereafter, and at autopsy in order to determine the organ-to-body weight ratios.

FOOD CONSUMPTION: Yes
Food consumption were determined once weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples of blood were collected from the tip of the tail of all animals at day 26
- Anaesthetic used for blood collection: No specified
- Animals fasted: Yes, before blood collection animals were deprived of food and water for a period of about 24 hours
- How many animals: all animals
- Parameters examined: Differential white blood cell count; haemoglobin; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); mean platelet volume (MPV); packed cell volume; platelet distribution width (PDW); prothrombin time; red blood cells; red blood cell distribution width (RDW-SD) and thrombocytes (platelets).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: glucose was determined in blood from fasted rats (24 hours) collected from the tip of the tail on day 26, the other parameters were determined in blood collected from the abdominal aorta at autopsy.
- Animals fasted: No (except for glucose; 24 hours fasted)
- How many animals: all animals
- Parameters examined: alanine aminotransferase (ALAT) / glutamic-pyruvic transaminase (GPT); albumin; alkaline phosphatase (ALP); aspartate amino-transferase (ASAT) / glutamic-oxalacetic transaminase (GOT); bilirubin direct; bilirubin total; chloride (Cl); creatinine; gamma glutamyl transferase (gammaGT); glucose (blood); inorganic phosphate; potassium (K); sodium (Na); total protein and urea.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

At the end of the treatment period, all rats were sacrificed according to a stratified-random method, so that, on the average, time of killing was approximately the same for each group. The animals were killed by exsanguination via the abdominal aorta under ether anaesthesia and then examined grossly for pathological changes.

GROSS PATHOLOGY: Yes, samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde: adrenals, heart, kidneys, liver, lungs with tranchea and larynx, nose, spleen, testes and all gross lesions. All but the heart, nose and gross lesions were weighed. Lungs were inflated with the fixative at 10 cm water pressure.

HISTOPATHOLOGY: Yes, the tissues required for microscopical examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed on the adrenals, heart, kidneys, liver, lungs with tranchea and larynx, nose, spleen, testes and all gross lesions of all animals of the control group and the top-concentration group. The nose was also examined of all animals from the intermediate concentration groups.

Statistics:

Body weights were analyzed by an alaysis of co-variance followed by the Dunnett's multiple comparion test. Incidences of histopathological changes were analyzed by the Fisher exact probability test. Analysis of variance followed by the Dunnett's multiple comparison tests or the Mann/Whitney u-test were applied to food consumption, organ weights, the haematological and clinical chemistry data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

see box "Details on results"

Mortality:

no mortality observed

Description (incidence):

see box "Details on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see box "Details on results"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see box "Details on results"

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

see box "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see box "Details on results"

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see box "Details on results"

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see box "Details on results"

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Exposure to ethyl lactate did not result in changes in condition, health or mortality. One male exposed to 600 and 2 females exposed to 2500 mg/m³ showed transient trembling.

BODY WEIGHT AND WEIGHT GAIN
All animals gained weight during the exposure period, but the rats of the 2500 mg/m³ group considerably less than the other rats. The differences with controls was statistically significant at all stages. Mean weight gain of the other test groups was comparable with that of the controls both in males and in females.

FOOD CONSUMPTION
Food consumption was depressed in rats exposed to 2500 mg/m³ throughout the study. In males the differences with the controls were statistically significant during all weeks, and in females during the first two weeks and during the last week. Food intake was statistically significantly lower in males exposed to 150 mg/m³ than in controls during the last week. During the first three weeks food consumption tended to be even higher than in controls. In addition, males of the 600 mg/m³ group showed a completely comparable food consumption pattern as the controls. Therefore, the decreased food consumption value in the low-concentration group is considered an isolated finding unrelated to the exposure.

HAEMATOLOGY
The haematological variables examined did not show differences amongst the groups attributable to treatment. The values showed the common variation between the individual animals and between the groups.

CLINICAL CHEMISTRY
Urea values were lower in rats exposed to 2500 mg/m³ than in controls, reaching a statistically significant extent in females only. Glucose values were statistically significantly higher in males of the highest-concentration group when compared with those of the controls. The other biochemical parameters examined did not show differences which could be related to the exposure to ethyl lactate. One rat exposed to 150 mg ethyl lactate/m³ air had very high levels of aspartate aminotransferase and alanine aminotransferase in plasma. These were considered isolated findings unrelated to treatment.

ORGAN WEIGHTS
Absolute liver weights were statistically significantly decreased in males and females exposed to 2500 mg/m³ in comparison with liver weights of the controls. However, when the liver weights were expressed relative to body weight, they did not differ statistically significantly from that of the controls. Relative weights of testes and adrenals were statistically significantly higher in male rats exposed to the highest concentration, whereas the absolute weights of these organs were completey comparable with those of the controls.

GROSS PATHOLOGY
Gross examination at autopsy revealed one control male with small testis and one male of the low dose group with two small testes. These gross changes are common findings in rats. They are, therefore, considered not to be related to treatment. No other gross findings were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment related changes were observed in the nose, both in the respiratory and the olfactory epithelium. Minimal changes occurred in the low dose group; incidence and severity increased with the concentration levels.
- Respiratory epithelium:
The changes consisted of hyperplasia and hypertrophy of goblet cells, mainly in epithelium of the nasal septum and ventrolateral parts of the nasal cavity and, in more severe cases, also of the nasoturbinates. Frequently, these changes were accompanied by nest-like infolds of goblet cells. The effect on the respiratory epithelium was found in all treatment groups and the severity was distinctly concentration-related. All animals of the 600 mg/m³ group and most animals of the 2500 mg/m³ group showed hyperplasia of the ciliated epithelium covering the nasal turbinates and the lateral walls. Two males and two females of the 600 mg/m³ group showed a few small cysts in the hyperplastic respiratory epithelium of the nasoturbinates.
- Olfactory epithelium:
The changes mainly occurred in the dorsal part of the nose and consisted of moderate to very severe atrophy with epithelial disarrangement, ingrowth in the lamina propria and replacement of olfactory epithelium by respiratory epithelium with nest-like infolds. The olfactory epithelial atrophy was characterized (in order of increasing severity) by: disarrangement, disappearing of the cellular apices, and flattening of the cells and thinning of the olfactory layer as a whole. In several animals exposed to 2500 mg/m³ locally there was hardly any epithelium recognizable.
In most animals of the 2500 mg/m³ group loss of nerve bundels and Bowman's glands was observed in the lamina properia of the dorsal part of the nasal cavity. The loss was most obvious in the caudal parts. Inflammatory changes occurred only in a few animals of the high dose group.
Apart from slight hypertrophy and hyperplasia of goblet cells in two males none of the control animals showed histopathological nasal changes.

The histopathological changes observed in the other organs examined are common findings in the strain of rats used. These lesions were about equally distributed amongst control and test groups, or occurred only in a single animal. Therefore, they are not considered to be related to the exposure to the test substance.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

< 150 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

2

Key result

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

2 500 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no indications of clear systemic effects associated with the treatment.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical results:

Mean daily actual concentrations turned out to be 145 (± 2), 603 (± 2) and 2451 (± 21) mg/m³. Between brackets the standard error of the mean (SEM) is given. The nominal concentrations were 138, 569, and 2092 mg/m³, which is slightly lower than the actual concentrations. The nominal concentration is calculated from the use of test material during exposure and the volume of air passed through the inhalation chamber. The determination of the volume of air through the chambers, however, is not very exact. This might be an explanation for the differences between the nominal and actual concentrations. Occasionally, the nebulizers did not work well during a short period, resulting in low mean daily concentrations with a high SEM.

Applicant's summary and conclusion

Conclusions:

In a sub-acute inhalation toxicity study, Ethyl (S)-lactate (99.8% purity) showed no effects on condition, health and behaviour. The most prominent finding consisted of histopathological changes to the lining epithelium of the nasal cavity of rats. Based on the results, the NOAEC (local) for rats is considered to be < 150 mg/m³ and as no clear systemic effects were noted, the NOAEC (systemic) for rats is considered to be 2500 mg/m³.

Executive summary:

In a sub-acute inhalation toxicity study ethyl lactate was administered to 5 male and 5 female Wistar derived rats/concentration by whole body exposure at concentrations of 0, 1.5, 0.6 and 2.5 mg/L (0, 150, 600 and 2500 mg/m³) for 6 hours per day, 5 days/week for a total of 28 days.

The exposure to ethyl lactate resulted in concentration-related adverse effects in the nose of all test groups and in growth retardation and decreased food consumption in rats exposed to 2500 mg ethyl lactate/ m³ air. Growth retardation might be explained by the impaired ability to smell and taste as a result of severe damage to the olfactory epithelium. The increased blood glucose value in males exposed to 2500 mg/m³ is considered an isolated finding unrelated to treatment. Further all observed effects can be explained from the reduced food intake and subsequent growth retardation. It was concluded that the NOEC (local) was lower than 150 mg/m³ and the NOEC (systemic) is considered to be 2500 mg/m³.

This sub-acute toxicity study in the rat is acceptable and satisfies the guideline requirement (OECD 412) for a sub-acute inhalation study in the rat.