Methyl (S)-(-)-lactate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996-04-04 to 1997-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Principles of method if other than guideline:

The sensory irritation study was carried out in male rats and mice to provide data on the irritating properties of both substances after a single inhalation exposure to the test material. In case significant reductions in the breathing rate were observed, the RD50 was calculated, viz. the concentration at which a 50% reduction of the respiratory rate is obtained (Alarie, 1973 and 1981; ASTM; 1984). Furthermore, clinical symptoms, body and lung weights and gross observation at necropsy seven days after exposure were used to examine the toxicity of the test material.

GLP compliance:

yes (incl. QA statement)

Test type:

other: sensory irritation

Limit test:

no

Test material

Test material information

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 the test substance: ethyl-(L)-lactate
- Product name: Ethyl Lactate Elect
- Batch no.: ET003DN
- Appearance: liquid
- Purity: 99.8%

Test animals

Species:

other: rat and mouse

Strain:

other: Wistar rats and Swiss CD1 mice

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation:rats and mice: 5-6 weeks old at the day of arrival, 10-11 weeks (group A) and 12-14 weeks (other groups) old at the day of exposure to the test substance
- Weight at study initiation: rats: average weight per rat in a group: 324-348 g, mice: average weight per mouse in a group: 38.7-45.3 g
- Fasting period before study: no
- Housing: the first 6 or 7 days in cages in quarantine, thereafter the rats were housed four per cage in stainless wire-mesh cages, the mice were individually housed in macrolon cages with sterilized saw dust bedding.
- Diet (e.g. ad libitum): ad libitum, cereal-based rodent diet prepared by SDS Special Diets Services, Witham, England
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: Group A: 28/29 days, other groups: 37-42 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): group A: 19.0-23.0, other groups: 19.5-26.0
- Humidity (%): group A: 40-70, other groups: 44-70%, however, higher relative humidity values of up to 84% were reached during a total period of about 7 days
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

other: vapour and aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

0.8 µm

Geometric standard deviation (GSD):

4

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in an experimental set-up of the TNO-Institute's design. The animals were secured in modified animal restrainers (Battelle) and placed individually in one of the four plethysmographs connected to a central exposure chamber (diameter 53 mm). The animals were allowed to breath freely from a stream of fresh test atmosphere. Pressure transducers connected to the plethysmographs were sensing changes caused by inhalation and exiperation. During exposure of the mice, a void filling was placed in each of the plethysmographs to reduce the chamber volume in order to maximize the signals. The amplified signals were transmitted to a polygraph recorder for off-line manual calculations.
- System of generating particulates/aerosols: The test atmospere was generated by passing metered amounts of the test material using a roller pump (Gilson, Villiers le Bel, France) to an all glass atomizer (Institute's design). In order to generate low concentrations of ethyl lactate, the resulting test atmosphere was mixed with a measured amount of dry pressurized air (relative humidity < 1%), using a rotameter. The resulting test atmosphere was directed to the exposure unit towards the location of the anmials and finally passed to the exhaust. Before and after exposure (during the acclimatization and recovery period) the animals breathed dry pressurized air.
- Source and rate of air: Air flow was 16.8 L/min during most ethyl lactate exposures However, at the lower concentrations tested, air flow was increased to 31.2 L/min (exposure C (mice) and exposure D (rats and mice)).
- Method of particle size determination: Particle size analysis was carried out at the location of the animals in absence of the animals. A 10-stage Andersen cascade impactor was used to measure particle size distribution in the test atmosphere. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometric standard deviation (gsd) were calculated.
- Temperature, humidity, pressure in air chamber: The temperature was between 21.4 and 22.9 °C. Except for the first exposure, relative humidity was low, viz. lower than 10% due to the use of dry pressurized air.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentrations in the test atmosphere was determined by means of gas chromatographic analysis. During each 30-min exposure period, a measured test atmosphere sample (0.91-0.95 L/min, total sampling time between approximately 19 and 25 min) was drawn through two impingers in series filled with acetone (p.a. quality). After sampling the content of each impinger was quantitatively transferred to a 50-ml volumetric flask. Each sample was injected once. To determine the concentration of the test atmosphere samples, the peak area of the ethyl lactate was compared with that of standard solutions containing ethyl lactate (0.0257, 0.1285, 0.6423 and 3.2116 mg/mL) in acetone. To prepare a calibration graph, each standard solution was injected twice. Besides, a second standard solution of 3.2116 mg/mL ethyl lactate was also injected twice and was in good agreement with the first standards. A calibration graph with a correlation coefficient of 0.9999 was obtained. Until analysis, standard solutions and samples were kept at approximately 4 °C.
- Samples taken from breathing zone: no, sampling ports for the actual concentration measurement and for the temperature and relative humidity were located in the exhaust pipe, just above the central exposure chamber.

TEST ATMOSPHERE:
- Particle size distribution: see Table 2 in box "Any other information on material and methods incl. tables". For ethyl lactate it was shown that from the particles still present, approximately 80% present at the animals' breathing zone was smaller than or equal to 5.0 µm. The total amount of aerosolized material captured was approximately 30 mg/m³, which was far less than the actual concentration of about 4.02 g/m³ or the nominal concentration of about 5.4 mg/m³.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

30 min

Concentrations:

Rats: 0.806, 1.626, 1.881 and 4.020 g/m³
Mice: 0.333, 0.812, 1.718 and 3.000 g/m³

No. of animals per sex per dose:

4 males per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 7 days

- Frequency of observations and weighing: The rats and mice were visually inspected before, during and after exposure, and up to seven days after exposure until necropsy for signs of ill health, reaction to treatment and mortality. However, clinical signs (or absence of clinical signs) were explicitly recorded before exposure and during the 7-day observation period only. During exposure, except for a single remark, only the breathing rate was recorded whereas at the end of the exposure day only a general statement was made in the animal log-book. Body weights were recorded just prior to exposure (day 0), one day after exposure and just before necropsy.

- Respiratory irritation: The groups of rat and mice were exposed by inhalation for a single 30-min period, after an acclimatization period of at least 30 minutes. A recovery perod of at least 20 minutes in the animal restrainers was allowed after exposure. The control respiratory rate was measured during the acclimatization period as the average of six 21-sec periods recordd at 1-min intervals immediately preceding the exposure period. Durig th exposure period, the respiratory rate was measured for 21-sec periods at 1-min intervals during the first eleven minutes of exposure and at 3-min intervals for the remainder of the exposure period. After exposure, rates were measured eleven times for 21 seconds at 1-min intervals after exposure stop, followed by 3-min intervals. The relative breathing rate was calculated as: (exposure value/ mean pre-exposure value) x 100%, or, (recovery value/ mean pre-exposure value) x 100%. Since the decrease in breathing rate was around or more than 50% at the first exposure in both species, three more groups of rats and mice were exposed to ethyl lactate. The minimum decrease considered significant was 12%.

- Necropsy of survivors performed: yes, seven days after exposure animals were killed by exsanguination from the abdominal aorta under ether anaesthesia. All animals were necropsied and examined for gross pathological changes.

- Other examinations performed: The lungs (with trachea and larynx) were weighed.

Statistics:

During all exposures of the mice a monotonic, asymptotic decrease in breathing frequency was observed with time. Consequently, minimum values (maximum decrease) were seen towards the end of the exposure period. The RD50 value was calculated using the maximum decreases, in this case the mean of the three last measurements during exposure (viz. at 23, 26 and 29 min). The regression line, R = a + b logC (in which R is response and C is concentration in mg/m³) through the mean response of the four murine exposure groups at 23, 26 and 29 min was determined according to Alarie (1973; 1981). In view of the monotonic, asymptotic decrease during almost the entire exposure period, the RD50 value was also calculated using the mean of the reactions through the exposure period, after the first 5 minutes. According to a method described by Bos et al. (1992), it was checked whether a concentration-response-relationship was present which would point at other (toxic) actions but sensory irritation.
For rats, maximal responses were observed at about 7, 8 and 9 minutes after the start of the exposure; thereafter increases in breathing rate were noted. The RD50 values was therefore calculated using the mean of the three measurements at 7, 8 and 9 minutes (Barrow et al., 1977).

Results and discussion

Effect levels

Effect levels 1

Sex:

male

Dose descriptor:

other: RD50, rat

Effect level:

791 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

30 min

Effect levels 2

Sex:

male

Dose descriptor:

other: RD50, mouse

Effect level:

772 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

30 min

Mortality:

None of the animals died.

Clinical signs:

other: No abnormalities were observed before, during and after exposure. During the 7-day observation period, no abnormalities were observed except for alopecia in the neck region of one rat.

Body weight:

The day after exposure, slight body weight gain or slight body weight loss was observed in all animals, which was explained by stress induced by the exposure conditions. Seven days after exposure all rats had gained weight whereas most mice had gained weight too.

Gross pathology:

In both species there was no increace in relative lung weight with increasing concentration. Macroscopic changes at necropsy were limited to grey discoloured or irregular surfaced lungs, with grey stains and/or grey/black, white or red. somewhat swollen spots or specks. A concentration-response relationship was not observed.

Other findings:

Respiratory irritation:
The RD50 was calculated using the maximum decreases, in this case the mean of the tree last measurements during exposure (viz. at 23, 26 and 29 min). The regression line through the mean response of the four exposure groups at 23, 26 and 28 min was R = 1.5663 - 0.3693 logC, with a correlation coefficient of -0.9047. From this regression equation for mice a RD50 of 772 mg/m³ was calculated. In view of the monotonic, asymptotic decrease during almost the entire exposure period, the RD50 value was also calculated using the mean of the reactions through the exposure period, after the first 5 minutes. An RD50 value of 886 mg/m³ was obtained which was slightly higher (as could be expected) than that obtained using the three last measurments, showing the maximum responses. According to a method described by Bos et al. (1992), it was checked whether a concentration-response-relationship was present which could point at other (toxic) actions but sensory irritation. In this study in mice with ethyl lactate there was no concentration-response relationship. After exposure to the three lowest levels, relative breathing frequencies increased and almost reached pre-exposure values (up to 95%); the recovery ion relative breathing rate after the first exposure to 3000 mg/m³ reached a highest level of 70% only.
For rats, maximal responses were observed at about 7, 8 and 9 minutes after the start of the exposure; thereafter increases in breathing rate were noted. The RD50 value was therefore calculated using the mean of the trhee measurements at 7, 8 and 9 minutes. The regression line was: R = 1.8160 - 0.4541 logC (C in mg/m³) with a correlation coefficient of 0.8981. From this regression equation an RD50 value of 791 mg/m³ was estimated which turned out to be in good agreement with that observed in mice. The Bos method again did not show any concentration-response relationship. After exposure, relative breathing frequencies increased and reached highest values of up to 80-87%.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The RD50 values of Ethyl (S)-lactate were 772 mg/m³ in mice and 791 mg/m³ in rats. In the same study an RD50 of 760 mg/m³ in mice and 701 mg/m³ in rats was found for n-butyl-L-lactate. The similarity in the results supports the conclusion, that the effects on breathing rate can be attributed to irritating effects from the lactic acid formed at hydrolysis of the substances, which is a rapid process during and after uptake in the organism. The sensory irritating effect is thus a pH effect and not a toxic effect.

Executive summary:

The airway irritating properties of Ethyl (S)-lactate were studied by exposing four groups of male rats and mice for a single period of 30 minutes. All groups consisted of four animals. Concentrations of ethyl lactate were between 0.333 and 4.020 g/m³. Animals were necropsied seven days after exposure. No abnormalities were observed before, during and after exposure, and during the 7-day observation period, no treatment-related abnormalities were observed. None of the animals died. The day after exposure, slight body weight gain or slight body weight loss was observed in all animals. Seven days after exposure all rats had gained weight whereas most mice had gained weight too.

In both species after exposure there was no increase in relative lung weight with increasing concentration. Macroscopic changes at necropsy were limited to grey discoloured or irregular surfaced lungs, with grey stains and/or grey/black, white or red, somewhat swollen spots or specks. A concentration-response relationship was not observed.

From the results of the present study, it was concluded that RD50 values of Ethyl (S)-lactate were 772 mg/m³ in mice and 791 mg/m³ in rats. In the same study an RD50 of 760 mg/m³ in mice and 701 mg/m³ in rats were found for n-butyl-L-lactate. The similarity in the results supports the conclusion, that the effects on breathing rate can be attributed to irritating effects from the lactic acid formed at hydrolysis of the substances, which is a rapid process during and after uptake in the organism. The irritating effect on breathing is thus a pH effect and not a toxic effect.