Ethyl (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:

1991-02-15 to 1991-04-12

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:

yes

Remarks:

not measured: food consumption, haematological and clinical chemistry, organ weight determinations, and microscopical examination of tissues and organs other than the nose.

Principles of method if other than guideline:

A sub-acute (28-day) inhalation toxicity study followed by a (28-day) recovery period was conducted in rats to provide data on recovery from, persistence of, or delayed occurrence of toxic effects on the nose. In a previous study (TNO report
no. V 90.322, see IUCLID section 7.5.2), ethyl lactate was irritating and toxic to the lining epithelium of the nasal cavity at levels of 150, 600 and 2500 mg/m³. Since it is necessary for the health risk evaluation of ethyl lactate to obtain a no-toxic effect level, an additional study had to be performed.

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 the test substance: etyhl-(S)(-)-lactate
- Trade name: Purasolv Elect
- Batch number no: EK 3003
- Appearance: clear liquid
- Expiry date: 31-01-1992
- Purity: 99.7%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rats (Hsd/Cpb:WU) were obtained from Charles River Wiga, Sulzfeld, FRG
- Age at study initiation: 5-6 weeks old at arrival in the Institute; 6-7 weeks old at the start of the treatment
- Weight at study initiation: 196.7 g (males) and 143.4 g (females)
- Fasting period before study: no, but during exposure both control and test animals had no access to food or water
- Housing: During the acclimatization period (days -7 until -1) and during the main part of the recovery period (days 31-56) the rats were housed in groups of five, separated by sex in suspended, stainless steel cages, fitted with wire mesh floor and front. During the exposure period the rats were housed individually in wire mesh stainless steel cages in a modified H 1000 multitiered inhalation chamber manufactured by Hazleton Systems Inc., USA.
- Diet (e.g. ad libitum): except during exposure, the rats were maintained on the Institute's cereal-based, powdered stock diet for rats, mice and hamsters.
- Water (e.g. ad libitum): except during exposure, community tap water was supplied in glass bottles which were cleaned once weekly
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): generally between 20-24 °C during exposure, and between 19 and 25 °C before and after exposure. Occasionelly, levels of up to 25.5 °C were reached.
- Humidity (%): The relative humidity generally fluctuated between 40-70%, and during several days in inhalation chamber C (75 mg/m³; values of up to 77%). In addition, due to wet cleaning activities the relative humidity exceeded the upper limit, the peaks eaching maxima up to 97% for about half an hour each time.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: vapour

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in modified H1000 multitiered inhalation chambers manufactured by Hazleton Systems Inc., USA. The chambers have a pyramidal top and bottom. The chambers have been constructed of stainless steel with glass doors on two sides which allow 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: To generate the test atmospheres, metered amounts of ethyl lactate were transported by roller pumps (Watson-Marlow 502S/R) to pressurized air driven nebulizers (Lee dispenser, type 110 K, Lee Co., USA). The generated mixture of air and test material was diluted with filtered air till the required concentration was achieved. To allow all particles to mix with air and to evaporate, the animals were located in the cage unit at the right bottom level of each inhalation chamber. Animals in the control group were exposed to freshly filtered air under similar conditions as the test groups.
- Temperature, humidity, pressure in air chamber: The temperature fluctuated between 20-24°C during exposure. The relative humidity generally fluctuated between 40-70%, but during several days in inhalation chamber C (75 mg/m³) values up to 77% were reached. In addition, due to wet cleaning activities the relative humidity exceeded the upper limit, the peeks reaching maxima up to 97% for about half an hour each time. Each chamber was fitted with a micromanometer which showed the negative pressure inside (1-4 mm water column).
- Air flow rate: Airflow was monitored by means of an anemometer (Thermo-Air type 442) and was recorded three times each exposure day. Total airflow through the chambers varied between 38 and 40 m³/hour.
- Air change rate: between 17 and 18 air changes per hour during exposure, otherwise 10 air changes per hour.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test material was determined by means of a total carbon analyser (Ratfisch RS 55, FRG). Test atmosphere samples were taken sequentially 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 were passed via a controlled valves system (Kuax-Control, Kuhnke 61.000) to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). To ensure that no test material would condensate during sampling transport the sampling lines and the valves system were heated. Each chamber was monitored approximately once each half an hour for about 7 minutes, resulting in about 12 measurements 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:

The concentration of the test material was determined by means of a total carbon analyser (Ratfisch RS 55, FRG). Before the start of the study the response of the flame ionization detector (FID) was calibrated by passing flows of 25, 75, and 200 mg/m³ over the FID. The calibration mixtures corresponding to these flows, were prepared by injecting known quantities of test material (0.7, 2.2, or 5.8 µL) in 40-L teflon bags, which were filled with 30 L of air. After evaporation of the test material and careful mixing with the air, samples were passed to the total carbon analyser. The response of the FID (in scale units) was recorded. The calibration procedure was repeated towards the end of the exposure period. Similar data were obtained. The nominal concentration was determined by dividing the total daily amount of test substance used per treatment group by the total volume of air passed through each exposure unit.

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

Doses / concentrations 2

Dose / conc.:

25 mg/m³ air (nominal)

Remarks:

Low concentration;
Mean daily concentration (analytical): 24 (+/- 1) mg/m³

Doses / concentrations 3

Dose / conc.:

75 mg/m³ air (nominal)

Remarks:

Mid concentration;
Mean daily concentration (analytical): 73 (+/- 5) mg/m³

Doses / concentrations 4

Dose / conc.:

200 mg/m³ air (nominal)

Remarks:

High concentration;
Mean daily concentration (analytical): 202 (+/- 3) mg/m³

No. of animals per sex per dose:

5, for the control and 200 mg/m³ exposure group 5 additional rats/sex were exposed (recovory group).

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: In a previous study, ethyl lactate was irritating and toxic to the lining epithelium of the nasal cavity at levels of 150, 600 and 2500 mg/m³. Clinical observations, growth, gross examination at autopsy and microscopical examination of the nose were used as criteria for disclosing possible harmful effects at lower concentrations of 25, 75 and 200 mg/m³.
- Rationale for animal assignment (if not random): One day before the start of the study, the rats were divided over the various groups in such a way that the mean initial body weight was about the same in all groups (< ± 20% of the mean weight). A few rats were replaced by reserve animals in order to equalize the mean initial body weight. Computer randomization according to body weight was not possible because the number of animals in each group was not the same (groups B and C comprised of less animals than groups A and B). Computer randomization was, however, performed upon arrival.
- Rationale for selecting satellite groups: To provide data on recovery from, persistence of, or delayed occurrence of toxic effects on the nose.
- Post-exposure recovery period in satellite groups: Following the exposure period, rats of the recovery groups (Control and 200 mg/m³) were kept for a 28-day period without treatment.
- Section schedule rationale (if not random): On nominal day 28, all rats of the main groups, and on day 56, all rats of the recovery groups, were killed in such a sequence that the average time of killing was about the same for each group.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The general condition and behaviour of all animals were checked at least once daily throughout the study. All signs of ill health, reaction to treatment or mortality were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was individually handled and carefully examined for clinical signs, abnormal behaviour or abnormal appearance at the weekly weighing.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights of all rats were recorded during the acclimatization period (day -4), one day before the start of the exposure (allocation procedure), just prior to the first exposure to the test substance (day 0), and subsequent at weekly intervals (including the day of autopsy).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

On nominal day 28, all rats of the main groups, and on nominal day 56, all rats of the recovery groups, were killed in such a sequence that the average time of killing was about the same for each group. The animals were anaesthetized by ether, bied to death by cannulating the abdominal aorta and then examined macroscopically for pathological changes.

GROSS PATHOLOGY: Yes, the animals were examined macroscopically for pathological changes.

HISTOPATHOLOGY: Yes, the nose of all animals was preserved in a neutral, aqueous, phosphate-buffered, 4% solution of formaldehyde, decalcified, embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed on 4 sections of the nose of each animal.

Statistics:

Body weight data of the main groups were analysed by one-way analysis of covariance using preexposure (day 0) weights as the covariate. When group means were significantly different (p< 0.05) individual pairwise comparisons were made using Dunnett's multiple comparison method. Body weight data of the recovery groups were analysed by Student t-test. Incidence of histopathological changes were analysed 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.

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:

no effects observed

Description (incidence and severity):

see box "Details on results"

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

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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

see box "Details on results"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see box "Details on results"

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND SURVIVAL
No clinical symptoms or signs of systemic toxicity were observed during the study. All study animals survived to the scheduled sacrifice.

BODY WEIGHTS
There were no statistically significant differences in mean body weights between the test groups and the controls.

GROSS PATHOLOGY
Gross examination of animals of the main groups at autopsy revealed a slightly oedematous and red discoloured thymus in one male of the control
group and in one male of the mid-concentration group. The kidneys of one male of the control group were slightly enlarged and soft. These gross changes are common findings in rats and they are not considered to have any toxicological relevance. One female rat of the mid-concentration group showed a subcutaneous haemorrhage between the eyes, which was most probably caused
by trauma and was not regarded to have any clinical significance. Gross examination of rats of the recovery groups did not reveal any abnormalities.

HISTOPATHOLOGY: NON-NEOPLASTIC
In a few rats of either sex of both the main and the recovery groups (controls included), the respiratory epithelium of the nasal cavity showed minimal changes, seen as nest-like infolds and slight hypertrophy/hyperplasia. The changes were about equally distributed amongst the various groups or occurred in a single animal only. Therefore, the changes were not ascribed to inhalation of ethyl lactate.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

200 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

2

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

200 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects observed at the high concentration of 200 mg/m³

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical results:

The mean daily concentrations of ethyl lactate in the test atmospheres during exposure were 24 (+/- 1) mg/m³, 73 (+/- 5) mg/m³ and 202 (+/- 3) mg/m³. The actual concentrations were generally close to the intended concentrations.

Applicant's summary and conclusion

Conclusions:

In a sub-acute inhalation toxicity study in rats, Ethyl (S)-lactate showed no effects adverse effects. There were no compound related effects in mortality, clinical signs, body weight or gross and histological pathology. Microscopic examination revealed minimal changes (nest-like infolds, slight hypertrophy, and slight hyperplasia) in the nasal respiratory epithelium of animals in all groups, the control group included. Type and incidence of the changes were similar in the various groups, or the changes occurred in a single animal only. These changes were not ascribed to treatment. Based on the results, the NOEC is considered to be 200 mg/m³.

Executive summary:

In a sub-acute inhalation toxicity study, Ethyl (S)-lactate was administered to 5 male and 5 female Wistar rats/concentration by whole body exposure at concentrations of 0, 0.025, 0.075 and 0.2 mg/L (0, 25, 75 and 200 mg/m³) for 6 hours per day, 5 days/week for a total of 28 days. Two additional (recovery) groups of 5 male and 5 female Wistar rats exposed to 0 or 0.2 mg/ were kept untreated for 28 post-exposure days. Clinical observations, growth, macroscopical observations at autopsy and microscopical examinations of the nose were used as criteria for disclosing possible harmful effects. There were no compound related effects in mortality, clinical signs, body weight or gross and histological pathology. Microscopic examination revealed minimal changes (nest-like infolds, slight hypertrophy, and slight hyperplasia) in the nasal respiratory epithelium of animals in all groups, the control group included. Type and incidence of the changes were similar in the various groups, or the changes occurred in a single animal only. These changes were not ascribed to treatment. Based on the results, the NOAEC is considered to be 200 mg/m³.

In contrast to a previously conducted study (TNO report V 90.322, 1995), in which rats were exposed to 150, 600 or 2500 mg ethyl lactate per m³ for 28 days, the results of the present study show that rats exposed to 25, 75 or 200 mg ethyl lactate per m³ for the same period did not develop compound-related histopathological changes in the nose. The histopathological changes observed at an exposure concentration of 150 mg/m³ (TNO report V 90.322), viz. replacement of olfactory epithelium by respiratory epithelium, goblet cell hypertrophy and moderate goblet cell hyperplasia, were considered to be in line with the changes observed at concentration-response relationship including the response seen at the 150 mg/m³ exposure concentration. The minimal changes observed in the present study consisting of nest-like infolds and slight hypertrophy and hyperplasia are fully comparable to the minimal changes observed at the 150 mg/m³ level of the previous study. However, since in the present study these minimal changes were observed in all groups, controls included, and their incidences in treatment groups were not different from those in the control group, or the changes occurred in a single animal only, these minimal alterations were not ascribed to treatment. Therefore, it is concluded that the no-adverse-effect concentration, as regards to local effects, in the present study is 200 mg/m³, indicating that the level of 150 mg/m³ used in the previous study (TNO report V 90.322, 1995), should be considered a no-adverse-effect concentration as well.