2-methylpropyl-(R)-2-hydroxypropanoate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

May 1992 to 1993-01-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Isobutyl-(S)-lactate is used as read-across partner to isobutyl-(R)-lactate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

77027-84-0

Cas Number:

77027-84-0

IUPAC Name:

77027-84-0

Constituent 2

Reference substance name:

Isobutyl-(S)-lactate

IUPAC Name:

Isobutyl-(S)-lactate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Isobutyl-(S)-lactate
- Physical state: Clear, colourless liquid
- Analytical purity: 99.7 %
- Lot/batch No.: PSP-IBL-01
- Maximum storage time: 1 year
- Storage condition of test material: Room temperature, absence of light
- Density: 0.979 kg/L at 293 K

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 5-6 weeks
- Housing: 5 animals of same sex/ stainless steel cage
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-22.5 °C
- Humidity (%): 50-70 %
- Air changes (per hr): 10
- 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: N.A.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Modified H 1000 multitiered inhalation chambers
- Method of holding animals in test chamber: The chambers are constructed of stainless steel with glass doors on two sides. The rats were housed individually in wire mesh stainless cages
- Source and rate of air: Pressurised air driven nebulizers (lee dispenser, type 110K, Lee Co., USA).
- System of generating particulates/aerosols: The two lower concentrations (100 and 200 mg/m³), metered amounts of isobutyl lactate were transported by roller pumps to pressurized air driven nebulisers. The two higher concentrations levels (400 and 800 mg/m³) were generated by atomising the test material into small droplets by using compressed air driven nebulizers of the institute's design. Each nebuliser consisted of an atomiser and a glass jar, containing the test material. The nebulisers were operated at pressures of between 0.72 and 1.5 bar (400 mg/m³) or 1.05 and 2.6 bar (800 mg/m³). During operation, the test material was drawn through a sucking pipe to the atomizer. The generated spray was blown against a baffle which was fitted approximately 8 cm below the nozzle orifice to remove the larger droplets. The generated mixtures of air and test material generated by either way were diluted with filtered air until the required concentrations were achieved.
- Temperature, humidity in air chamber: air temperature delivered to the exposure units: 19.5-23.5 °C, relative humidity ranged between 40 and 70 %
- Air flow rate: the total air flow through the exposure units was monitored by means of an anemometer and ranged between 23.7 and 26.7 m³/hour

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatographic measurements. Representative samples were obtained by passing 30 L test atmosphere at 2 L/min through an impinger filled with acetone p.a. quality. After sampling, the content of the impinger was quantitatively transferred to a 50 mL volumetric flask. The impinger was rinsed with acetone, each rinsing was transferred to the volumetric flask as well. Finally, the flask was filled up to the 50 mL mark with acetone. To determine the concentration of the test atmosphere samples, the peak area of the isobutyl lactate peak was compared with that of standard solutions containing isobutyl lactate in acetone corresponding to concentration levels up to 800 mg isobutyl lactate per m³ air
- Samples taken from breathing zone: Yes. Test atmosphere samples were taken sequentially from each of the exposure units at the animals breathing zone and were analysed by a total carbon analyser.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual mass concentrations of isobutyl lactate in the test atmospheres were calculated using the calibration graphs obtained by plotting the concentrations measured by GC analysis (in mg/m³) against the total carbon output (in mm). 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:

Six hours/day

Frequency of treatment:

Five days a week during a period of 28 days, resulting in a total of 20 exposure days

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 200, 400 and 800 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale:
The concentrations were selected based on the results of sub-acute inhalation studies with ethyl lactate and an acute inhalation study with isobutyl lactate (TNO-Nutrition and Food Research report numbers V 90.322, V 91.416, V 92.344 respectively). In the acute study with isobutyl lactate it was shown that rats could be exposed to a level of 6.16 g/m³ for a period of 4-hours followed by a 14-day observation period without mortalities or severe clinical signs.

Positive control:

N.A.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once per day throughout the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once per day throughout the study. In addition, 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 acclimatisation period, one day before the start of the exposure (allocation procedure), just prior to the first exposure to the test substance (day 0), and subsequently at weekly intervals (including the day of autopsy). For each animal, the weekly body weight gain was calculated.

FOOD CONSUMPTION:
Food intake was measured weekly per cage, and the efficiency of food utilisation was calculated and expressed as gram weight gain per gram food
consumed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy on day 28
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: All rats
- the following parameters were examined: Haemoglobin concentration, packed cell volume, red blood cell count, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count.
- the following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 26 and at autopsy on day 28
- Animals fasted: Yes (day 26)
- How many animals: All rats
- The following parameters were examined: The blood taken from animals on day 26 was used for the determination of glucose. The blood taken from animals on day 28 was used for: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, total bilirubin, sodium, potassium, calcium, chloride and inorganic phosphate

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Samples of the following tissues and organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4 % solution of formaldehyde:
adrenals, liver, heart, lungs with trachea and larynx, nose, spleen, testes, kidneys.

HISTOPATHOLOGY: Yes
Histopathological examination was performed on all organs and tissues mentioned above of all animals of the control and the high concentration group. Histopathological examination of the nose was extended to all rats of the low- and mid-concentration groups.

Other examinations:

N.A.

Statistics:

Body weight data were analysed by one-way analysis of covariance using pre-exposure (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. Food intake was analysed by analysis of variance followed by the LSD test. Red blood cell- and coagulation variables, absolute white blood cell counts and data on clinical chemistry, and organ weights were analysed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. Percentages of white blood cell counts were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. Incidences 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):

One male rat exposed to 200 mg/m³ showed nasal encrustations on day 8 of the study. All females of the low-concentration group showed alopecic areas on the skin in the last week of the experiment. These clinical signs are incidentally found in rats of this strain and age of rats and are not considered to be treatment-related.

Mortality:

no mortality observed

Description (incidence):

None of the rats died.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights did not show statistically significant differences between test groups and controls in either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Description (incidence and severity):

Food intake or food conversion efficiency were not significantly changed in rats exposed to isobutyl lactate.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Mean corpuscular haemoglobin (MCH) was slightly increased in female rats exposed to 200, 400 or 800 mg/m³ isobutyl lactate. Prothrombin time (PTT) was increased in female rats exposed to 100, 400 or 800 mg/m³. Both effects were however not dose-related. Since the haemoglobin concentration and the erythrocyte count were not affected in these rats upon isobutyl lactate exposure, the slight effect on MCH was not considered to be of toxicological significance. In addition the slight changes observed in PTT were neither considered to be of relevance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Except for a significant decrease in GGT activity in female rats exposed to 100, 200 or 800 mg/m³, there were no consistent differences between the controls and the rats exposed to isobutyl lactate. Since the decrease in GGT was not concentration-related this finding was not considered to be of toxicological significance.

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):

Female rats exposed to 100, 200 or 800 mg/m³ showed a small increase in the absolute, but not in the relative kidney weight. Since the effect on the absolute weight of the kidney was not dose-related this effect is not thought to be exposure-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination at autopsy did not reveal any abnormalities.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See box "Details on results" below.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

See box "Details on results" below.

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
One male rat exposed to 200 mg/m³ showed nasal encrustations on day 8 of the study. All females of the low-concentration group showed alopecic areas on the skin in the last week of the experiment. These clinical signs are incidentally found in rats of this strain and age of rats and are not considered to be treatment-related. None of the rats died.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights did not show statistically significant differences between test groups and controls in either sex.

FOOD CONSUMPTION FOOD EFFICIENCY
Food intake or food conversion efficiency were not significantly changed in rats exposed to isobutyl lactate.

HAEMATOLOGY
Mean corpuscular haemoglobin (MCH) was slightly increased in female rats exposed to 200, 400 or 800 mg/m³ isobutyl lactate. Prothrombin time (PTT) was increased in female rats exposed to 100, 400 or 800 mg/m³. Both effects were however not dose-related. Since the haemoglobin concentration and the erythrocyte count were not affected in these rats upon isobutyl lactate exposure, the slight effect on MCH was not considered to be of toxicological significance. In addition the slight changes observed in PTT were neither considered to be of relevance.

CLINICAL CHEMISTRY
Except for a significant decrease in GGT activity in female rats exposed to 100, 200 or 800 mg/m³, there were no consistent differences between the controls and the rats exposed to isobutyl lactate. Since the decrease in GGT was not concentration-related this finding was not considered to be of toxicological significance.

ORGAN WEIGHTS
Female rats exposed to 100, 200 or 800 mg/m³ showed a small increase in the absolute, but not in the relative kidney weight. Since the effect on the absolute weight of the kidney was not dose-related this effect is not thought to be exposure-related.

GROSS PATHOLOGY
Gross examination at autopsy did not reveal any abnormalities.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the nasal cavity revealed treatment-related histopathological changes in all animals of the 800 mg/m³ group and the
majority of the animals of the 400 mg/m³ isobutyl lactate group. These treatment-related nasal changes comprised:
a) an increased number of nest-like infolds in the respiratory epithelium lining the septum (goblet-cell hyperplasia)
b) minimal to slight hyperplasia of the respiratory epithelium lining the nasoturbinates and the septum.

In 2 out of 5 males of both groups, this hyperplasia was accompanied by rhinitis. Respiratory epithelium hyperplasia was more pronounced in the 800mg/m³ than in the 400 mg/m³ group. The difference in incidence between control and high-concentration group attained the level of statistical significance (p < 0.05). Very slight respiratory nest-like infolds were seen in one or a few animals in the control, 100 and 200 mg/m³ groups.
Therefore, only in the 400 and 800 mg/m³ groups these nasal changes are considered to be related to treatment and point to an irritating effect of
lactate on the nasal epithelium at levels higher than 200 mg/m³. In addition, minimal to slight disarrangement of the olfactory epithelium
was found in 6 out of 10 rats (3/5, both males and females) of the high concentration group (800 mg/m³). The hyperplasia of the respiratory epithelium as well as the disarrangement of the olfactory epithelium were restricted to the anterior segment of the nasal cavity. The former lesion was seen only on the inner part of the nasoturbinates and the dorsal part of the septum. The olfactory epithelial disarrangement was restricted to a small region,viz. the most anterior dorso-medial part of the nose.
Microscopic examination of the other organs that were examined did not reveal any treatment-related histopathological changes. The lesions
observed were approximately equally distributed amongst the test groups and controls, or they occurred in one or a few animals only. Therefore, they are not ascribed to the inhalation of the test substance.

ANALYTICAL RESULTS
The actual concentrations were generally close to the intended concentrations and the overall mean (± SD) of 20 daily mean values turned out to be 104 ± 14, 223 ± 27, 420 ± 41 and 844 ± 105 mg/m³.
The nominal concentrations of isobutyl lactate were calculated from the total daily amount of test substance used per treatment group and the total volume of air passed through each exposure unit. The generation of the test atmosphere showed an efficiency of ca. 64 % for the low concentration level (100 mg/m³), of ca. 75 % for the mid-concentration l (200 mg/m³), of ca. 78 % for the mid-concentration 2 (400 mg/m³), and of ca. 84 % for the high concentration level (800 mg/m³).

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

200 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

> 800 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a subacute inhalation toxicity study, isobutyl-(S)-lactate (99.7 % purity) showed no effects on mortality, body weight, food consumption, haematology, clinical chemistry and organ weights. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 400 and 800 mg/m³ isobutyl-(S)-lactate. Therefore, the NOAEC (local) for male and female rats is considered to be 200 mg/m³ and as no systemic effects were noted the NOAEC (systemic) for both sexes is considered to exceed 800 mg/m³.

Executive summary:

In a subacute inhalation toxicity study, isobutyl-(S)-lactate (99.7% purity) was administered to 5 Wistar rats/sex/concentration by whole body exposure at concentrations of 0, 100, 200, 400 and 800 mg/m³ for 6 hours/day for 5 days/week for a total of 20 exposures during a 4 week study. No adverse effects were observed on mortality, body weight, food consumption, haematology, clinical chemistry and organ weights. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 400 and 800 mg/m³ isobutyl-(S)-lactate. The local effects were characterised by goblet cell and respiratory epithelial hyperplasia. These nasal changes are considered to be related to treatment and point to an irritating local effect of isobutyl-(S)-lactate on the nasal epithelium. Therefore, the NOAEC (local) for male and female rats is considered to be 200 mg/m³ and as no systemic effects were noted the NOAEC (systemic) for both sexes is considered to exceed 800 mg/m³.

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