2-methylpropyl-(R)-2-hydroxypropanoate

Endpoint summary

Description of key information

Based on a set of studies performed with either isobutyl-S-lactate, being the optical antipode of isobutyl-R-lactate, or on the primary metabolites isobutanol and lactate (test substance: calcium lactate), isobutyl-R-lactate is considered to be devoid of any systemic toxicity up to the limit dose of 1000 mg/kg bw/d. However, local effects (irritation of nasal epithelium) were identified in the 28-day inhalation study on isobutyl-S-lactate, resulting in an NOAEL of 200 mg/m³. Accordingly, isobutyl-R-lactate is considered (by read-across) as irritating to the respiratory tract.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication. Study conducted in accordance to OECD guideline 408. 2-methyl-1-propanol was used for read-across to isobutyl-R-lactate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: mean body weight male: 172 g, female: 147 g
- Housing: Individually in stainless steel wire mesh cages (type DK III)
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The drinking water solutions were prepared freshly twice a week. To ensure homogenity of the solutions of the test substance in the drinking water, each mixture was stirred for about 30 min using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of the test substance in the drinking water was analysed over a period of 6 days. To check for the applied concentrations a sample of each concentration was taken for analysis by capillary gas chromatography at the beginning and at the end of the application period.

Duration of treatment / exposure:

90 days

Frequency of treatment:

ad libitum (as drinking water solutions)

Remarks:

Doses / Concentrations:
0, 1000, 4000, 16000 ppm
Basis:
nominal in water

Remarks:

Doses / Concentrations:
0, 80, 340, 1450 mg/kg/d
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of of a dose range finding study to check for palatability 16000 ppm was selected as maximum dose to avoid this complication.

Positive control:

N.A.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded at the beginning of the study and weekly throughout the study.

FOOD CONSUMPTION :
Food consumption were determined once a week

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once a week for a period of 4 days. The mean daily intake of the test substance (in mg per kg body weight) was calculated at the intervals at which water consumption was determined

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the beginning of treatment and at the termination of the animals by using an ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 87 of the study. Blood was taken from the retroorbital venous plexus
- Animals fasted: No data
- How many animals: 10 animals/sex/dose
- Parameters examined were: white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, reticulocytes, differential blood count and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 87 of the study. Blood was taken from the retroorbital venous plexus
- Animals fasted: No data
- How many animals: 10 animals/sex/dose
- Parameters examined were: sodium, potassium, chloride, inorganic phosphate, calcium, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-glutamyltransferase, urea, albumin, blood creatinine, total bilirubin, total protein, globulins, triglycerides, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After sacrification, the animals were necropsied and assessed by gross pathology. The weight of the anesthetized animals and the weights of their livers, kidneys, adrenal glands and testes were determined. Organs or tissues required by guidelines as well as gross lesions were fixed in a 4% formaldehyd solution.

HISTOPATHOLOGY: Yes
Histological examinations and assessment of the findings were carried out after histotechnical processing and staining with hematoxylin and eosin.

Other examinations:

None

Statistics:

Mean values and standard deviations were calculated for body weight, food and water consumption, intake of the test substances, hematological and clinical chemistry parameters as well as for absolute and relative organ weights. The organ weights were statistically evaluated using the DUNNETT’s test for comparison of the dose groups with the control groups. The analysis of variance (ANOVA) with subsequent DUNNETT’s test was used to compare the body weights as well as the hematological and clinical biochemistry data of the dose groups with those of the control groups.

Clinical signs:

no effects observed

Description (incidence and severity):

No effects on mortality and clinical signs occurred.

Mortality:

no mortality observed

Description (incidence):

No effects on mortality and clinical signs occurred.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related effects. Histopathological changes of the testes (tubular degeneration and diffuse hyperplasia of Leydig’s cells), the spleen (minimal increase in extramedullary hematopoiesis) or the kidneys (dilation of the renal pelvis) occurred sporadically in control and/or animals treated with MEP.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

16 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at highest dose.

Critical effects observed:

not specified

Table 1: Effective test substance intake
Test group		MEP concentration in drinking water (p.p.m.)		Mean daily substance intake (mg/kg bw)
				males		females
1		1000		75		91
2		4000		300		385
3		16000		1251		1657

Conclusions:

The NOAEL of 2-methyl-1-propanol after oral administration via the drinking water over a period of 90 days is considered to be 16000 ppm for both sexes.

Executive summary:

A repeated subchronic oral dose toxicity study (OECD 408) was conducted using male and female Wistar rats at doses of 0, 1000 ppm (about 80 mg/kg/d), 4000 ppm (about 340 mg/kg/d), and 16000 p.p.m. (about 1450 mg/kg/day) of 2-methyl-1-propanol (99.8% purity). The animals received the test item daily over a period of 90 days via the drinking water. The test substance had no effect on mortality, food consumption, water consumption, body weight, haematological and clinical chemistry examinations and on pathological findings. Based on the results reported the NOAEL for orally administered 2-methyl-1-propanol via the drinking water is considered to be 16000 p.p.m. (about 1450 mg/kg bw/day) for both sexes.

This study in rats is acceptable and satisfies the principle requirement for a repeated oral dose toxicity study according to OECD 408 in rats. Due to the fact that isobutanol is a degradation product of isobutyl lactate, this result is relevaant for risk assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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.

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

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

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

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.

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.

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).

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'

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

800 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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.

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

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

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

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.

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.

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).

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'

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data is available for the target substance isobutyl-R-lactate. Therefore, available data on the optical antipode, isobutyl-S-lactate, and on the relevant primary metabolite, isobutanol, were used in a read-across approach. Moreover, to assess the toxic potential of the other primary metabolite, lactic acid, the calcium salt of lactic acid, calcium lactate, was also used as read-across partner.

A repeated dose inhalation toxicity study in rats with isobutyl-S-lactate was conducted in accordance with OECD guideline 412. No systemic effects were reported after 28 days. The NOAEC (systemic) is thus 800 mg/m³. Local irritation of the nasal epithelium was reported in animals of the high (800 mg/m³) and middle dose groups (400 mg/m³). The NOAEC (local) is considered to be 200 mg/m³.

A subchronic oral repeated dose toxicity study with isobutanol was conducted in rats in accordance with OECD guideline 408. Isobutanol is the product of enzyme catalysed hydrolysis of the target substance isobutyl-R-lactate and therefore a suitable read-across partner. No toxic effects were noted after oral administration of 1450 mg/kg bw/d via drinking water in both sexes.

No adverse effects were reported in a subchronic study (equivalent to OECD 408) conducted with the calcium salt of lactic acid, calcium lactate in rats by oral administration via drinking water. The NOAEL in this study is considered to be 4500 mg/kg bw/day for both sexes (calculated from 50,000 mg/L by applying the conversion factor of 0.09 for rats as recommended in the EFSA guidance document). Therefore, lactic acid/free lactate is of no toxicological concern.

Moreover, based on the data presented in IUCLID chapter 7.9.1 the source substance isobutanol does not elicit neurotoxic effects.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study conducted with the primary metabolite isobutanol.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
GLP guideline study conducted with the optical antipode isobutyl-S-lactate (read-across).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
GLP guideline study conducted with the optical antipode isobutyl-S-lactate (read-across).

Justification for classification or non-classification

Based on the available data from the three read-across substances isobutanol, isobutyl-S-lactate and calcium lactate, no classification according to the CLP Regulation is warranted for the target substance isobutyl-R-lactate.