Butyl (S)-2-hydroxypropionate

Endpoint summary

Description of key information

Suitable data is available for butyl-S-lactate. A repeated dose inhalation toxicity study in male Wistar rats with butyl-S-lactate revealed no systemic effects and thus the NOAEC (systemic) is considered to be greater than 600 mg/m³. Local irritation of the nasal epithelium was reported only in animals of the high (600 mg/m³) concentration group. Therefore, the NOAEC (local) is considered to be 200 mg/m³.

To further assess the repeated dose toxicity of the butyl-S-lactate, a read-across approach was pursued. Suitable data from the source substances n-butanol and n-butyl acetate were used. 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 a read-across partner.

In a sub-chronic oral repeated dose toxicity study with n-butanol the oral NOAEL in rats was considered to be 125 mg/kg bw/day (corresponding to 247 mg/kg bw/d butyl-S-lactate).

In a sub-chronic repeated dose inhalation toxicity study, the NOAEC for male and female rats was considered to be 500 ppm after exposure to n-butyl acetate (equals 2400 mg/m³, corresponding to 3020 mg/m³ butyl-S-lactate).

No adverse effects were reported in a sub-chronic 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.

Moreover, based on the data presented in IUCLID chapter 7.9.1 the source substances n-butyl acetate and n-butanol do not elicit neurotoxic effects.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related ataxia first appeared in the high-dose group (500 mg/kg bw/day) during week 8. Ataxia and hypoactivity occurred infrequently during week 9 and 10. These signs increased to a weekly incidence of 32 and 29 % for ataxia and hypoactivity, respectively. At week 11 and continued at approximately the same frequency during weeks 12 and 13. Onset of ataxia and hypoactivity was about 2-3 minutes after dosing and duration was less than one hour. Other clinical signs observed did not appear to be directly related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three rats died during the study. Two of these deaths were the result of the rubber catheter slipping off the metal dosing cannula. And a mid-dose (125 mg/kg bw/day) male died during week 1.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effect was present on body weight or weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effect was present on food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The pathology observed was considered to be within normal limits for the age, sex and strain of the animal.

Haematological findings:

no effects observed

Description (incidence and severity):

Only one alteration in clinical pathologic parameters occurred that was suggestive if a treatment-related effect. At the interim sacrifice, RBC (p < 0.05), PVC (p < 0.01) and HGB (p < 0.01) averages in the high dose (500 mg/kg bw/d) females were 5 % less than the corresponding control average. The RBC and PCV (p < 0.05) averages for the middle-dose (125 mg/kg bw/d) females were also slightly (4 % and 3 % respectively) below those of the controls. However, RBC PVC and HGB averages were similar for control and treated groups of males at the interim evaluation and for control and treated groups of both sexes at the final evaluation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Small yet statistically significant differences between control and treated groups averages occurred in one sex and at one evaluation only, and there was no dose response relationship. Thus, they were not considered to be treatment related. They were: a lower (p < 0.05) cholesterol average in the high-dose males at the interim evaluation, a higher (p < 0.05) absolute neutrophil count in the middle-dose males at the interim evaluation, a higher (p < 0.05) relative segmented neutrophil count and a lower (p < 0.05) relative lymphocyte count in the low-dose females at the final evaluation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Small yet statistically significant differences between control and treated groups averages occurred in one sex and at one evaluation only, and there was no dose response relationship. Thus, they were not considered to be treatment related. They were: higher(p < 0.05) urine pH values in the low dose males at the interim and low-dose females at the final evaluations.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant difference between control and treated group averages was a slightly (p < 0.05) higher thyroid weight average in the high-dose males. No dose response relationship was present, as the absolute thyroid weights were similar for all three treated groups of males. Moreover, they were only 14% above control average. Thyroid weight averages of the treated females were not above those of the controls. Thus, this difference appears to be a chance occurrence rather than a treatment-related effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related lesion was observed in gross necropsy at the interim or final sacrifices or of the rats found dead or sacrificed in extremis. The lesions present were those commonly observed in laboratory rats and they were present in control and treated groups at similar frequency or were one-time occurrences. The enlarged uterine horns are related to the stage of the oestrus cycle. Three rats died during the study no gross lesions were seen in rat no 224. Rat no 202 (which had a catheter in its stomach) had dark area on the glandular mucosa of the stomach. In rat no. 133 (middle-dose) the left lobe of the lung was red, and the cranial and middle lobes were shrivelled.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related lesion was observed at the histopathologic evaluation. The lesions that were observed were one-time occurrences or were present in the control and treated groups at a similar frequency. The diffuse subacute lymphadenitis of the mandibular lymph node was visible grossly as red or enlarged lymph nodes. This is a commonly observed lesion on laboratory rats. The cause of death of the mid-dose rat (no. 133) that died during the study was determined to be a gavaging accident since a perforated oesophagus was found at histopathologic examination.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Critical effects observed:

no

Test material analysis:

The results of the analysis of the samples revealed that the stability and concentration of the samples were acceptable.

Conclusions:

The NOAEL of n-butanol after oral administration via gavage over a period of 90 days is considered to be 125 mg/kg bw/day for both sexes.

Executive summary:

A repeated sub-chronic dose toxicity study was conducted equivalent to OECD 408. Male and female rats (20/sex/group) at doses of 0, 30, 125 or 500 mg/kg bw/day were treated orally with n-butanol. The animals received the test item daily over a period of ca. 90 days via gavage. The only unequivocal effects produced by n-butanol were ataxia and hypoactivity at the 500 mg/kg bw/day dose level. The maximum weekly incidence was 32 % and 29 %, respectively. No dose-related differences were observed between treatment or control rats in body or organ weight changes, food consumption or mortality, gross pathology and histopathological and ophthalmic evaluations. Ataxia and hypoactivity (lasting less than 1 hour) were observed 2–3 minutes after dosing in both sexes of the high dose group during the last 6 weeks of dosing. Such ataxia and hypoactivity are typically seen following high oral doses of alcohols. No treatment-related signs were observed in the 30 and 125 mg/kg bw/day treatment groups. Based on the results reported the NOAEL for orally administrated n-butanol via gavage is 125 mg/kg bw/day for both sexes (corresponding to 247 mg/kg bw/day Butyl-S-lactate).

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

247 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Comparable to guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997-03-13 to 2000-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 12th May, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Systemic name: butyl-S-(-)-2-hydroxypropionate
- CAS Reg. No.: 34451-19-9
- Molecular formula: C7H14O3
- Molecular weight: 146 g/mol
- Purity: 98.9 %
- Batch no.: BU-6001K and BU 5001H
- Appearance: liquid/ water white
- Boiling point: 189 °C
- Flash pomt: 79 °C
- Vapour pressure: 0.4 mbar at 20 °C
- Storage conditions: ambient temperature

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species is used because it is considered the most suitable for this type of study, and is usually required by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Females (if applicable) Not applicable
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: mean body weight of 255 g
- Housing: housed in groups of three, in suspended, stainless steel cages, fitted with wire mesh floor and front. The cages were randomly divided over the cage racks according to a computer randomization program and each cage was provided with a coloured card showing the animal identification numbers, the cage number, the group letter and the study number
- Diet (e.g. ad libitum): ad libitum, commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RMS) obtained from SDS, Special Diets Services, Witham, England
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 35-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

other: humidified air

Remarks on MMAD:

Particle size was not measured

Details on inhalation exposure:

EXPOSURE LEVELS:
The exposure levels of the test item have been established based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other local toxic effects at levels of 400 or 600 mg/m³ and higher.

EXPOSURE UNITS:
The animals were exposed to the test atmosphere in nose-only inhalation units. Each unit consisted of a cylindrical column surronded by a transparent cylinder. The column has a volume of ca. 50 L and consisted of a top assembly with the entrance of the chamber, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animals exposure. Several empty ports were used for test atmosphere sampling, particle size analysis, and for measurement of temperature and relative humidity. The animals were secured in plastic animal holders, positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column.

GENERATION OF THE TEST ATMOSPHERE:
To generate the test atmospheres complete evaporation was used, using a rotating liquid film evaporator. The glass flask containg the test item was placed in an oil bath at a temperature of 125 °C. A small metered flow of dry clean air was passed through the test material and subsequently mixed with a secondary metered flow of dry clean air (main flow). Appropriate fractions of this main flow were taken using eductors and were diluted with metered amounts of humidified air and passed to the inhalation units. The remaining part of the main flow was passed directly to the exhaust. The test material was changed daily.

TEMPERATURE AND RELATIVE HUMIDITY:
The temeprature and the realtive hunidity of the test atmospheres were recorded at least three times per exposure day using a RH/T device (Testo 610).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total Carbon Analysis:
The amount of test item vapozr was monitored semi-continuously by means of a total carbon analyser using flame ionisation detection (FID). The settings of the total carbon analyser were as follows: oven temperature: 150 °C, H2 flow: 0.5 bar, air flow: 0.8 bar, sampling back pressure: 200 mbar, sampling temperature: 130 °C. The samples were taken sequentially from each of the units at the animals breathing zone. The samples were drawn through sampling lines and were passed via a controlled valves system to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). Each unit was monitored approximately once each half hour for about 7 min, resulting in about 13-14 measurements per concentration level per day.

Duration of treatment / exposure:

20 days

Frequency of treatment:

6 hours a day, 5 days a week for a period of 4 weeks

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

Dose / conc.:

75 mg/m³ air

Remarks:

Low concentration

Dose / conc.:

200 mg/m³ air

Remarks:

Mid concentration

Dose / conc.:

600 mg/m³ air

Remarks:

High concentration

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The exposure levels have been established in consultation with the sponsor. They are based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other (local) toxic effects at levels of 400 or 600 mg/m³ and higher.

Positive control:

n.a.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon (shortly after exposure) for dead or moribund animals, to minimize loss of animals from the study. At weekends only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the day of the start of the study (allocation procedure), just prior to the start of the first exposure (day 0), on nominal days 7, 14, 21, 28 and on the day of scheduled necropsy (day 29). In addition, weekly body weight gain was calculated. Body weights were also recorded on day -3 to monitor adequate growth during the acclimatization period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 29, the day after the last exposure, all rats were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia. Subsequently they were examined macroscopically for pathological changes.

HISTOPATHOLOGY: Yes
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. The respiratoty tract (nose, larynx, trachea and lungs) was processed as follows: The nose (nasal cavity) was cut at 6 levels. Levels of cross sections through the nasal cavity were assigned according to international standards (Woutersen et al., 1994). The larynx was cut longitudinally at three levels. The trachea with the bifurcation was cut longitudinally/transversally at three levelsEach lung lobe was cut at one sagittal level. Histopathological examination was performed on the nose of all animals of all groups. The other respiratory tract organs were examined in the control and high concentration group.

ORGAN WEIGHTS:
The following organs were weighed and the relative organ weights (g/kg bw) were calculated based on the final body weight of the rats: adrenals, heart, kidneys, lungs with trachea and larynx, liver, spleen and testes. Samples of these organs and the nose of all animals were preserved in a neutral aqueous phosphate-buffered solution of formaldehyde.

Statistics:

Body weight data were analysed by one-way analysis of covariance (COVAR) using pre-exposure (day 0) weights as the covariate. Organ weights were analysed by one-way analysis of variance (ANOVA). When group means were significantly different (p < 0.05), individual pairwise comparisons were made using Dunnett's multiple comparison method. The incidence of histopathological changes was evaluated 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. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) may be greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

General condition and behaviour were not adversely affected by exposure to the test material. Clinical signs included a visually slightly increased superficial breathing pattern in animals exposed to the highest concentration during exposure and areas of sparsely haired skin in one control rat.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed until the scheduled autopsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in mean body weights and mean body weight gain between the treatment groups and the controls although body weight gain in animals exposed to the high concentration vapour test atmosphere appeared slightly lower than those in the other groups.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant difference in the mean absolute and relative organ weights between the treatment groups and the controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examination at autopsy revealed findings in the epididymides, kidneys, mediastinal lymph nodes, skin/subcutis and testes in one or two animals only. Moreover, the findings are common for rats of this strain and age. Most animals exhibited gross findings in the lungs. However, the incidences of these findings were about equally distributed between the groups, including the control group. Therefore, the changes were considered not to be related to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the nasal tissues showed very slight to slight transitional epithelium hyperplasia at level 2 in 5 of 6 animals exposed to 600 mg/m³. Slight focal transitional epithelium hyperplasia was also observed in one animal exposed to 600 mg/m³ (nasal level 1) and in one control animal (nasal level 2). One animal exposed to 600 mg/m³ additionally showed very slight focal squamous epithelial hyperplasia (nasal level 1). Very slight focal goblet cell hyperplasia (nasal level 3) was seen in two animals exposed to 600 mg/m³. No nasal tissue changes were observed in animals exposed to 75 or 200 mg/m³.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

600 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse systemic effects observed in any test group

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Very slight to slight transitional epithelium hyperplasia of the nasal passages.

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/L air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Analytical results on actual concentration:

The overall mean daily concentrations of the test item vapour in the test atmospheres were 73 ± 7, 199 ± 11 and 591 ± 16 mg/m³ for the low, mid and high concentration test atmosphere, respectively.

Airflow:

Mean airflow through the control unit was 33.2 L/min, mean airflow through the low concentration unit was 28.5 L/min. The total airflow through the mid and high concentration unit could not be established, but was minimally 20.5 and 28.5 L/min, respectively.

Nominal concentration:

Nominal concentrations of the mid and high concentration test atmosphere could not be calculated. The mean nominal concentration of the low BL vapour test atmosphere was 112 ± 29 mg/m³, which was of the same order of magnitude as the actual concentration.

Temperature and relative humidity:

The daily mean temperatures in the test atmospheres were between 20.8 and 21.2 °C, the daily mean relative humidities were between 38 and 43%.

Conclusions:

In a sub-acute inhalation toxicity study, Butyl-S-lactate (98.9% purity) showed no effects on survival, body weight, organ weights and gross pathology. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 600 mg/m³. Therefore, the NOAEC (local) for male rats is considered to be 200 mg/m³ and as no systemic effects were noted, the NOAEC (systemic) for male rats is considered to exceed 600 mg/m³.

Executive summary:

In a sub-acute inhalation toxicity study, toxicity of the test item (purity 98.9 %) was examined in male Wistar rats. Groups of 6 male rats were exposed nose-only to target vapour concentrations of 0, 75, 200 or 600 mg/m³ test item for 6 hours a day, 5 days a week during a period of 4 weeks (a total of 20 exposure days). No adverse effects were observed on survival, body and organ weights, and gross pathology. The most prominent finding consisted of histopathological changes in the nasal tissues, which showed transitional epithelium hyperplasia in almost all animals exposed to 600 mg/m³ test item but not in animals exposed to lower concentrations. These nasal changes are related to treatment and point to an irritating effect of Butyl-S-lactate on the nasal epithelium. Therefore, the local NOAEC is considered to be 200 mg/m³. The systemic NOAEC is considered to exceed 600 mg/m³ as no systemic effects were observed. This sub-acute toxicity study in the rat is acceptable and satisfies the guideline requirement according to OECD 412 for a sub-acute inhalation study in the rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

600 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997-03-13 to 2000-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 12th May, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Systemic name: butyl-S-(-)-2-hydroxypropionate
- CAS Reg. No.: 34451-19-9
- Molecular formula: C7H14O3
- Molecular weight: 146 g/mol
- Purity: 98.9 %
- Batch no.: BU-6001K and BU 5001H
- Appearance: liquid/ water white
- Boiling point: 189 °C
- Flash pomt: 79 °C
- Vapour pressure: 0.4 mbar at 20 °C
- Storage conditions: ambient temperature

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species is used because it is considered the most suitable for this type of study, and is usually required by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Females (if applicable) Not applicable
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: mean body weight of 255 g
- Housing: housed in groups of three, in suspended, stainless steel cages, fitted with wire mesh floor and front. The cages were randomly divided over the cage racks according to a computer randomization program and each cage was provided with a coloured card showing the animal identification numbers, the cage number, the group letter and the study number
- Diet (e.g. ad libitum): ad libitum, commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RMS) obtained from SDS, Special Diets Services, Witham, England
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 35-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

other: humidified air

Remarks on MMAD:

Particle size was not measured

Details on inhalation exposure:

EXPOSURE LEVELS:
The exposure levels of the test item have been established based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other local toxic effects at levels of 400 or 600 mg/m³ and higher.

EXPOSURE UNITS:
The animals were exposed to the test atmosphere in nose-only inhalation units. Each unit consisted of a cylindrical column surronded by a transparent cylinder. The column has a volume of ca. 50 L and consisted of a top assembly with the entrance of the chamber, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animals exposure. Several empty ports were used for test atmosphere sampling, particle size analysis, and for measurement of temperature and relative humidity. The animals were secured in plastic animal holders, positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column.

GENERATION OF THE TEST ATMOSPHERE:
To generate the test atmospheres complete evaporation was used, using a rotating liquid film evaporator. The glass flask containg the test item was placed in an oil bath at a temperature of 125 °C. A small metered flow of dry clean air was passed through the test material and subsequently mixed with a secondary metered flow of dry clean air (main flow). Appropriate fractions of this main flow were taken using eductors and were diluted with metered amounts of humidified air and passed to the inhalation units. The remaining part of the main flow was passed directly to the exhaust. The test material was changed daily.

TEMPERATURE AND RELATIVE HUMIDITY:
The temeprature and the realtive hunidity of the test atmospheres were recorded at least three times per exposure day using a RH/T device (Testo 610).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total Carbon Analysis:
The amount of test item vapozr was monitored semi-continuously by means of a total carbon analyser using flame ionisation detection (FID). The settings of the total carbon analyser were as follows: oven temperature: 150 °C, H2 flow: 0.5 bar, air flow: 0.8 bar, sampling back pressure: 200 mbar, sampling temperature: 130 °C. The samples were taken sequentially from each of the units at the animals breathing zone. The samples were drawn through sampling lines and were passed via a controlled valves system to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). Each unit was monitored approximately once each half hour for about 7 min, resulting in about 13-14 measurements per concentration level per day.

Duration of treatment / exposure:

20 days

Frequency of treatment:

6 hours a day, 5 days a week for a period of 4 weeks

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

Dose / conc.:

75 mg/m³ air

Remarks:

Low concentration

Dose / conc.:

200 mg/m³ air

Remarks:

Mid concentration

Dose / conc.:

600 mg/m³ air

Remarks:

High concentration

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The exposure levels have been established in consultation with the sponsor. They are based on similar studies with ethyl lactate and isobutyl lactate showing moderate to severe nasal irritation and other (local) toxic effects at levels of 400 or 600 mg/m³ and higher.

Positive control:

n.a.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon (shortly after exposure) for dead or moribund animals, to minimize loss of animals from the study. At weekends only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the day of the start of the study (allocation procedure), just prior to the start of the first exposure (day 0), on nominal days 7, 14, 21, 28 and on the day of scheduled necropsy (day 29). In addition, weekly body weight gain was calculated. Body weights were also recorded on day -3 to monitor adequate growth during the acclimatization period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On day 29, the day after the last exposure, all rats were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia. Subsequently they were examined macroscopically for pathological changes.

HISTOPATHOLOGY: Yes
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. The respiratoty tract (nose, larynx, trachea and lungs) was processed as follows: The nose (nasal cavity) was cut at 6 levels. Levels of cross sections through the nasal cavity were assigned according to international standards (Woutersen et al., 1994). The larynx was cut longitudinally at three levels. The trachea with the bifurcation was cut longitudinally/transversally at three levelsEach lung lobe was cut at one sagittal level. Histopathological examination was performed on the nose of all animals of all groups. The other respiratory tract organs were examined in the control and high concentration group.

ORGAN WEIGHTS:
The following organs were weighed and the relative organ weights (g/kg bw) were calculated based on the final body weight of the rats: adrenals, heart, kidneys, lungs with trachea and larynx, liver, spleen and testes. Samples of these organs and the nose of all animals were preserved in a neutral aqueous phosphate-buffered solution of formaldehyde.

Statistics:

Body weight data were analysed by one-way analysis of covariance (COVAR) using pre-exposure (day 0) weights as the covariate. Organ weights were analysed by one-way analysis of variance (ANOVA). When group means were significantly different (p < 0.05), individual pairwise comparisons were made using Dunnett's multiple comparison method. The incidence of histopathological changes was evaluated 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. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) may be greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

General condition and behaviour were not adversely affected by exposure to the test material. Clinical signs included a visually slightly increased superficial breathing pattern in animals exposed to the highest concentration during exposure and areas of sparsely haired skin in one control rat.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed until the scheduled autopsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in mean body weights and mean body weight gain between the treatment groups and the controls although body weight gain in animals exposed to the high concentration vapour test atmosphere appeared slightly lower than those in the other groups.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant difference in the mean absolute and relative organ weights between the treatment groups and the controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examination at autopsy revealed findings in the epididymides, kidneys, mediastinal lymph nodes, skin/subcutis and testes in one or two animals only. Moreover, the findings are common for rats of this strain and age. Most animals exhibited gross findings in the lungs. However, the incidences of these findings were about equally distributed between the groups, including the control group. Therefore, the changes were considered not to be related to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the nasal tissues showed very slight to slight transitional epithelium hyperplasia at level 2 in 5 of 6 animals exposed to 600 mg/m³. Slight focal transitional epithelium hyperplasia was also observed in one animal exposed to 600 mg/m³ (nasal level 1) and in one control animal (nasal level 2). One animal exposed to 600 mg/m³ additionally showed very slight focal squamous epithelial hyperplasia (nasal level 1). Very slight focal goblet cell hyperplasia (nasal level 3) was seen in two animals exposed to 600 mg/m³. No nasal tissue changes were observed in animals exposed to 75 or 200 mg/m³.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

600 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse systemic effects observed in any test group

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: Very slight to slight transitional epithelium hyperplasia of the nasal passages.

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/L air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Analytical results on actual concentration:

The overall mean daily concentrations of the test item vapour in the test atmospheres were 73 ± 7, 199 ± 11 and 591 ± 16 mg/m³ for the low, mid and high concentration test atmosphere, respectively.

Airflow:

Mean airflow through the control unit was 33.2 L/min, mean airflow through the low concentration unit was 28.5 L/min. The total airflow through the mid and high concentration unit could not be established, but was minimally 20.5 and 28.5 L/min, respectively.

Nominal concentration:

Nominal concentrations of the mid and high concentration test atmosphere could not be calculated. The mean nominal concentration of the low BL vapour test atmosphere was 112 ± 29 mg/m³, which was of the same order of magnitude as the actual concentration.

Temperature and relative humidity:

The daily mean temperatures in the test atmospheres were between 20.8 and 21.2 °C, the daily mean relative humidities were between 38 and 43%.

Conclusions:

In a sub-acute inhalation toxicity study, Butyl-S-lactate (98.9% purity) showed no effects on survival, body weight, organ weights and gross pathology. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 600 mg/m³. Therefore, the NOAEC (local) for male rats is considered to be 200 mg/m³ and as no systemic effects were noted, the NOAEC (systemic) for male rats is considered to exceed 600 mg/m³.

Executive summary:

In a sub-acute inhalation toxicity study, toxicity of the test item (purity 98.9 %) was examined in male Wistar rats. Groups of 6 male rats were exposed nose-only to target vapour concentrations of 0, 75, 200 or 600 mg/m³ test item for 6 hours a day, 5 days a week during a period of 4 weeks (a total of 20 exposure days). No adverse effects were observed on survival, body and organ weights, and gross pathology. The most prominent finding consisted of histopathological changes in the nasal tissues, which showed transitional epithelium hyperplasia in almost all animals exposed to 600 mg/m³ test item but not in animals exposed to lower concentrations. These nasal changes are related to treatment and point to an irritating effect of Butyl-S-lactate on the nasal epithelium. Therefore, the local NOAEC is considered to be 200 mg/m³. The systemic NOAEC is considered to exceed 600 mg/m³ as no systemic effects were observed. This sub-acute toxicity study in the rat is acceptable and satisfies the guideline requirement according to OECD 412 for a sub-acute inhalation study in the rat.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

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

Suitable data is available for Butyl-S-lactate. A repeated dose inhalation toxicity study in male Wistar rats with the target substance at vapour concentrations of 75, 200 and 600 mg/m³ was conducted in accordance with OECD guideline 412. No systemic effects were observed. The NOAEC (systemic) is thus considered to exceed 600 mg/m³. Local irritation of the nasal epithelium was reported only in animals of the high (600 mg/m³) concentration group. Therefore, the NOAEC (local) is considered to be 200 mg/m³. Accordingly, Butyl-S-lactate is considered as irritating to the respiratory tract.

To further assess the repeated dose toxicity of the primary metabolite butanol, suitable data from the source substances n-butanol and n-butyl acetate were used. 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 sub-chronic oral repeated dose toxicity study with n-butanol was conducted in rats. Ataxia and hypoactivity were observed 2-3 minutes after dosing in both sexes of the high dose group. Based on these effects, the oral NOAEL was considered to be 125 mg/kg bw/day (corresponding to 247 mg/kg bw/day Butyl-S-lactate).

In a sub-chronic inhalation toxicity study, n-butyl acetate showed no effects on mortality, haematology and clinical chemistry. Decreased body weight and feed consumption were noted for the 1500 and 3000 ppm groups, but there was no systemic or organ-specific toxicity. The most prominent finding consisted of histopathological changes in the nasal cavity of rats exposed to 1500 and 3000 ppm n-butyl acetate. It is most likely, that the observed systemic effects are secondary to local respiratory effects, as irritation of the olfactory epithelium is causing pain which might lead to a reduced food uptake and subsequently to a reduced body weight. Based on the study results, the NOAEC for male and female rats is considered to be 500 ppm (equals 2400 mg/m³, corresponding to 3020 mg/m³ Butyl-S-lactate).

No adverse effects were reported in a sub-chronic 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 substances n-butyl acetate and n-butanol does not elicit neurotoxic effects.

Justification for classification or non-classification

Based on the available data, classification of butyl-S-lactate is not warranted according to the CLP Regulation 1272/2008.