2-ethylhexyl lactate

Endpoint summary

Description of key information

Inhalation is the only relevant exposure pathway for 2-ethylhexyl lactate under REACH.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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:

supporting study

Study period:

March 1997 - October 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, but only male rats tested at one dose level. Rats were exposed to the test substance either in the form of aerosols or as vapour.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

; one exposure concentration only, tested both as aerosols and as vapour. Only male rats used.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: young, male Wistar rats (Crl:(WI)WU BR) were obtained from a collony maintained under SPF-conditions at Carles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 255 g
- Fasting period before study: no
- Housing: In groups of three, in suspended, stainless steel cages, fitted with wire mesh floor and front
- Diet (e.g. ad libitum): Commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS, Special Diets Services, Witham, England, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-24.0 °C
- Humidity (%): 35-60 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

IN-LIFE DATES: From: 12 March 1997-22 April 1997

Route of administration:

other: inhalation aerosol or vapour

Type of inhalation exposure:

nose only

Vehicle:

other: air and nitrogen

Remarks on MMAD:

MMAD / GSD: The MMAD of the 2-ethylhexyl lactate aerosol test atmosphere varied between 0.85 and 1.15 µm, indicating that particles were respirable. The gsd varied between 1.3 and 2.5

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmosphere in nose-only inhalation units. Each unit consisted of a cylindrical column surrounded by a transparant 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 animal exposure.
- Method of holding animals in test chamber: the animals were secured in plastic animal holders (Battelle)
- Source and rate of air: humidified pressurized air
- Method of conditioning air: humidified pressurized air
- System of generating particulates/aerosols: To generate the 2-ethylhexyl lactate vapour test atmosphere, a heating mantle and cooler were used. The test material in the flask was kept at a temperature between 132 and 172 °C, the cooler was kept at a temperature between 17.3 and 18.6 °C. The resulting flow was subsequently diluted with metered amounts of humidified air and passed to the inhalation unit. Test material was changed daily. After the first day's exposure it was noticed that the test material remaining in the glass bulb had turned yellow, which might suggest oxidation. The next day, a small flow of nitrogen instead of air was used, which did not result in discolouration. GC analysis carried out by the sponsor indeed showed a considerable loss of test material when using air instead of nitrogen. Nitrogen was, therefore, used during the remainder of the study. The flow of nitrogen used was so small in comparison to the dilution flow that depletion of oxygen in the test atmosphere was considered to be neglegible
The 2-ethylhexyl lactate aerosol test atmosphere was generated by passing metered amounts of the test material using a roller pump (Gilson France SA, Villiers le Bel, France) to an all glass atomizer (Institute's design). The aerosolized material was subsequently mixed with metered amounts of humidified clean air and directed towards animals. The test atmosphere was exhausted at the bottom of the unit. Before the start of the first exposure, the rate of airflow through the atomizer was established at the pressure used. The pressure settings and the settings of the rotameter (dilution airflow) were recorded at regular intervals (at least three times per day). In this way, the total exposure airflow was monitored indirectly through the aerosol generation system.
- Temperature, humidity, pressure in air chamber: The daily mean temperatures in the test atmosphere were between 20.8 and 21.2 °C, the daily mean relative hunidities were between 38 and 43 %. A positive pressure in the central column and a slightly negative pressure in the outer cylinder, which enclosed the entire animal holder, so that dilution of test atmosphere by air leaking form the animals' thorax to the nose was prevented.
- Air flow rate: Mean airflow through the control unit was 33.2 L/min, and those through the 2-ethylhexyl lactate vapour and aerosol unit were 15.3 and 44.3 L/min,respectively.
- Air change rate: Calculated from the air flow rates and the volume of the column of 50 L, air exhange rates for control, vapour and aerosol exposure are 0.66, 0.31 and 0.89 air changes per minute, respectively.
- Method of particle size determination: For the 2-ethylhexyl lactate aerosol exposure, particle size distribrution measurement was carried out once per week using a 10-stage cascade impactor (Andersen, Atlanta, USA) with a largest cut off size of 32.0 µm. The Mass Median Aeordynamic Diameter (MMAD) and the geometric standard deviation (GSD) were calculated (Lee, 1972).
- Treatment of exhaust air: none

TEST ATMOSPHERE
- Brief description of analytical method used: FID
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle: nitrogen and/or air were needed to create aerosols or vapour in the desired concentrations
- Composition of vehicle: compressed air and/or nitrogen

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total carbon analysis:
The amount of 2-ethylhexyl lactate vapour was monitored semi-continuously by means of a total carbon analyser (Ratfisch RS 55, Munich, Germany) using flame ionisation detection (FID). Before the start of the study (21 March 1997), the response of the FID was calibrated by passing flows containing 75 mg/m³ 2-ethylhexyl lactate over the FID. The calibration mixtures corresponding to these flows were prepared by injecting a known quantity of test material (6.0 µl 2-ethylhexyl lactate) in Tedlar bags, with an amount of 75 nL (normal liters) of preheated dry clean air. After evaporation of the test material and careful mixing, samples were passed to the total carbon analyser. The response of the FID (in scale units) was recorded.
The calibration procedure for 2-ethylhexyl lactate was repeated on 26 march 1997, using 6.0 µl of test material and 70 L of air, corresponding to a concentration of 81 mg/m³. This sample was reused on 2 April 1997 due to slightly changed settings of the total carbon analyser.
Test atmospeheres were taken from the unit at the animals' breathing zone. The samples were drawn through sampling lines and were passed via a controlled valves system (KUAX-Control, Kuhnke 61.000) to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). To ensure that no test material would condensate during sampling transport, the sampling lines and the valves system were heated. The unit was monitored, approximately once each half hour for about 7 min, resulting in about 13-14 measurements per concentration level per day.
Gravimetrical analysis:
As a result of the low vapour pressure of 2-ethylhexyl lactate, it was proved in a former study (TNO-report V96.391) that gravimetrical analysis could be used to monitor the concentration of 2-ethylhexyl lactate aerosol in the test atmosphere. The gravimetrical analysis was carried out at least three times per day. Measured test atmosphere samples (50L at 5L/min) were drawn through fibre glass filters. Before and immediately after sampling the filters were weighed (micro balance, Mettler Toledo, Switzerland). The actual concentration was calculated by dividing the amount of test material present on each filter by the volume of test atmosphere sample taken.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Remarks:

Doses / Concentrations:
0, 75 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, vapour: 77 ± 16 mg/m³, aerosol: 78 ± 3 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

6 males per dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The level of 75 mg/m³ corresponds to the lowest level that still induced nasal changes in the 2-ethylhexyl lactate aerosol study performed earlier (TNO-report V96.391). One group of animals was exposed to about 75 mg/m³ 2-ethylhexyl lactate vapour, the other group to the same level of 2-ethylhexyl lactate aerosol. In this way, it could be tested within one study, whether or not an aerosol is more toxic than a vapour at the same concentration, viz. whether a 75 mg/m³ vapour level will be a NOEL in contrast to the 75 mg/m³ aerosol level which proved to be a Minimum-Observed-Adverse-Effect Level (MOAEL; TNO-report V96.391).
- Rationale for animal assignment (if not random): the rats were allocated to the various groups proportionately to weight class by a computer randomization program
- Section schedule rationale (if not random): all rats were killed in such a sequence that the average time of killing was approximately the same for each group.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was observed dail in the morning hours by cage-side observations. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon (shortly after exposure). At weekends only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: If it appeared necessary at cage-side observations, the animals were handeled to detect signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: on day -3, on day 0, just prior to the start of the first exposure, on nominal days 7, 14, 21, 28 and on the day of scheduled necropsy (day 29).

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All animals were examined macroscopically for pathological changes. Adrenals, heart, kidneys, lungs with trachea and larynx, liver, spleen and testes were weighed and relative organ weights (g/kg body weight) were calculated based on the final body weight of the rats. Samples of the abovementioned organs an the nose of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde. The lungs were first inflated with the fixative under ca. 15 cm water pressure.

HISTOPATHOLOGY: Yes
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
The respiratory 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; Young, 1986). The larynx was cut longitudinally at three levels. The trachea with the bifurcation was cut longitudinally/transversally at three levels. Each lung lobe was cut at one sagittal level.
Histopathological examination was performed on the nose and the other respiratory tract organs of all animals.

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 (Cochran, 1957; Steel and Torrie, 1960; Dunnett, 1955 and 1964).
The incidence of histopathological changes was evaluated by Fisher's exact probability test (Siegel, 1956).
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 aanalysis, 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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
General condition and behaviour were not adversely affected by exposure to the test material. Areas of sparsely haired skin in one control rat and one animal exposed to 2-ethylhexyl lactate vapour, but this was not considered to be treatment-related. All rats survived until their scheduled autopsy.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in mean body weights and mean body weight gain between the treatment groups and the controls.

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

GROSS PATHOLOGY
Gross examination at autopsy revealed findings in the kidneys, mediastinal lymph nodes and skin/subcutis 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 euqally distributed between the groups, including the control group. Therefore, the changes were not considered to be related to the treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidences of slight histopathological changes in the larynx, trachea and lungs were about equally distributed amongst the groups, including the control group. Therefore, the changes were considered not to be related to the treatment.
Aerosol:
2-Ethylhexyl lactate aerosol at 75 mg/m³, induced very slight respiratory epithelium hyperplasia in all animals at nasal level 4 and in 4 of 6 animals at nasal level 5. Very slight goblet cell hyperplasia was seen in animals of this group at nasal levels 3-5. Very slight to slight focal transitional epithelial hyperplasia was observed in one animal at nasal level 1 and in two animals at level 2.
Vapour:
The effect of 75 mg/m³ 2-ethylhexyl lactate vapour was restricted to very slight focal goblet cell hyperplasia at nasal levels 3-5.

Dose descriptor:

NOAEL

Effect level:

< 75 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Conclusions:

2-Ethylhexyl lactate aerosol induced stronger effects than 2-ethylhexyl lactate vapour. No NOAEL was identified; the MOAEL is 75 mg/m³ 2-ethylhexyl lactate.

Executive summary:

In a sub-acute inhalation toxicity study 2-ethylhexyl lactate was administered to 6 male Wistar derived rats/concentration by nose-only exposure at concentrations of 0 and 75 mg/m³ as aerosol, and of 75 mg/m³ as vapour, for 6 hours per day, 5 days/week for a total of 28 days.

In a previously conducted study with 2-ethylhexyl-L-lactate aerosol (TNO-report V96.391), minimal respiratory effects were already observed at a concentration of 75 mg/m³ EHL aerosol, which was much lower thant the effect levels in the vapour studies of the other lactate esters. This result suggested a difference in effect level between vapour and aerosol exposure. Therefore, in the present study one group was exposed to 75 mg/m³ EHL vapour and the other to EHL aerosol at the same level. In this way it was tested within one study whether or not aerosol would be more toxic than a vapour at the same concentration.

No exposure-related effects could be found based on clinical signs, body and organs weights. Microscopic evaluation of the respiratory tract showed slight respiratory epithelium hyperplasia, including goblet cell hyperplasia, of the nasal passages of all animals exposed to 75 mg/m³ EHL aerosol, as seen in the previous aerosol study (TNO-report V96.391). Effects of EHL vapour, observed in almost all animals exposed to 75 mg/m³, were restricted to goblet cell hyperplasia. These results indicate that EHL aerosol induced a slightly stronger effect than dit the EHL vapour.

No NOAEL was identified, instead the minimal-observed-adverse-effect level (MOAEL) was 75 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

600 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:

supporting study

Study period:

March 1997 - October 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, but only male rats tested at one dose level. Rats were exposed to the test substance either in the form of aerosols or as vapour.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

; one exposure concentration only, tested both as aerosols and as vapour. Only male rats used.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: young, male Wistar rats (Crl:(WI)WU BR) were obtained from a collony maintained under SPF-conditions at Carles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 255 g
- Fasting period before study: no
- Housing: In groups of three, in suspended, stainless steel cages, fitted with wire mesh floor and front
- Diet (e.g. ad libitum): Commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS, Special Diets Services, Witham, England, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-24.0 °C
- Humidity (%): 35-60 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

IN-LIFE DATES: From: 12 March 1997-22 April 1997

Route of administration:

other: inhalation aerosol or vapour

Type of inhalation exposure:

nose only

Vehicle:

other: air and nitrogen

Remarks on MMAD:

MMAD / GSD: The MMAD of the 2-ethylhexyl lactate aerosol test atmosphere varied between 0.85 and 1.15 µm, indicating that particles were respirable. The gsd varied between 1.3 and 2.5

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmosphere in nose-only inhalation units. Each unit consisted of a cylindrical column surrounded by a transparant 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 animal exposure.
- Method of holding animals in test chamber: the animals were secured in plastic animal holders (Battelle)
- Source and rate of air: humidified pressurized air
- Method of conditioning air: humidified pressurized air
- System of generating particulates/aerosols: To generate the 2-ethylhexyl lactate vapour test atmosphere, a heating mantle and cooler were used. The test material in the flask was kept at a temperature between 132 and 172 °C, the cooler was kept at a temperature between 17.3 and 18.6 °C. The resulting flow was subsequently diluted with metered amounts of humidified air and passed to the inhalation unit. Test material was changed daily. After the first day's exposure it was noticed that the test material remaining in the glass bulb had turned yellow, which might suggest oxidation. The next day, a small flow of nitrogen instead of air was used, which did not result in discolouration. GC analysis carried out by the sponsor indeed showed a considerable loss of test material when using air instead of nitrogen. Nitrogen was, therefore, used during the remainder of the study. The flow of nitrogen used was so small in comparison to the dilution flow that depletion of oxygen in the test atmosphere was considered to be neglegible
The 2-ethylhexyl lactate aerosol test atmosphere was generated by passing metered amounts of the test material using a roller pump (Gilson France SA, Villiers le Bel, France) to an all glass atomizer (Institute's design). The aerosolized material was subsequently mixed with metered amounts of humidified clean air and directed towards animals. The test atmosphere was exhausted at the bottom of the unit. Before the start of the first exposure, the rate of airflow through the atomizer was established at the pressure used. The pressure settings and the settings of the rotameter (dilution airflow) were recorded at regular intervals (at least three times per day). In this way, the total exposure airflow was monitored indirectly through the aerosol generation system.
- Temperature, humidity, pressure in air chamber: The daily mean temperatures in the test atmosphere were between 20.8 and 21.2 °C, the daily mean relative hunidities were between 38 and 43 %. A positive pressure in the central column and a slightly negative pressure in the outer cylinder, which enclosed the entire animal holder, so that dilution of test atmosphere by air leaking form the animals' thorax to the nose was prevented.
- Air flow rate: Mean airflow through the control unit was 33.2 L/min, and those through the 2-ethylhexyl lactate vapour and aerosol unit were 15.3 and 44.3 L/min,respectively.
- Air change rate: Calculated from the air flow rates and the volume of the column of 50 L, air exhange rates for control, vapour and aerosol exposure are 0.66, 0.31 and 0.89 air changes per minute, respectively.
- Method of particle size determination: For the 2-ethylhexyl lactate aerosol exposure, particle size distribrution measurement was carried out once per week using a 10-stage cascade impactor (Andersen, Atlanta, USA) with a largest cut off size of 32.0 µm. The Mass Median Aeordynamic Diameter (MMAD) and the geometric standard deviation (GSD) were calculated (Lee, 1972).
- Treatment of exhaust air: none

TEST ATMOSPHERE
- Brief description of analytical method used: FID
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle: nitrogen and/or air were needed to create aerosols or vapour in the desired concentrations
- Composition of vehicle: compressed air and/or nitrogen

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total carbon analysis:
The amount of 2-ethylhexyl lactate vapour was monitored semi-continuously by means of a total carbon analyser (Ratfisch RS 55, Munich, Germany) using flame ionisation detection (FID). Before the start of the study (21 March 1997), the response of the FID was calibrated by passing flows containing 75 mg/m³ 2-ethylhexyl lactate over the FID. The calibration mixtures corresponding to these flows were prepared by injecting a known quantity of test material (6.0 µl 2-ethylhexyl lactate) in Tedlar bags, with an amount of 75 nL (normal liters) of preheated dry clean air. After evaporation of the test material and careful mixing, samples were passed to the total carbon analyser. The response of the FID (in scale units) was recorded.
The calibration procedure for 2-ethylhexyl lactate was repeated on 26 march 1997, using 6.0 µl of test material and 70 L of air, corresponding to a concentration of 81 mg/m³. This sample was reused on 2 April 1997 due to slightly changed settings of the total carbon analyser.
Test atmospeheres were taken from the unit at the animals' breathing zone. The samples were drawn through sampling lines and were passed via a controlled valves system (KUAX-Control, Kuhnke 61.000) to the total carbon analyser. The response of the total carbon analyser was recorded in scale units and converted into concentration values (mg/m³). To ensure that no test material would condensate during sampling transport, the sampling lines and the valves system were heated. The unit was monitored, approximately once each half hour for about 7 min, resulting in about 13-14 measurements per concentration level per day.
Gravimetrical analysis:
As a result of the low vapour pressure of 2-ethylhexyl lactate, it was proved in a former study (TNO-report V96.391) that gravimetrical analysis could be used to monitor the concentration of 2-ethylhexyl lactate aerosol in the test atmosphere. The gravimetrical analysis was carried out at least three times per day. Measured test atmosphere samples (50L at 5L/min) were drawn through fibre glass filters. Before and immediately after sampling the filters were weighed (micro balance, Mettler Toledo, Switzerland). The actual concentration was calculated by dividing the amount of test material present on each filter by the volume of test atmosphere sample taken.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours a day, 5 days a week

Remarks:

Doses / Concentrations:
0, 75 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, vapour: 77 ± 16 mg/m³, aerosol: 78 ± 3 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

6 males per dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The level of 75 mg/m³ corresponds to the lowest level that still induced nasal changes in the 2-ethylhexyl lactate aerosol study performed earlier (TNO-report V96.391). One group of animals was exposed to about 75 mg/m³ 2-ethylhexyl lactate vapour, the other group to the same level of 2-ethylhexyl lactate aerosol. In this way, it could be tested within one study, whether or not an aerosol is more toxic than a vapour at the same concentration, viz. whether a 75 mg/m³ vapour level will be a NOEL in contrast to the 75 mg/m³ aerosol level which proved to be a Minimum-Observed-Adverse-Effect Level (MOAEL; TNO-report V96.391).
- Rationale for animal assignment (if not random): the rats were allocated to the various groups proportionately to weight class by a computer randomization program
- Section schedule rationale (if not random): all rats were killed in such a sequence that the average time of killing was approximately the same for each group.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was observed dail in the morning hours by cage-side observations. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon (shortly after exposure). At weekends only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: If it appeared necessary at cage-side observations, the animals were handeled to detect signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: on day -3, on day 0, just prior to the start of the first exposure, on nominal days 7, 14, 21, 28 and on the day of scheduled necropsy (day 29).

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All animals were examined macroscopically for pathological changes. Adrenals, heart, kidneys, lungs with trachea and larynx, liver, spleen and testes were weighed and relative organ weights (g/kg body weight) were calculated based on the final body weight of the rats. Samples of the abovementioned organs an the nose of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde. The lungs were first inflated with the fixative under ca. 15 cm water pressure.

HISTOPATHOLOGY: Yes
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
The respiratory 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; Young, 1986). The larynx was cut longitudinally at three levels. The trachea with the bifurcation was cut longitudinally/transversally at three levels. Each lung lobe was cut at one sagittal level.
Histopathological examination was performed on the nose and the other respiratory tract organs of all animals.

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 (Cochran, 1957; Steel and Torrie, 1960; Dunnett, 1955 and 1964).
The incidence of histopathological changes was evaluated by Fisher's exact probability test (Siegel, 1956).
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 aanalysis, 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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
General condition and behaviour were not adversely affected by exposure to the test material. Areas of sparsely haired skin in one control rat and one animal exposed to 2-ethylhexyl lactate vapour, but this was not considered to be treatment-related. All rats survived until their scheduled autopsy.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in mean body weights and mean body weight gain between the treatment groups and the controls.

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

GROSS PATHOLOGY
Gross examination at autopsy revealed findings in the kidneys, mediastinal lymph nodes and skin/subcutis 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 euqally distributed between the groups, including the control group. Therefore, the changes were not considered to be related to the treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidences of slight histopathological changes in the larynx, trachea and lungs were about equally distributed amongst the groups, including the control group. Therefore, the changes were considered not to be related to the treatment.
Aerosol:
2-Ethylhexyl lactate aerosol at 75 mg/m³, induced very slight respiratory epithelium hyperplasia in all animals at nasal level 4 and in 4 of 6 animals at nasal level 5. Very slight goblet cell hyperplasia was seen in animals of this group at nasal levels 3-5. Very slight to slight focal transitional epithelial hyperplasia was observed in one animal at nasal level 1 and in two animals at level 2.
Vapour:
The effect of 75 mg/m³ 2-ethylhexyl lactate vapour was restricted to very slight focal goblet cell hyperplasia at nasal levels 3-5.

Dose descriptor:

NOAEL

Effect level:

< 75 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Conclusions:

2-Ethylhexyl lactate aerosol induced stronger effects than 2-ethylhexyl lactate vapour. No NOAEL was identified; the MOAEL is 75 mg/m³ 2-ethylhexyl lactate.

Executive summary:

In a sub-acute inhalation toxicity study 2-ethylhexyl lactate was administered to 6 male Wistar derived rats/concentration by nose-only exposure at concentrations of 0 and 75 mg/m³ as aerosol, and of 75 mg/m³ as vapour, for 6 hours per day, 5 days/week for a total of 28 days.

In a previously conducted study with 2-ethylhexyl-L-lactate aerosol (TNO-report V96.391), minimal respiratory effects were already observed at a concentration of 75 mg/m³ EHL aerosol, which was much lower thant the effect levels in the vapour studies of the other lactate esters. This result suggested a difference in effect level between vapour and aerosol exposure. Therefore, in the present study one group was exposed to 75 mg/m³ EHL vapour and the other to EHL aerosol at the same level. In this way it was tested within one study whether or not aerosol would be more toxic than a vapour at the same concentration.

No exposure-related effects could be found based on clinical signs, body and organs weights. Microscopic evaluation of the respiratory tract showed slight respiratory epithelium hyperplasia, including goblet cell hyperplasia, of the nasal passages of all animals exposed to 75 mg/m³ EHL aerosol, as seen in the previous aerosol study (TNO-report V96.391). Effects of EHL vapour, observed in almost all animals exposed to 75 mg/m³, were restricted to goblet cell hyperplasia. These results indicate that EHL aerosol induced a slightly stronger effect than dit the EHL vapour.

No NOAEL was identified, instead the minimal-observed-adverse-effect level (MOAEL) was 75 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

75 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

At concentrations up to and including 600 mg/m³, only local irritation effects are seen in the lungs. At the highest concentration of 1800 mg/m³, signs of systemic toxicity were observed. Therefore, a systemic toxicity NOAEC of 600 mg/m³ can be set.