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Administrative data

Description of key information

Suitable data is available for 2-ethylhexyl-S-lactate.

A repeated dose oral toxicity study in rats and mice with 2-ethylhexanol at concentrations of 0, 25, 125, 250 and 500 mg/kg bw/day for 90 days similar with OECD guideline 408. In both studies, a NOAEL of 125 mg/kg bw/day was determined, which corresponds to 195 mg/kg bw/day 2-ethylhexyl-S-lactate.

In a subchronic inhalation toxicity study conducted in accordance with OECD guideline 413, 2-ethylhexanol was administered to 10 male and 10 female Wistar rats by dynamic whole body exposure at concentrations of 15, 40 and 120 ppm (79.8, 212.8 and 638.4 mg/m³) for 6 hours per day, 5 days/week for a total of 90 days. No adverse effects have been observed. Thus, a NOAEC of 638.4 mg/m³ which corresponds to 998 mg/m³ of 2-ethylhexyl-S-lactate has been established.

In a further inhalation study, conducted according to OECD guideline 412 with the target substance 2-ethylhexyl-S-lactate was administered to 5 male and 5 female Wistar derived rats/concentration by nose only exposure at concentrations of 0, 75, 200, 600 and 1800 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days. In this study, local effects have been observed from 75 mg/m³ and systemic toxicity consisting of growth retardation, decreased food intake, and slight induction of hepatic peroxisome proliferation was noted at 1800 mg/m³. Thus, the NOAEC for systemic toxicity is considered to be 600 mg/m³.

In the subacute inhalation study, there is a big distance between the doses. The systemic NOAEC in the subchronic study is between the high dose (1800 mg/m³) and the dose below (600 mg/m³) of the subacute inhalation study. Thus, the NOAEC of the subchronic study (998 mg/m³) is considered to be more precise and therefore, it is used for the calculation of the DNEL.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Ethylhexyl-S-lactate is systemically rapidly transformed into lactic acid and 2-ethylhexanol, the latter being the relevant moiety for toxicological assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
2-ethylhexanol (99.8% purity)

3. ANALOGUE APPROACH JUSTIFICATION
Metabolism data are provide in the chapter "basic toxicokinetics".

4. DATA MATRIX
Reason / purpose:
read-across source
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female mouse at 250 mg/kg died during treatment.
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:
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:
effects observed, treatment-related
Description (incidence and severity):
There were 14 and 13% increases in relative stomach weight at 250 and 500 mg/kg, respectively, and 6.9 and 5.9% increases in relative liver weight at 250 and 500 mg/kg, respectively. See organ weight changes in the mouse in table 2 in "Any other information on results incl. tables".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were dark red foci in the glandular stomach of 2/10 females at 500 mg/kg. There were no other gross findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related findings were a moderate focal or multifocal acanthosis in the forestomach of 2/10 males and 1/10 females at 500 mg/kg.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hepatic peroxisome proliferation:
No increases in pCoA activity were seen in male or female mice at any dose level. See results in table 3 ("Any other information on results incl. tables").
Details on results:
Effects in the 13-week mouse study were limited to increases in relative stomach and liver weight in males at 250 and 500 mg/kg and a low incidence of inflammatory lesions in the forestomach at 500 mg/kg.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
not specified

Table 2: Relative Organ Weights in Mice at a Termination in 13-week Oral Gavage Dosing Studies with 2 -ethylhexanol

Males [Dose (mg/kg/day)] Females [Dose (mg/kg/day)]
0 250 500 0 250 500
Mice (n=10)
Liver 3.43 (0.23) 3.82 (0.38)* 4.23 (0.38)** 3.48 (0.34) 3.90 (0.39) 4.16 (0.61)**
Stomach 0.76 (0.09) 0.88 (0.10) 1.03 (0.08)** 0.87 (0.06) 1.03 (0.13)** 1.12 (0 16)**

Values are mean organ/body weight ratios (±SD).

*p< 0.05.

**p< 0.01.

Table 3: Hepatic CN-lnsensitive Palmitoyl Coenzyme A Activities after 13 Weeks of Oral Gavage Dosing of 2-ethylhexanol

Dose (mg/kg/day)
0 25 125 250 500
Mice (n=3) Mean PCoA activity m nanomol/min/mg of protein (±SD)
Males 4.35 (0.73) 4.76 (0.29) 3.67 (0.42) 3 87 (1.98) 4.33 (1.89)
Females 3.17 ( 1.58) 4.66 (0.82) 5.4 (1.78) 5.31 (1.54) 4.49 (2.27)

*p < 0.001 and **p < 0.05 by ANOVA/Students t test.

Conclusions:
For the oncogenicity study, 750 mg/kg bw/day was selected as a high dose in the oncogenicity study likely to produce minimal stomach inflammation and yet produce toxicity without seriously affecting the lifespan. 50 mg/kg bw/day was selected as a dose level probably free of treatment-effects.
Executive summary:

In a subchronic toxicity study, 2-ethylhexanol (99.8% purity) was administered to 10 male and 10 female B6C3F1 mice by gavage dose levels of 0, 25, 125, 250 and 500 mg/kg bw/day.

One female mouse died at 250 mg/kg bw but no other mortalities occured during treatment.

No clinical signs and body weight changes were observed as well as hematological and biochemistry findings.

Organ weight changes were observed in the stomach and liver at 250 and 500 mg/kg bw/day.

At 500 mg/kg bw/day dark red foci in the glandular stomach were found. Histopathological findings were multifocal acanthosis in the stomach.

No increases in pCoA activity were seen in male or female mice at any dose level.

Based on these results, the NOAEL is 125 mg/kg bw/day which corresponds to 195 mg/kg bw/day for 2 -ethylhexyl-S-lactate and a high dose level of 750 mg/kg bw/day was selected for an oncogenicity study.

This subchronic toxicity study in the mouse is acceptable and satisfies the principle guideline requirement for a subchronic oral study (OECD 408).

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 attached read-across report (see IUCLID section 13).

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 details and justification of read-across please refer to the attached report in section 13 of IUCLID.
Reason / purpose:
read-across source
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a decreased weight gain in male and female rats at 500 mg/kg bw, starting at week 4 in males and week 11 in females amounting to weight losses of 7% in males and 6% in females by week 13.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no differences from controls at any treatment level in food consumption in rats
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a 25% increase in reticulocyte numbers in male and female rats at 500 mg/kg bw.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females at 250 and 500 mg/kg bw had 30 and 36% decreases in serum ALT activities, respectively. Females at 500 mg/kg bw had a 16% decrease in serum cholesterol concentration and males at 500 mg/kg bw had 13% decreases in total protein and albumin concentrations.
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):
Significant differences from controls in rats were moderate and limited to the brain, kidneys, liver, stomach, and testes at 250 and 500 mg/kg bw. Male rat relative brain weights increased by 6% at 500 mg/kg bw, male kidney weights by 8% at 250 and 16% at 500 mg/kg bw, male liver weights by 8% at 250 and 29% at 500 mg/kg bw, male stomach weights by 11 % at 500 mg/kg bw, and testis weights by 5.5% at 500 mg/kg bw. Female rat kidney weights increased by 5% at 250 and 6% at 500 mg/kg bw, female liver weights by 8% at 250 and 15% at 500 mg/kg bw, and female stomach weights by 6% at 250 and 16% at 500 mg/kg. Please refer to detailed results in table 2 in box "Any other information on results incl. tables".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions differing from controls were only seen at 500 mg/kg bw. 2/10 males and 4/10 females exhibited single or multiple slightly elevated foci in the forestomach. There were no other gross findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related findings in rats were limited to the forestomach and liver at 500 mg/kg bw. There was a generalized acanthosis of the forestomach mucosa in 1/10 males with ballooning degeneration of the epithelial wall and acanthosis of the forestomach mucosa in 2/10 males and 5/10 females. There was a moderate decrease in hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal, 8-cell hyperplasia in 3/10 female rats.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hepatic peroxisome proliferation:
Hepatic peroxisome proliferation was determined in an ancillary 13-weeks study by measuring the activity of hepatic cyanide-insensitive palmitoyl Coenzyme A in livers at termination. Increases in pCoA activity were 6.5-fold in male rats and 3.4-fold in females at 500 mg/kg bw, with decreases in body weight gain similar to those in the main study. Please refer to detailed results in table 3 in box "Any other information on results incl. tables".
Details on results:
Effects in the 13-week rat study were limited to dose levels of 250 and 500 mg/kg bw, the principal effects of significance being mild reductions in weight gain at 500 mg/kg bw, moderate increases in relative stomach, liver, and kidney weights at 250 and 500 mg/kg bw, and inflammatory changes in the forestomach at 500 mg/kg bw. The target organs identified subchronically were thus the liver, forestomach, kidneys, and possibly the adrenals. The central nervous system, the thymus, and the spleen, identified as target organs at dose levels of 1000 and 1500 mg/kg bw in the dose range finding study, are not target organs at the dose levels in the sub-chronic study.
Key result
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
not specified

Table 2: Relative Organ Weights in Rats

Males [Dose (mg/kg bw/day)] Females [Dose (mg/kg bw/day)]
0 250 500 0 250 500
Rats (n=10)
Brain 0.68 (0.03) 0 70 (0.02) 0.72 (0.02)** 1.07 (0.03) 1.1 (0.06) 1.1 (0.04)
Kidneys 0.69 (0.02) 0.75 (0.02)** 0.81 (0.04 )** 0.77 (0.02) 0.81 (0.03)* 0.82 (0.03)**
Liver 2.77 (0.11) 2.98 (0.08)** 3.57 (0.22)** 2.67 (0.11) 2.88 (0.08)** 3.07 (0.07)**
Stomach 0.57 (0.03) 0.58 (0.03) 0.63 (0.02)** 0.71 (0.03) 0.75 (0.03)* 0.82 (0.04)**
Testes/Ovaries 1.11 (0.05) 1.16 (0.07) 1.17 (0.06)* 0.041 (0.003) 0.037 (0.005)* 0.039 (0.004)

Values are mean organ/body weight ratios (± SD)

*p< 0.05.

**p< 0.01.

Table 3: Hepatic CN-lnsensitive Palmitoyl Coenzyme A activities after 13 weeks

Dose (mg/kg bw/day)
0 25 125 250 500
Rats (n=3) Mean pCoA activity m nanomol/min/mg of protein (±SD)
Males 3.38 (1.11) 4.49 (0.78) 5.21 (0.70) 6.24 (1.83) 22.0 (0.56)*
Females 2.95 (2.36) 4.54 (0.69) 5.01 (0.74) 6.6 (1.43) 9.92 (2.26)**

*p< 0.001 and **p< 0.05 by ANOVA/Students t-test.

Conclusions:
In conclusion, in a 13-week subchronic toxicity study (conducted similar to OECD 408), 2-ethylhexanol was adminstered orally to ten F344 rats/sex/group at dose levels of 0, 25, 125, 250 and 500 mg/kg bw/day. Based on the results, the NOEL can be considered to be 125 mg/kg bw/day.
Executive summary:

In a 13-week subchronic toxicity study (conducted similar to OECD 408), 2-ethylhexanol (99.8% purity) was administered orally to ten F344 rats/sex/group at dose levels of 0, 25,125, 250 and 500 mg/kg bw/day. Under the conditions of this study, there was no mortality and no adverse clinical signs of toxicity were noted during the treatment period.

There was a decreased weight gain in male and female rats at 500 mg/kg bw, starting at week 4 in males and week 11 in females amounting to weight losses of 7% in males and 6% in females by week 13. No effects were noted on food consumption. At 500 mg/kg bw a 25% increase in reticulocyte numbers were observed. Clinical biochemistry analysis revealed that females at 250 and 500 mg/kg bw had 30 and 36% decreases in serum ALT activities, respectively. Females at 500 mg/kg bw had a 16% decrease in serum cholesterol concentration and males at 500 mg/kg bw had 13% decreases in total protein and albumin concentrations. In addition, significant differences in organ weights from controls in rats were moderate and limited to the brain, kidneys, liver, stomach, and testes at 250 and 500 mg/kg bw. Male rat relative brain weights increased by 6% at 500 mg/kg bw, male kidney weights by 8% at 250 and 16% at 500 mg/kg bw, male liver weights by 8% at 250 and 29% at 500 mg/kg bw, male stomach weights by 11 % at 500 mg/kg bw, and testis weights by 5.5% at 500 mg/kg bw. Female rat kidney weights increased by 5% at 250 and 6% at 500 mg/kg bw, female liver weights by 8% at 250 and 15% at 500 mg/kg bw, and female stomach weights by 6% at 250 and 16% at 500 mg/kg. Gross pathology showed that 2/10 males and 4/10 females exhibited single or multiple slightly elevated foci in the forestomach. There were no other gross findings. Histopathology showed that dose-related findings in rats were limited to the forestomach and liver at 500 mg/kg bw. There was a generalized acanthosis of the forestomach mucosa in 1/10 males with ballooning degeneration of the epithelial wall and acanthosis of the forestomach mucosa in 2/10 males and 5/10 females. There was a moderate decrease in hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal, 8-cell hyperplasia in 3/10 female rats. Additionally, hepatic peroxisome proliferation was determined in an ancillary 13-weeks study by measuring the activity of hepatic cyanide-insensitive palmitoyl Coenzyme A in livers at termination. Increases in pCoA activity were 6.5-fold in male rats and 3.4-fold in females at 500 mg/kg bw, with decreases in body weight gain similar to those in the main study. Based on the results obtained from this study, the NOEL can be considered to be 125 mg/kg bw/day which corresponds to 195 mg/kg bw/day 2 -ethylhexyl-S-lactate.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
195 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:
key study
Study period:
February 1996-October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar derived (Hsd Cpb.WU) rats were obtained from Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: on average 229.5 g for males and 170.1 g for females
- Fasting period before study: none
- Housing: in groups of five per sex in suspended, stainless steel cages fitted with wire-mesh floor and front
- Diet (e.g. ad libitum): cereal-based TNO rodent diet, ad libithum
- Water (e.g. ad libitum): potable water supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN), ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-24.0 °C
- Humidity (%): 43-72 %
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES: From: 7 February 1996-21 March 1996
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Particle size measurement showed that almost all particles in the animal breathing zone were respirable, viz. they were smaller than or equal to 4.2 µm. The mean Mass Median Aerodynamic Diameter (MMAD) was 1.4, 2.6, 1.7, and 2.5 µm for the low, intermediate and high exposure levels, respectively. The geometric standard deviation was 1.8 for the low concentration level and 1.5 for the other three levels.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmospheres in nose-only inhalation units. Each unit consisted of a cylindrical teflon-coated column, surrounded by a transparent cylinder. The column had a volume of ca. 50 L and consisted of a top assembly, with two rodent tube sections underneath and an exhaust section at the bottom. The rodent tube sections had 40 ports for animal exposure.
- Method of holding animals in test chamber: plastic animal holders (Batelle)
- Source and rate of air: humidified pressurized air
- Method of conditioning air: humidified
- System of generating particulates/aerosols: intermediate- and high-concentration test atmosphere were generated by nebulizing the test material using compressed air driven steel nebulizers (Institute's design). These nebulizers consisted of an atomizer (Lechler Informatic Buro Holland, Oudorp, the Netherlands) and a glass jar containing the test materials. During operation, the test material was drawn through a suction tube to the atomizer and was blown against a baffle which was fitted below the nozzel orifice in such a way that the larger droplets were removed by impaction. The impacted test material drained back into the test material supply at the bottom of the jar. The resulting aerosol was subsequently mixed with metered amounts of humidified pressurized air at the entrance of the exposure unit and directed towards the animals.
The low-concentration test atmosphere was generated by passing metered amounts of the test material using a roller pump (Golson, Villiers le Bel, France) to a compressed air driven, all glass nebulizer (Institute's design). This device was chosen in order to generate the test atmosphere at a low concentration of 75 mg/m³. The generated mixture of test material and air was subsequently mixed with metered amounts of pressurized air and was again mixed at the entrance of the exposure unit with metered amounts of pressurized air. The resulting test atmosphere was directed downward through the mixing chamber towards the animals' noses.
- Temperature, humidity, pressure in air chamber: the daily mean temperature was 22.5 ± 0.7 °C, 22.5 ± 0.7 °C, 22.5 ± 0.6 °C, 22.4 ± 0.7 °C and 22.4 ± 0.7 °C for the control, low, intermediate and high concentration levels, respectively. The daily relative humidity was 56 ± 8 %, 50 ± 7 %, 52 ± 7 %, 50 ± 7 % and 53 ± 7 %, repectively. A positive pressure in the central column and a slightly negative pressure in the outer cylinder, which enclosed the entire animal holder, were maintained to prevent dilution of the test atmosphere by air leaking from the animal's thorax to the nose.
- Air flow rate: the mean daily airflows were 26.5, 73.1, 55.7, 71.4 and 45.2 L/min for the control, low, intermediate and high concentration level, respectively.
- Air change rate: given the volume of ca 50 L of each inhalation unit, the air change rate was calculated to be 0.53, 1.46, 1.11 1.43 and 0.90 air changes per minute for the control, low, intermediate and high concentration level, respectively.
- Method of particle size determination:once per week (except for the control test atmosphere) using an 11-stage cascade impacter (Institute's design) with the largest cut off size of 4.2 µm. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometric standard deviation (gsd) were calculated (Lee, 1972).
- Treatment of exhaust air: The test atmosphere was exhausted at the bottom of the unit.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrical analysis was used to monitor the concentration of 2-ethylhexyl lactate in the test atmosphere. Preliminary experiments showed that 2-ethylhexyl lactate did hardly evaporate, even after using forced evaporation, showing the method suitable for measurement of the actual concentration in air. The acutal concentration of 2-ethylhexyl lactate in the test atmosphere was determined at least 3 times per exposure per day. Measured test atmosphere samples were obtained by passing approximately 100, 50, 30 or 10 liters of test atmosphere for the low-, intermediate-, and high-concentration level, respectively, at sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed. The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: compressed air was used to generate aerosols
- Composition of vehicle: compressed air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As a result of the low vapour pressure of the test material, it was checked whether gravimetrical analysis could be used as a means to monitor the concentration of 2-ethylhexyl lactate in the test atmosphere. Hereto, the degree of evaporation was determined during the preliminary phase of the study as follows:
- drying of a known amount of the test materials on a glass fiber filters (Sartorius) at ambient temperature and humidity (19.2 °C/46.4 %). This procedure was carried out twice, for up to 70 and 73 minutes, respectively.
- after forced evaporation of a known amount of the test material on a glass fiber fliter using dried pressurized air (5L/min). This procedure was also carried out twice, for up to 70 and 105 minutes, respectively.
The first procedure showed thath no evaporation had occurred sionce slight increases in weight (up to 0.7 %) rather than decreases in weight were observed. It was condluded that the test material was slightly hygroscopic and did not evapirate at ambient conditions.
The second procedure, using forced evaporation, showed that decreases in weight up to 0.5% were observed after 10 minutes, up to 1 % after 20 minutes, and up to 2.5 % after 30 minutes. After 70 and 105 minutes, respectively, the decreases in weight amounted to approximately 5 %.
These preliminary ecperiments therefore, showed, that 2-ethylhexyl lactate did hardly evaporate, even after using forced evaporation. It was, therefore, concluded that gravimetrical analysis was a suitable method to measure the actual concentration in air.
The acutal concentration of 2-ethylhexyl lactate in the test atmosphere was determined at least 3 times per exposure per day. Measured test atmosphere samples were obtained by passing approximately 100, 50, 30 or 10 liters of test atmosphere for the low-, intermediate-, and high-concentration level, respectively, at sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed.The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
Duration of treatment / exposure:
28 days (4 weeks)
Frequency of treatment:
6 hours a day, 5 days a week
Remarks:
Doses / Concentrations:
0, 244, 392, 784 and 2144 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 77 (± 12), 221 (± 27), 627 (± 91) and 1791 (± 111) mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The exposure levels were based on similar studies with ethyl lactate showing moderate to severe nasal irritating and toxic (local) effects at levels of 600 and 2500 mg/m³, whereas slight systemic toxicity (reduced food consumption and growth retardation) was observed at the 2500 mg/m³ level only. The No-Oberserved-Adverse-Effect Level (NOAEL) in these studies was 200 mg/m³. In addition, the in vitro hydrolysing activity of these compounds by nasal olfactory epithelium was taken into account with 2-ethylhexyl lactate showing an approximately 6.5 times higher affinity and a 2.5 times higher velocity than ethyl lactate.
In view of the higher in vitro hydrolysing acticity, the target levels chosen for the present study, therefore, were slightly lower than those for ethyl lactate, viz.: 0, 75, 200, 600 and 1800 mg/m³. The highest concentration level was intended to result in local toxic effects but not to produce overt systemic toxicity nor an incidence of fatalities which would prevent meaningful evaluation. The lowest concentration was expected not to produce any evidence of toxicity. The intermediate concentrations were expected to produce no or minimal observable local toxic effects.
- Rationale for animal assignment (if not random): The animals were allocated to five groups of 5 rats/sex, proportionally by weight class, by a computer randomization programme.
- Section schedule rationale (if not random): At the end of the treatment period (day 28) all rats were killed in such a way that, on average, time of killing was the same for each group.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the general condition and behaviour of all individual animals were visually inspected daily in the morning hours. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon, shortly after exposure. In weekends only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the general condition and behaviour of all individual animals were visually inspected daily in the morning hours. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon, shortly after exposure. In weekends only one check per day was carried out. All abnormalities, signs of ill health or reaction to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once during the acclimatization period (day -7), one day before the start of the study (allocation procedure), just prior to the start of the first exposure to the test materials (day 0), on days 7, 14 and 21, and on the day of necropsy (day 28) in order to determine the organ-to-body weight ratios. In addition, weekly body weight gain was calculated.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food intake was measured weekly per cage over 4 successive 1-week periods, by weighing the feeders.

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: Yes, the efficiency of food utilization was calculated and expressed as gram weight per gram food consumed.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy, the day after the last exposure
- Anaesthetic used for blood collection: Yes (identity), under ether anaesthesia
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Haemoglobin concentration, packed cell volume, red blood cell count, reticulocytes (blood smears were prepared and stained; those of the control group and two highest exposure groups were counted since changes in red blood cell variables were observed in these two groups only), total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Fasting glucose was determined on day 26 of the study in blood collected from the tip of the tail of all rats after deprivation of food for ca. 24 hours. The other clinico-chemical examinations were conducted in blood collected at necropsy from the abdominal aorta of all, non-fasted rats.
- Animals fasted: Yes for glucose, no for the other examinations
- How many animals: all animals
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALAT; glutamic-pyruvic transaminase), aspartate aminotransferase (ASAT; glutamic-oxaloacctic transaminase), gamma glutamyl transferase (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, total bilirubin, cholesterol, triglycerides, phospholipids, sodium (Na), potassium (K), calcium (Ca), chloride (Cl) and inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Samples of the following tissues/organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde: liver (small representative samples were taken from the two largest liver lobes; the remainder of the livers was preserved for biochemical examinations), adrenals, spleen, heart, gross lesions, kidneys, testes, lungs with tranchae and larynx, and nose (nasal cavity). All but the gross lesions and the nose were weighed. The lungs were inflated with the fixative under 15 cm water pressure.

HISTOPATHOLOGY: Yes. All tissues/organs listed above (gross pathology) were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
The respiratory tract (nose, larynx, tranchea 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) and are indicated in Figure 1 (see section 'attached background material'). Each level is reported seperately.
The larynx was cut longitudinally. The three regions reported are: (1) the cranial part extending from the epiglottis to the cricoid cartillage, (2) the caudal part at the level of the cricoid cartillage, and (3) the transition between the caudal part of the larynx and the first trancheal ring.
The tranchea with the bifurcation was cut longitudinally/transversally. Five regions are reported: (1) the longitudinally-cut cranial part comprising the first 3 to 7 tracheal rings, (2) the transversally-cut medial part, adjacent to the caudal part, (3) the longitudinally-cut caudal part comprising the last 5 tracheal rings cranial to the bifurcation, (4) the longitudinally-cut bifurcation, and (5) the longitudinally-cut extrapulmonary bronchi.
Each lung lobe was cut at three sagittal levels. The left lung lobe and the right lung lobe are reported seperately.
Histopathological examinations were performed on heart, kidneys, liver, spleen, and testes of all animals of the control and high-concentration group. The respiratory tract (nose, larynx, tranchea and lungs) was examined in all rats of all groups since treatment-related changes were seen in rats of both sexes of the high-concentration group.
Other examinations:
Biochemical examination:
The remainder of the livers from each rat, after weighing and samples had been taken into formalin, were immediately deep frozen in liquid nitrogen and stored at -70 to -80 °C. At analysis, the livers were homogenised using a potter Elvejhem tissue homogenizer at 0-4 °C 0.25 M sucrose/ 2mM EDTA and 10 mM Tris-HCl (20 % w/v). A part of each homogenate was then frozen and thawed three times. After centrifugation the supernatant was stored at -70 to -80 °C prior to determination of acyl-CoA oxidase activities with palmitoyl-CoA as substrate as described by Reubsaet et al. (1988). Protein concentrations were determined using the Biorad method on a Cobas-bio analyzer. The acyl-CoA oxidase activity was expressed as nmol H2O2/min per mg protein.
Biochemical examinations were, in first instance, performed on the liver of all animals of the control and high concentration group. Since statistically significant changes were observed in animals of the high concentration group, the biochemcial examinations were extended to all groups.
Statistics:
Body weights and body weight gain data, organ weights, haematological (absolute data), clinical chemistry data and acyl-CoA oxidase activity in the liver were evaluated by one-way analysis of covariance (for body weights) or analysis of variance, followed by the Dunnett's multiple comparison test (Cochran, 1957; Steel and Torrid, 1960); Dunnett, 1955 and 1964).
Relative haematological data were analysed by Kruskal-Wallis non-parametric analysis of variance followed by the Mann/Whitney U-test (Siegel, 1956).
Incidences of histopathological changes were analysed by the Fisher exact probability test (Siegel, 1956).
No statistical analyses were performed on food consumption data since only one measurement was made per sex per group. 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) will be greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as concentration-response relationships and whether the results were significant in the light of other biological and pathological findings.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
Abnormalities seen during exposure were confined to visually increased breathing rate in animals exposed to 600 or 1800 mg/m³ at the beginning of the treatment period until nominal day 11.
All animals of the high-concentration group showed nasal discharge towards the end of the treatment period. In addition, 9/10 rats of this group were sniffing during the last three days.
A dermal wound, followed by dermal encrustations and alopecic areas on the skin/fur was observed in one female of the low concentration group. This finding was not considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Male rats of the high concentration group showed statistically significant decreased body weights when compared to controls throughout the treatment period. The mean weekly body weight gain in this group was also significantly lower than that of controls during the first two weeks. Male rats exposed to 75 or 600 mg/m³ showed a decreased body weight gain during the last exposure week.
Female rats did not show any significant differences in mean body weight or weekly body weight gain when compared to controls.

FOOD CONSUMPTION
Mean food intake was reduced in males of the high concentration group relative to controls during the entire period. In females, mean food was comparable amongst all groups.

FOOD EFFICIENCY
Mean food conversion efficiency was reduced in males of the high concentration group relative to controls during the entire period. In males exposed to 75 or 600 mg/m³, food conversion efficiency was low during the last exposure week

HAEMATOLOGY
Changes in red blood cell or coagulation variables in rats exposed to 2-ethylhexyl lactate were limited to a statistically significant increase in haemoglobin content and packed cell volume in males exposed to 600 mg/m³ and an increase in red blood cell count and packed cell volume and a decrease in mean corpuscular haemoglobin concentration in females of the high-concentration group.
An increase in the absolute and relative number of neutrophils was observed in males exposed to 200 or 1800 mg/m³. The differences with the controls were statistically significant for the absolute number in males exposed to 1800 mg/m³ and in the relative number of this cell type in males exposed to 200 mg/m³. Males of the latter group concomitantly showed a significant decrease in the relative number of lymphocytes.

CLINICAL CHEMISTRY
Slightly though significantly increased ALAT levels were observed in females of the low and high concentration group. Since this change was not seen in the intermediate concentration groups, was not concentration-related and not observed in males, this finding was considered to be an incidental finding, unrelated to the treatment.
The slight, though statistically significant decrease in calcium level in males of the high concentration group was considered to be a fortuitous finding.

ORGAN WEIGHTS
In animals exposed to 2-ethylhexyl lactate statistically significant changes in spleen, liver and lung weights were observed relative to the controls:
- absolute spleen weight decreased in rats of both sexes of the 1800 mg/m³ group and in males exposed to 600 mg/m³; relative spleen weight decreased in females of the high concentration group only,
- relative liver weight increased in both sexes of the high concentration group,
- relative lung weight increased in male rats of the high concentration group.

GROSS PATHOLOGY
At autopsy, most gross changes were observed in the lungs of animals exposed to the test compound while only one control animal exhibited lungs with a gross change. However, concentration-response relationships were absent. In addition, a few changes were observed in organs outside the respiratory tract. They occurred in a single animal only and are common for rats of this strain and age. Therefore, the changes in the lungs and other organs were considered to be unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination revealed treatment-related changes in the respiratory tract. No other target organs were identified.
Animals of the 200, 600 and 1800 mg/m³ groups exhibited changes througout the respiratory tract. In animals of the 75 mg/m³ group, the changes were restricted to the nasal cavity.
Nasal cavity:
All types of epithelium lining the nasal cavity (squamous, transitional, respiratory and olfactory) were affected in animals of the 1800 mg/m³ group. In animals of the 200 and 600 mg/m³ groups, changes were observed in squamous, transitional and respiratory epithelium, whereas in animals exposed to 75 mg/m³, changes were largely restricted to the respiratory epithelium including goblet cell hyperplasia.
In animals exposed to 75 mg/m³, very slight to slight goblet cell hyperplasia was observed at levels 2-6, predominantly in the middle (medial) part of the septum and in the ventral meatus. In addition, some animals exhibited minimal focal hyperplasia of the respiratory epithelium at levels 2-5. Minimal focal transitional epithelial hyperplasia was observed in one male and one female. Rhinitis was not observed.
In animals exposed to 200 mg/m³, the squamous epithelium at level 1 was focally hyperplastic. The transitional epithelium was focally hyperplastic at levels 1 and 2. At level 2, the epithelial hyperplasia included disorganized appearance with a flattened surface, and a decreased number of goblet cells. This type of epithelial hyperplasia was denoted 'atypical hyperplasia' (Schwartz et al., 1984). Focal respiratory epithelial and goblet cell hyperplasia were largely comparable to that found in 75 mg/m³ exposed animals, although the incidence of the respiratory epithelial hyperplasia had increased in the 200 mg/m³ group, and the lesions extended to level 6. One female exhibited very slight focal olfcatory epithelial athrophy at level 3. In addition, very slight rhinitis occurred at all levels.
In the 600 mg/m³ exposed animals, minimal atrophy of the squamous epithelium at level 1 was observed in one male and one female. No atrophy of the olfactory epithelium was observed. In general, the hyperplastic changes were more severe and more extensive throughout the nasal cavity than in animals exposed to 200 mg/m³. The incidence and severity of rhinitis tended to be slightly increased when compared to the 200 mg/m³ group.
In the animals exposed to 1800 mg/m³, almost the entire squamous, transitional and respiratory epithelial lining was affected and demonstrated (atypical) hyperplasia, metaplasia, rhinitis and atrophy with ulcerations. In addition, a large part of the olfactory epithelial lining was atrophic with ulcerations. Where the olfactory epithelium was mostly affected, when compared to controls, the lamina propria was less cellular with loss of nerves and Bowman's glands, while the thickness suggested oedema. Rhinitis was observed thoughout levels 1-6. Rhinitis included intraepithelial and submucosal inflammatory cell infiltrates and intraepithelial micorabscesses. Especially with regard to the degenerative lesions and rhinitis, the incidence and severity of the lesions had markedly increased when compared to the other exposure groups. Goblet cell hyperplasia was not observed at the highest concentration level.
Larynx and trachea:
Slight epithelial hyperplasia and slight squamous metaplasia were observed in larynx and trachea of animals exposed to 200, 600 or 1800 mg/m³. All three regions of the larynx, the cranial part of the trachea and the tip of the carina at the bifurcation were sensitive sites. A concentration-related severity, however, was not observed.
Some animals exposed to 75 mg/m³ exhibited slight squamous metaplasia at the bifurcation, but a statistically significant difference in incidence with te controls was not present. Moreover, squamous metaplasia was also seen in one female control. The changes at the location of the bifurcation as seen in these animals, therefore, might be considered not to be treatment-related.
Lungs:
Changes in the lungs were observed in animals exposed to 200, 600 or 1800 mg/m³ and consisited of focal interstitial collagen deposits ('septal fibrosis') in the walls of alveolar ducts. The affected sites were covered with cuboidal epithelial cells. Septal fibrosis progressed from very slight or slight in female animals exposed to 200 or 600 mg/m³ to slight or moderate in the 1800 mg/m³ exposed females. An increase in severity with increasing concentration was less prominent in male rats.
Other organs:
All other organs exhibited histopathological changes which were about equally distributed among the groups or occurred in a single animal only. Moreover, they are common for the strain and age of the rats used. They were, therefore, not attributed to treatment.

OTHER FINDINGS
Compared to the control group, the hepatic acyl-CoA oxidase activity had significantly increased in rats exposed to 1800 mg/m³, indicative of peroxisome proliferating activity. In rats exposed to 75, 200 or 600 mg/m³, acyl-CoA oxidase activity was comparable to or slightly lower than that of controls.
Dose descriptor:
NOAEL
Effect level:
< 75 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The lowest level tested, viz. 75 mg/m³ was associated with minimal histopathological nasal changes in rats of both sexes. The concentration of 75 mg/m³ was, therefore, considered to be a minimum-observed-adverse-effect level (MOAEL).
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified
Conclusions:
2-Ethylhexyl lactate is irritating to the respiratory tract. At the highest level tested, viz. 1800 mg/m³, also systemic toxicity consisting of growth retardation, decreased food intake, and slight induction of hepatic peroxisome proliferation was noted. Since minimal histopathological nasal changes were observed in rats of both sexes exposed to the lowest level tested, viz. 75 mg/m³, this concentration was considered to be a minimum-observed-adverse-effect level (MOAEL).
Executive summary:

In a subacute inhalation toxicity study 2-ethylhexyl lactate was administered to 5 male and 5 female Wistar derived rats/concentration by nose only exposure at concentrations of 0, 75, 200, 600 and 1800 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

The lowest level tested, viz. 75 mg/m³ was associated with minimal histopathological nasal changes in rats of both sexes. The concentration of 75 mg/m³ was, therefore, considered to be a minimum-observed-adverse-effect concentration (MOAEC). The next higher levels tested, viz. 200 and 600 mg/m³, resulted in changes throughout the respiratory tract. At the highest level tested, viz. 1800 mg/m³, besides local changes in the respiratory tract, systemic toxicity consisting of growth retardation, decreased food intake, and slight induction of hepatic peroxisome proliferation was noted.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
998 mg/m³
Study duration:
subchronic
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:
key study
Study period:
February 1996-October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar derived (Hsd Cpb.WU) rats were obtained from Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: on average 229.5 g for males and 170.1 g for females
- Fasting period before study: none
- Housing: in groups of five per sex in suspended, stainless steel cages fitted with wire-mesh floor and front
- Diet (e.g. ad libitum): cereal-based TNO rodent diet, ad libithum
- Water (e.g. ad libitum): potable water supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN), ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-24.0 °C
- Humidity (%): 43-72 %
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES: From: 7 February 1996-21 March 1996
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Particle size measurement showed that almost all particles in the animal breathing zone were respirable, viz. they were smaller than or equal to 4.2 µm. The mean Mass Median Aerodynamic Diameter (MMAD) was 1.4, 2.6, 1.7, and 2.5 µm for the low, intermediate and high exposure levels, respectively. The geometric standard deviation was 1.8 for the low concentration level and 1.5 for the other three levels.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmospheres in nose-only inhalation units. Each unit consisted of a cylindrical teflon-coated column, surrounded by a transparent cylinder. The column had a volume of ca. 50 L and consisted of a top assembly, with two rodent tube sections underneath and an exhaust section at the bottom. The rodent tube sections had 40 ports for animal exposure.
- Method of holding animals in test chamber: plastic animal holders (Batelle)
- Source and rate of air: humidified pressurized air
- Method of conditioning air: humidified
- System of generating particulates/aerosols: intermediate- and high-concentration test atmosphere were generated by nebulizing the test material using compressed air driven steel nebulizers (Institute's design). These nebulizers consisted of an atomizer (Lechler Informatic Buro Holland, Oudorp, the Netherlands) and a glass jar containing the test materials. During operation, the test material was drawn through a suction tube to the atomizer and was blown against a baffle which was fitted below the nozzel orifice in such a way that the larger droplets were removed by impaction. The impacted test material drained back into the test material supply at the bottom of the jar. The resulting aerosol was subsequently mixed with metered amounts of humidified pressurized air at the entrance of the exposure unit and directed towards the animals.
The low-concentration test atmosphere was generated by passing metered amounts of the test material using a roller pump (Golson, Villiers le Bel, France) to a compressed air driven, all glass nebulizer (Institute's design). This device was chosen in order to generate the test atmosphere at a low concentration of 75 mg/m³. The generated mixture of test material and air was subsequently mixed with metered amounts of pressurized air and was again mixed at the entrance of the exposure unit with metered amounts of pressurized air. The resulting test atmosphere was directed downward through the mixing chamber towards the animals' noses.
- Temperature, humidity, pressure in air chamber: the daily mean temperature was 22.5 ± 0.7 °C, 22.5 ± 0.7 °C, 22.5 ± 0.6 °C, 22.4 ± 0.7 °C and 22.4 ± 0.7 °C for the control, low, intermediate and high concentration levels, respectively. The daily relative humidity was 56 ± 8 %, 50 ± 7 %, 52 ± 7 %, 50 ± 7 % and 53 ± 7 %, repectively. A positive pressure in the central column and a slightly negative pressure in the outer cylinder, which enclosed the entire animal holder, were maintained to prevent dilution of the test atmosphere by air leaking from the animal's thorax to the nose.
- Air flow rate: the mean daily airflows were 26.5, 73.1, 55.7, 71.4 and 45.2 L/min for the control, low, intermediate and high concentration level, respectively.
- Air change rate: given the volume of ca 50 L of each inhalation unit, the air change rate was calculated to be 0.53, 1.46, 1.11 1.43 and 0.90 air changes per minute for the control, low, intermediate and high concentration level, respectively.
- Method of particle size determination:once per week (except for the control test atmosphere) using an 11-stage cascade impacter (Institute's design) with the largest cut off size of 4.2 µm. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometric standard deviation (gsd) were calculated (Lee, 1972).
- Treatment of exhaust air: The test atmosphere was exhausted at the bottom of the unit.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetrical analysis was used to monitor the concentration of 2-ethylhexyl lactate in the test atmosphere. Preliminary experiments showed that 2-ethylhexyl lactate did hardly evaporate, even after using forced evaporation, showing the method suitable for measurement of the actual concentration in air. The acutal concentration of 2-ethylhexyl lactate in the test atmosphere was determined at least 3 times per exposure per day. Measured test atmosphere samples were obtained by passing approximately 100, 50, 30 or 10 liters of test atmosphere for the low-, intermediate-, and high-concentration level, respectively, at sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed. The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: compressed air was used to generate aerosols
- Composition of vehicle: compressed air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As a result of the low vapour pressure of the test material, it was checked whether gravimetrical analysis could be used as a means to monitor the concentration of 2-ethylhexyl lactate in the test atmosphere. Hereto, the degree of evaporation was determined during the preliminary phase of the study as follows:
- drying of a known amount of the test materials on a glass fiber filters (Sartorius) at ambient temperature and humidity (19.2 °C/46.4 %). This procedure was carried out twice, for up to 70 and 73 minutes, respectively.
- after forced evaporation of a known amount of the test material on a glass fiber fliter using dried pressurized air (5L/min). This procedure was also carried out twice, for up to 70 and 105 minutes, respectively.
The first procedure showed thath no evaporation had occurred sionce slight increases in weight (up to 0.7 %) rather than decreases in weight were observed. It was condluded that the test material was slightly hygroscopic and did not evapirate at ambient conditions.
The second procedure, using forced evaporation, showed that decreases in weight up to 0.5% were observed after 10 minutes, up to 1 % after 20 minutes, and up to 2.5 % after 30 minutes. After 70 and 105 minutes, respectively, the decreases in weight amounted to approximately 5 %.
These preliminary ecperiments therefore, showed, that 2-ethylhexyl lactate did hardly evaporate, even after using forced evaporation. It was, therefore, concluded that gravimetrical analysis was a suitable method to measure the actual concentration in air.
The acutal concentration of 2-ethylhexyl lactate in the test atmosphere was determined at least 3 times per exposure per day. Measured test atmosphere samples were obtained by passing approximately 100, 50, 30 or 10 liters of test atmosphere for the low-, intermediate-, and high-concentration level, respectively, at sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed.The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
Duration of treatment / exposure:
28 days (4 weeks)
Frequency of treatment:
6 hours a day, 5 days a week
Remarks:
Doses / Concentrations:
0, 244, 392, 784 and 2144 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 77 (± 12), 221 (± 27), 627 (± 91) and 1791 (± 111) mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The exposure levels were based on similar studies with ethyl lactate showing moderate to severe nasal irritating and toxic (local) effects at levels of 600 and 2500 mg/m³, whereas slight systemic toxicity (reduced food consumption and growth retardation) was observed at the 2500 mg/m³ level only. The No-Oberserved-Adverse-Effect Level (NOAEL) in these studies was 200 mg/m³. In addition, the in vitro hydrolysing activity of these compounds by nasal olfactory epithelium was taken into account with 2-ethylhexyl lactate showing an approximately 6.5 times higher affinity and a 2.5 times higher velocity than ethyl lactate.
In view of the higher in vitro hydrolysing acticity, the target levels chosen for the present study, therefore, were slightly lower than those for ethyl lactate, viz.: 0, 75, 200, 600 and 1800 mg/m³. The highest concentration level was intended to result in local toxic effects but not to produce overt systemic toxicity nor an incidence of fatalities which would prevent meaningful evaluation. The lowest concentration was expected not to produce any evidence of toxicity. The intermediate concentrations were expected to produce no or minimal observable local toxic effects.
- Rationale for animal assignment (if not random): The animals were allocated to five groups of 5 rats/sex, proportionally by weight class, by a computer randomization programme.
- Section schedule rationale (if not random): At the end of the treatment period (day 28) all rats were killed in such a way that, on average, time of killing was the same for each group.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the general condition and behaviour of all individual animals were visually inspected daily in the morning hours. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon, shortly after exposure. In weekends only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the general condition and behaviour of all individual animals were visually inspected daily in the morning hours. A groupwise observation was made once during each day's exposure. All animals were checked again in the afternoon, shortly after exposure. In weekends only one check per day was carried out. All abnormalities, signs of ill health or reaction to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once during the acclimatization period (day -7), one day before the start of the study (allocation procedure), just prior to the start of the first exposure to the test materials (day 0), on days 7, 14 and 21, and on the day of necropsy (day 28) in order to determine the organ-to-body weight ratios. In addition, weekly body weight gain was calculated.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food intake was measured weekly per cage over 4 successive 1-week periods, by weighing the feeders.

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: Yes, the efficiency of food utilization was calculated and expressed as gram weight per gram food consumed.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy, the day after the last exposure
- Anaesthetic used for blood collection: Yes (identity), under ether anaesthesia
- Animals fasted: No
- How many animals: all animals
- Parameters examined: Haemoglobin concentration, packed cell volume, red blood cell count, reticulocytes (blood smears were prepared and stained; those of the control group and two highest exposure groups were counted since changes in red blood cell variables were observed in these two groups only), total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Fasting glucose was determined on day 26 of the study in blood collected from the tip of the tail of all rats after deprivation of food for ca. 24 hours. The other clinico-chemical examinations were conducted in blood collected at necropsy from the abdominal aorta of all, non-fasted rats.
- Animals fasted: Yes for glucose, no for the other examinations
- How many animals: all animals
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALAT; glutamic-pyruvic transaminase), aspartate aminotransferase (ASAT; glutamic-oxaloacctic transaminase), gamma glutamyl transferase (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, total bilirubin, cholesterol, triglycerides, phospholipids, sodium (Na), potassium (K), calcium (Ca), chloride (Cl) and inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Samples of the following tissues/organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde: liver (small representative samples were taken from the two largest liver lobes; the remainder of the livers was preserved for biochemical examinations), adrenals, spleen, heart, gross lesions, kidneys, testes, lungs with tranchae and larynx, and nose (nasal cavity). All but the gross lesions and the nose were weighed. The lungs were inflated with the fixative under 15 cm water pressure.

HISTOPATHOLOGY: Yes. All tissues/organs listed above (gross pathology) were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
The respiratory tract (nose, larynx, tranchea 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) and are indicated in Figure 1 (see section 'attached background material'). Each level is reported seperately.
The larynx was cut longitudinally. The three regions reported are: (1) the cranial part extending from the epiglottis to the cricoid cartillage, (2) the caudal part at the level of the cricoid cartillage, and (3) the transition between the caudal part of the larynx and the first trancheal ring.
The tranchea with the bifurcation was cut longitudinally/transversally. Five regions are reported: (1) the longitudinally-cut cranial part comprising the first 3 to 7 tracheal rings, (2) the transversally-cut medial part, adjacent to the caudal part, (3) the longitudinally-cut caudal part comprising the last 5 tracheal rings cranial to the bifurcation, (4) the longitudinally-cut bifurcation, and (5) the longitudinally-cut extrapulmonary bronchi.
Each lung lobe was cut at three sagittal levels. The left lung lobe and the right lung lobe are reported seperately.
Histopathological examinations were performed on heart, kidneys, liver, spleen, and testes of all animals of the control and high-concentration group. The respiratory tract (nose, larynx, tranchea and lungs) was examined in all rats of all groups since treatment-related changes were seen in rats of both sexes of the high-concentration group.
Other examinations:
Biochemical examination:
The remainder of the livers from each rat, after weighing and samples had been taken into formalin, were immediately deep frozen in liquid nitrogen and stored at -70 to -80 °C. At analysis, the livers were homogenised using a potter Elvejhem tissue homogenizer at 0-4 °C 0.25 M sucrose/ 2mM EDTA and 10 mM Tris-HCl (20 % w/v). A part of each homogenate was then frozen and thawed three times. After centrifugation the supernatant was stored at -70 to -80 °C prior to determination of acyl-CoA oxidase activities with palmitoyl-CoA as substrate as described by Reubsaet et al. (1988). Protein concentrations were determined using the Biorad method on a Cobas-bio analyzer. The acyl-CoA oxidase activity was expressed as nmol H2O2/min per mg protein.
Biochemical examinations were, in first instance, performed on the liver of all animals of the control and high concentration group. Since statistically significant changes were observed in animals of the high concentration group, the biochemcial examinations were extended to all groups.
Statistics:
Body weights and body weight gain data, organ weights, haematological (absolute data), clinical chemistry data and acyl-CoA oxidase activity in the liver were evaluated by one-way analysis of covariance (for body weights) or analysis of variance, followed by the Dunnett's multiple comparison test (Cochran, 1957; Steel and Torrid, 1960); Dunnett, 1955 and 1964).
Relative haematological data were analysed by Kruskal-Wallis non-parametric analysis of variance followed by the Mann/Whitney U-test (Siegel, 1956).
Incidences of histopathological changes were analysed by the Fisher exact probability test (Siegel, 1956).
No statistical analyses were performed on food consumption data since only one measurement was made per sex per group. 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) will be greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as concentration-response relationships and whether the results were significant in the light of other biological and pathological findings.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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
Abnormalities seen during exposure were confined to visually increased breathing rate in animals exposed to 600 or 1800 mg/m³ at the beginning of the treatment period until nominal day 11.
All animals of the high-concentration group showed nasal discharge towards the end of the treatment period. In addition, 9/10 rats of this group were sniffing during the last three days.
A dermal wound, followed by dermal encrustations and alopecic areas on the skin/fur was observed in one female of the low concentration group. This finding was not considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Male rats of the high concentration group showed statistically significant decreased body weights when compared to controls throughout the treatment period. The mean weekly body weight gain in this group was also significantly lower than that of controls during the first two weeks. Male rats exposed to 75 or 600 mg/m³ showed a decreased body weight gain during the last exposure week.
Female rats did not show any significant differences in mean body weight or weekly body weight gain when compared to controls.

FOOD CONSUMPTION
Mean food intake was reduced in males of the high concentration group relative to controls during the entire period. In females, mean food was comparable amongst all groups.

FOOD EFFICIENCY
Mean food conversion efficiency was reduced in males of the high concentration group relative to controls during the entire period. In males exposed to 75 or 600 mg/m³, food conversion efficiency was low during the last exposure week

HAEMATOLOGY
Changes in red blood cell or coagulation variables in rats exposed to 2-ethylhexyl lactate were limited to a statistically significant increase in haemoglobin content and packed cell volume in males exposed to 600 mg/m³ and an increase in red blood cell count and packed cell volume and a decrease in mean corpuscular haemoglobin concentration in females of the high-concentration group.
An increase in the absolute and relative number of neutrophils was observed in males exposed to 200 or 1800 mg/m³. The differences with the controls were statistically significant for the absolute number in males exposed to 1800 mg/m³ and in the relative number of this cell type in males exposed to 200 mg/m³. Males of the latter group concomitantly showed a significant decrease in the relative number of lymphocytes.

CLINICAL CHEMISTRY
Slightly though significantly increased ALAT levels were observed in females of the low and high concentration group. Since this change was not seen in the intermediate concentration groups, was not concentration-related and not observed in males, this finding was considered to be an incidental finding, unrelated to the treatment.
The slight, though statistically significant decrease in calcium level in males of the high concentration group was considered to be a fortuitous finding.

ORGAN WEIGHTS
In animals exposed to 2-ethylhexyl lactate statistically significant changes in spleen, liver and lung weights were observed relative to the controls:
- absolute spleen weight decreased in rats of both sexes of the 1800 mg/m³ group and in males exposed to 600 mg/m³; relative spleen weight decreased in females of the high concentration group only,
- relative liver weight increased in both sexes of the high concentration group,
- relative lung weight increased in male rats of the high concentration group.

GROSS PATHOLOGY
At autopsy, most gross changes were observed in the lungs of animals exposed to the test compound while only one control animal exhibited lungs with a gross change. However, concentration-response relationships were absent. In addition, a few changes were observed in organs outside the respiratory tract. They occurred in a single animal only and are common for rats of this strain and age. Therefore, the changes in the lungs and other organs were considered to be unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination revealed treatment-related changes in the respiratory tract. No other target organs were identified.
Animals of the 200, 600 and 1800 mg/m³ groups exhibited changes througout the respiratory tract. In animals of the 75 mg/m³ group, the changes were restricted to the nasal cavity.
Nasal cavity:
All types of epithelium lining the nasal cavity (squamous, transitional, respiratory and olfactory) were affected in animals of the 1800 mg/m³ group. In animals of the 200 and 600 mg/m³ groups, changes were observed in squamous, transitional and respiratory epithelium, whereas in animals exposed to 75 mg/m³, changes were largely restricted to the respiratory epithelium including goblet cell hyperplasia.
In animals exposed to 75 mg/m³, very slight to slight goblet cell hyperplasia was observed at levels 2-6, predominantly in the middle (medial) part of the septum and in the ventral meatus. In addition, some animals exhibited minimal focal hyperplasia of the respiratory epithelium at levels 2-5. Minimal focal transitional epithelial hyperplasia was observed in one male and one female. Rhinitis was not observed.
In animals exposed to 200 mg/m³, the squamous epithelium at level 1 was focally hyperplastic. The transitional epithelium was focally hyperplastic at levels 1 and 2. At level 2, the epithelial hyperplasia included disorganized appearance with a flattened surface, and a decreased number of goblet cells. This type of epithelial hyperplasia was denoted 'atypical hyperplasia' (Schwartz et al., 1984). Focal respiratory epithelial and goblet cell hyperplasia were largely comparable to that found in 75 mg/m³ exposed animals, although the incidence of the respiratory epithelial hyperplasia had increased in the 200 mg/m³ group, and the lesions extended to level 6. One female exhibited very slight focal olfcatory epithelial athrophy at level 3. In addition, very slight rhinitis occurred at all levels.
In the 600 mg/m³ exposed animals, minimal atrophy of the squamous epithelium at level 1 was observed in one male and one female. No atrophy of the olfactory epithelium was observed. In general, the hyperplastic changes were more severe and more extensive throughout the nasal cavity than in animals exposed to 200 mg/m³. The incidence and severity of rhinitis tended to be slightly increased when compared to the 200 mg/m³ group.
In the animals exposed to 1800 mg/m³, almost the entire squamous, transitional and respiratory epithelial lining was affected and demonstrated (atypical) hyperplasia, metaplasia, rhinitis and atrophy with ulcerations. In addition, a large part of the olfactory epithelial lining was atrophic with ulcerations. Where the olfactory epithelium was mostly affected, when compared to controls, the lamina propria was less cellular with loss of nerves and Bowman's glands, while the thickness suggested oedema. Rhinitis was observed thoughout levels 1-6. Rhinitis included intraepithelial and submucosal inflammatory cell infiltrates and intraepithelial micorabscesses. Especially with regard to the degenerative lesions and rhinitis, the incidence and severity of the lesions had markedly increased when compared to the other exposure groups. Goblet cell hyperplasia was not observed at the highest concentration level.
Larynx and trachea:
Slight epithelial hyperplasia and slight squamous metaplasia were observed in larynx and trachea of animals exposed to 200, 600 or 1800 mg/m³. All three regions of the larynx, the cranial part of the trachea and the tip of the carina at the bifurcation were sensitive sites. A concentration-related severity, however, was not observed.
Some animals exposed to 75 mg/m³ exhibited slight squamous metaplasia at the bifurcation, but a statistically significant difference in incidence with te controls was not present. Moreover, squamous metaplasia was also seen in one female control. The changes at the location of the bifurcation as seen in these animals, therefore, might be considered not to be treatment-related.
Lungs:
Changes in the lungs were observed in animals exposed to 200, 600 or 1800 mg/m³ and consisited of focal interstitial collagen deposits ('septal fibrosis') in the walls of alveolar ducts. The affected sites were covered with cuboidal epithelial cells. Septal fibrosis progressed from very slight or slight in female animals exposed to 200 or 600 mg/m³ to slight or moderate in the 1800 mg/m³ exposed females. An increase in severity with increasing concentration was less prominent in male rats.
Other organs:
All other organs exhibited histopathological changes which were about equally distributed among the groups or occurred in a single animal only. Moreover, they are common for the strain and age of the rats used. They were, therefore, not attributed to treatment.

OTHER FINDINGS
Compared to the control group, the hepatic acyl-CoA oxidase activity had significantly increased in rats exposed to 1800 mg/m³, indicative of peroxisome proliferating activity. In rats exposed to 75, 200 or 600 mg/m³, acyl-CoA oxidase activity was comparable to or slightly lower than that of controls.
Dose descriptor:
NOAEL
Effect level:
< 75 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The lowest level tested, viz. 75 mg/m³ was associated with minimal histopathological nasal changes in rats of both sexes. The concentration of 75 mg/m³ was, therefore, considered to be a minimum-observed-adverse-effect level (MOAEL).
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified
Conclusions:
2-Ethylhexyl lactate is irritating to the respiratory tract. At the highest level tested, viz. 1800 mg/m³, also systemic toxicity consisting of growth retardation, decreased food intake, and slight induction of hepatic peroxisome proliferation was noted. Since minimal histopathological nasal changes were observed in rats of both sexes exposed to the lowest level tested, viz. 75 mg/m³, this concentration was considered to be a minimum-observed-adverse-effect level (MOAEL).
Executive summary:

In a subacute inhalation toxicity study 2-ethylhexyl lactate was administered to 5 male and 5 female Wistar derived rats/concentration by nose only exposure at concentrations of 0, 75, 200, 600 and 1800 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

The lowest level tested, viz. 75 mg/m³ was associated with minimal histopathological nasal changes in rats of both sexes. The concentration of 75 mg/m³ was, therefore, considered to be a minimum-observed-adverse-effect concentration (MOAEC). The next higher levels tested, viz. 200 and 600 mg/m³, resulted in changes throughout the respiratory tract. At the highest level tested, viz. 1800 mg/m³, besides local changes in the respiratory tract, systemic toxicity consisting of growth retardation, decreased food intake, and slight induction of hepatic peroxisome proliferation was noted.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
77 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

Justification for classification or non-classification

Based on the available data from a sub-acute inhalation study, classification of 2-ethylhexyl-S-lactate as H335 is warranted according to the CLP Regulation.