Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters

Endpoint summary

Description of key information

90-day oral study with the metabolite 2-ethylhexanol (CAS No. 104-76-7) in rats: NOAEL 125 mg/kg bw/d, corresponding to an equimolar NAEL for Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters of 646 mg/kg bw/d.
90-day oral study with read across substance Fatty acids, C18-unsatd., dimers (CAS No. 61788-89-4) in rats: NOAEL 741 mg/kg bw/d, corresponding to a NAEL for Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters of 889 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

no neurology, opthalmoscopy and urine analysis performed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 36-37 days
- Weight at study initiation: Mean body weight ranges at dosing were (male) 105-114 g and (female) 86-97 g.
- Housing: singly in stainless steel wire cages
- Diet: ad libitum (Kliba rats/mice/hamster maintenance diet, "A" 343 Meal, Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 6 days

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

Route of administration:

oral: gavage

Vehicle:

other: Cremophor EL

Details on oral exposure:

Doses were prepared daily by dispersing 2EH in an aqueous solution of Cremophor EL (5 µg/100 mL) by ultra high speed sonication for 1 min. Homogeneity was maintained by magnetic stirring throughout dosing. Dose volumes were 10 mL/kg, based on weekly body weights. Controls were given 5.0 ml/kg of vehicle.
The dose delivery system, established by prior studies in rats and mice (Astill et al., 1993), was oral gavage of an aqueous emulsion of 2EH stabilized by a nonionic surfactant. This system provided the most stable dose formulation and minimized gastrointestinal tract irritation and inflammation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations and homogeneity were checked by gas chromatographic analysis of samples from each dose level periodically.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily on 5 consecutive days per week

Remarks:

Doses / Concentrations:
0, 25, 125, 250, and 500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In preliminary subacute studies (11 days) rats were given 0, 100, 330, 1000 and 1500 mg/kg bw/d. Mortality occurred at 1000 and 1500 mg/kg bw/d. The only effects observed at 330 mg/kg were increased relative kidney weights in females, inflammatory edema in the forestomach of one female rat, and a decreased thymus size in males and females. The 11-day dose level free of any treatment-related effects was thus 100 mg/kg.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, or once daily on non-treatment days

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 29 and 84
- Animals fasted: Yes
- How many animals:
- Parameters checked: leucocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential leucocytes, and reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 29 and 84
- Animals fasted: Yes
- How many animals:
- Parameters checked: ALAT, ASAT, gamma-GT, AP, Glucose, Urea, total protein, albumin, Creatinine, Cholesterol, Triglycerides, total bilirubin, sodium, potassium, calcium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS: Yes, at termination

Sacrifice and pathology:

Moribund animals were euthanized; dead and euthanized animals were immediately necropsied and assessed grossly.
GROSS PATHOLOGY: Yes, adrenals, brains, kidneys, livers, stomachs, testes, and ovaries from all animals were weighed, and with other organs and tissues listed in U.S. EPA Health Effects Guidelines (1987b) fixed in 4% formalin.
HISTOPATHOLOGY: Yes, All tissues from high dose and control animals were stained with hematoxylin—eosin and examined microscopically.
Lungs, livers (including gallbladders in mice), spleens, kidneys, stomachs, sternums, femurs, and femur bone marrows were examined microscopically at intermediate dose levels. Skin, eyes, female mammary glands, thigh musculatures, and extraorbital lacrymatory glands were not examined in the absence of signs of toxicity. Livers were also stained with oil red for lipid content and examined microscopically.

Statistics:

Means and standard deviations were calculated for body weights, food and water consumption, clinical pathology results, and organ weights. Values for test groups were compared with controls in the main study by ANOVA followed by Dunnett's test (Dunnett, 1955, 1964).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease in males and females at 500 mg/kg bw/d

Food consumption and compound intake (if feeding study):

no effects observed

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):

at 500 mg/kg bw/d increase of reticulocytes

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

females at 250 mg/kg bw/d decrease in serum ALAT

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

changes at 250 mg/kg bw/d

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

at 500 mg/kg bw/d in forestomach

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 500 mg/kg bw/d in liver

Details on results:

CLINICAL SIGNS AND MORTALITY
One female mouse at 250 mg/kg died during treatment; there were no other mortalities or clinical findings differing from controls in rats at any treatment level.

BODY WEIGHT AND WEIGHT GAIN
There was decreased weight gain in male and female rats at 500 mg/kg, 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)
There were no differences from controls at any treatment level in food consumption in rats.

HAEMATOLOGY
There was a 25% increase in reticulocyte numbers in male and female rats at 500 mg/kg.

CLINICAL CHEMISTRY
Differences from controls in treated rats were seen mostly at 84 days (data not shown). Females at 250 and 500 mg/kg/d had 30 and 36% decreases in serum ALT activities, respectively. Females at 500 mg/kg had a 16% decrease in serum cholesterol concentration and males at 500 mg/kg had 13% decreases in total protein and albumin concentrations.

ORGAN WEIGHTS
Significant differences from controls in rats were moderate and limited to the brain, kidneys, liver, stomach, and testes at 250 and 500 mg/kg. Male rat relative brain weights inincreased by 6% at 500 mg/kg, male kidney weights by 8% at 250 and 16% at 500 mg/kg, male liver weights by 8% at 250 and 29% at 500 mg/kg, male stomach weights by 11% at 500 mg/kg, and testis weights by 5.5% at 500 mg/kg. Female rat kidney weights increased by 5% at 250 and 6% at 500 mg/kg, female liver weights by 8% at 250 and 15% at 500 mg/kg, and female stomach weights by 6% at 250 and 16% at 500 mg/kg.

GROSS PATHOLOGY
Gross lesions differing from controls in both species were seen at 500 mg/kg only. In rats 2/10 males and 4/10 females exhibited single
or multiple slightly elevated foci in the forestomach. There were no other gross findings in either species.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dose-related findings in rats (data not shown) were limited to the forestomach and liver at 500 mg/kg. 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 beta-cell hyperplasia in 3/10 female rats.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on: clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased serum ALAT and changes in organ weights

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

646 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substances, the source substance being a product of the hydrolysis of the target substance (refer to endpoint discussion for further details).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions Publication with summarized results

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

no neurobehaviour, no urine analysis performed, results only given as summary, no detailed data

Principles of method if other than guideline:

A 90-day subchronic inhalation toxicity study was performed on Wistar rats in accordance to OECD testing guidelines to evaluate the toxicological profile of 2-ethylhexanol, potential target organs, and a no-observable-adverse-eect-level (NOAEL).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr K. Thomae GmbH, D-88400 Biberach/Riss, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: males 238 g +/- max 2.3 g; females 170 g +/- max. 2.2 g
- Housing: The animals were kept individually in wire cages in the inhalation chambers. To accustom the animals to the exposure conditions, they were exposed in the inhalation chambers to supply air for 5 days before the exposure period.

During the exposure-free periods, the animals were housed individually in wire cages (type D III, Becker and Co., D-44579 Castrop-Rauxel, Germany)
KLIBA rat/mouse laboratory diet 24-343-4 (Klingentalmühle, AG, CH-4303 Kaiseraugst, Switzerland) and tap water were provided ad lib. during the exposure-free period.

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

The animals were randomly (WTALOC randomization program supplied by Instem) allocated to the test groups and identified by ear tattoos (Klimisch, 1986).

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

The different EH inhalation chamber concentrations were achieved by conveying the substance via continuously operating metering pumps to evaporators. For 15, 40 and 120 ppm EH the vapour generation was carried out in a thermostated glass container (maintained at 46.4±50.48C) with a downstream mixing unit. 40 and 120 ppm atmospheres were generated by means of a two-component atomizer using compressed air and evaporation of the EH-aerosol. The warmed air of the control group (45.78C) and the vapour-air mixture of the EH groups were then mixed with the overall stream and distributed to a horizontal-flow whole-body exposure system (inhalation chamber glass/steel construction with volumes of approx. 1.1 m3, manufactured by BASF AG, Ludwigshafen, Germany). In the inhalation chamber of the control group, the exhaust air system was set lower (positive pressure), in the EH inhalation chambers the exhaust air system was set higher than the supply air system (negative pressure). Pressure (mean chamber pressure from -10.2 to 10.1 Pascal) and temperature (mean temperature 23.1ºC to 23.8ºC) were measured continuously. The relative humidity (mean relative humidity 41.8% to 46.2%) was checked and recorded once daily. Samples of the inhalation atmospheres were analysed at intervals of about 15 min by gas chromatography (Hewlett-Packard gas chromatograph Model HP 5880 A with automatic sampler HP 7671 A, FID, column:1 m x 2 mm with 10% Triton_305 on Supelcoport, 102/120 mesh, oven temperature: 120ºC. C15-paraffin was used as the internal standard).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the daily inhalation chamber concentrations revealed that the values obtained closely fit the desired nominal level.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 6 hours per day, 5 days a week
The test groups were exposed to the different concentrations for 6 hr on workdays over a period of 90 days (65 exposures).

Remarks:

Doses / Concentrations:
15, 40 and 120 ppm
Basis:

No. of animals per sex per dose:

10

Control animals:

other: concurrent clean air

Details on study design:

- Dose selection rationale: The highest dose corresponded to the vapour saturation at 20ºC. Higher concentrations are only obtainable as aerosols but are not relevant for occupational areas. The intermediate concentration of 40 ppm and the low concentration of 15 ppm were selected to obtain a concentration-effect relationship and a NOAEL.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for clinical signs and mortality during exposure and daily in the non-exposure times.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at the beginning of the preflow period, 1 day before commencement of the exposure period and then weekly throughout the study. The difference between the body weight on the day of weighing and that of the preceding weighing was calculated as group mean average (body weight gain). This value was defined as body weight change.

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were carried out prior to the beginning of the preflow period and at the termination of the study using an ophthalmoscope.

HAEMATOLOGY: Yes, blood for clinical pathology testing was collected in randomized order by retro-orbital bleeding.
- Time schedule for collection of blood: on day 94 of the study
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked: white blood cells, red blood cells, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, differential blood count, thromboplastin time

CLINICAL CHEMISTRY: Yes, blood for clinical pathology testing was collected in randomized order by retro-orbital bleeding.
- Time schedule for collection of blood: on day 94 of the study
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked : (sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

At the end of the 90-day exposure period all animals were necropsied and assessed by gross pathology postmortem.
The body weight and the weight of the lungs, liver, kidneys, adrenal glands and testes were determined.
Organs or tissues required by guidelines to be tested as well as all gross lesions were fixed in a 4% formaldehyde solution.
Histological examination and assessment of findings were carried out after histotechnical processing and staining with haematoxylin and eosin.

Other examinations:

For determination of cyanide-insensitive palmitoyl-CoA oxidation (Lazarow, 1981), which was carried out in liver homogenates taken after the animals had been killed, an automatic enzyme analyser (ACP 5040, Eppendorf, Hamburg, Germany) was used. Protein concentrations in liver homogenates were determined (Lowry et al., 1951).

Statistics:

Mean values and standard deviation were calculated for body weight and body weight change, haematological and clinical biochemistry parameters as well as for absolute and relative organ weights. The organ weights were statistically evaluated using the Dunnett's test (Dunnett, 1955 and 1964) for comparison of the exposure groups with the control group. The analysis of variance (Cochran, 1957) with subsequent Dunnett's test (Dunnett, 1955 and 1964) was used to compare body weight, body weight change as well as haematological and clinical biochemistry data of the treatment groups with those of the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

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

Description (incidence and severity):

non-adverse effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs related to the treatment with EH were observed throughout the study period. No deaths were recorded during the study.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of the female animals of the 40 ppm and 120 ppm exposure groups was decreased in comparison to the control group on day 37. The males of the 15 ppm exposure group showed a statistically significantly higher increase in body weight on day 93 compared to the control group. The differences in body weight/ body weight gain were incidental not dose-related and therefore of no toxicological relevance.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmological examinations revealed no effects associated with the exposure of the animals to the chemical.

HAEMATOLOGY and CLINICAL CHEMISTRY
The haematological or clinical biochemistry investigations showed no changes related to exposure to the chemical (data not shown). The only differences observed were in the values for bilirubin in males exposed to 120 ppm (4.07 mmol/litre compared with 2.99 mmol/litre in the controls) and for glucose in females exposed to 15 ppm (6.98 mmol/litre compared with 7.81 mmol/litre in the controls). Both findings, however, are considered to be of no toxicological significance, since they occurred only in one sex. The changes in the values for bilirubin were only registered in males, for glucose only in females. Additionally, with regard to the value for glucose, no relationship to the administered substance concentration was observed.

ORGAN WEIGHTS
No exposure-related organ weight changes were observed (data not shown).

GROSS PATHOLOGY
There were no macroscopic findings that could be attributed to the treatment with the chemical.

HISTOPATHOLOGY: NON-NEOPLASTIC
All the histomorphological findings were considered to have occurred incidentally and were not associated with the exposure to EH.

Dose descriptor:

NOAEC

Effect level:

120 ppm

Based on:

test mat.

Remarks:

corresponding to the vapour saturation

Sex:

male/female

Basis for effect level:

other: No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time, necropsy and histological examination.

Dose descriptor:

NOAEC

Effect level:

638.4 mg/m³ air

Based on:

test mat.

Remarks:

corresponding to the vapour saturation

Sex:

male/female

Basis for effect level:

other: No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time, necropsy and histological examination.

Critical effects observed:

not specified

Cyanide-insensitive palmitoyl-CoA oxidation, a marker for peroxisome proliferation, was found elevated in a subchronic study in Fischer 344 rats after gavage application of 500 mg/kg but not under the conditions of this 90-day subchronic inhalation study.

Executive summary:

A 90-day subchronic inhalation toxicity study was performed on Wistar rats with 2-ethylhexan-1-ol (CAS No. 104-76-7) in accordance to OECD guideline 413 (Klimisch, 1998). 10 males and 10 females per group were exposed to 2-ethylhexanol vapours at concentrations of 15, 40 and 120 ppm (the latter corresponding to the vapour saturation at 20ºC) 6 hours/day on working days for 90 days. The respective controls inhaled clean air under the same conditions. No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time. There were no findings related to the treatment with 2-ethylhexanol either at necropsy or at histological examination. The highest concentration tested under these conditions (120 ppm) was found to be the NOAEL for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 2) study from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substances, the source substance being a product of the hydrolysis of the target substance (refer to endpoint discussion for further details).

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions Publication with summarized results

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

no neurobehaviour, no urine analysis performed, results only given as summary, no detailed data

Principles of method if other than guideline:

A 90-day subchronic inhalation toxicity study was performed on Wistar rats in accordance to OECD testing guidelines to evaluate the toxicological profile of 2-ethylhexanol, potential target organs, and a no-observable-adverse-eect-level (NOAEL).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr K. Thomae GmbH, D-88400 Biberach/Riss, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: males 238 g +/- max 2.3 g; females 170 g +/- max. 2.2 g
- Housing: The animals were kept individually in wire cages in the inhalation chambers. To accustom the animals to the exposure conditions, they were exposed in the inhalation chambers to supply air for 5 days before the exposure period.

During the exposure-free periods, the animals were housed individually in wire cages (type D III, Becker and Co., D-44579 Castrop-Rauxel, Germany)
KLIBA rat/mouse laboratory diet 24-343-4 (Klingentalmühle, AG, CH-4303 Kaiseraugst, Switzerland) and tap water were provided ad lib. during the exposure-free period.

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

The animals were randomly (WTALOC randomization program supplied by Instem) allocated to the test groups and identified by ear tattoos (Klimisch, 1986).

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

The different EH inhalation chamber concentrations were achieved by conveying the substance via continuously operating metering pumps to evaporators. For 15, 40 and 120 ppm EH the vapour generation was carried out in a thermostated glass container (maintained at 46.4±50.48C) with a downstream mixing unit. 40 and 120 ppm atmospheres were generated by means of a two-component atomizer using compressed air and evaporation of the EH-aerosol. The warmed air of the control group (45.78C) and the vapour-air mixture of the EH groups were then mixed with the overall stream and distributed to a horizontal-flow whole-body exposure system (inhalation chamber glass/steel construction with volumes of approx. 1.1 m3, manufactured by BASF AG, Ludwigshafen, Germany). In the inhalation chamber of the control group, the exhaust air system was set lower (positive pressure), in the EH inhalation chambers the exhaust air system was set higher than the supply air system (negative pressure). Pressure (mean chamber pressure from -10.2 to 10.1 Pascal) and temperature (mean temperature 23.1ºC to 23.8ºC) were measured continuously. The relative humidity (mean relative humidity 41.8% to 46.2%) was checked and recorded once daily. Samples of the inhalation atmospheres were analysed at intervals of about 15 min by gas chromatography (Hewlett-Packard gas chromatograph Model HP 5880 A with automatic sampler HP 7671 A, FID, column:1 m x 2 mm with 10% Triton_305 on Supelcoport, 102/120 mesh, oven temperature: 120ºC. C15-paraffin was used as the internal standard).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the daily inhalation chamber concentrations revealed that the values obtained closely fit the desired nominal level.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 6 hours per day, 5 days a week
The test groups were exposed to the different concentrations for 6 hr on workdays over a period of 90 days (65 exposures).

Remarks:

Doses / Concentrations:
15, 40 and 120 ppm
Basis:

No. of animals per sex per dose:

10

Control animals:

other: concurrent clean air

Details on study design:

- Dose selection rationale: The highest dose corresponded to the vapour saturation at 20ºC. Higher concentrations are only obtainable as aerosols but are not relevant for occupational areas. The intermediate concentration of 40 ppm and the low concentration of 15 ppm were selected to obtain a concentration-effect relationship and a NOAEL.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for clinical signs and mortality during exposure and daily in the non-exposure times.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded at the beginning of the preflow period, 1 day before commencement of the exposure period and then weekly throughout the study. The difference between the body weight on the day of weighing and that of the preceding weighing was calculated as group mean average (body weight gain). This value was defined as body weight change.

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were carried out prior to the beginning of the preflow period and at the termination of the study using an ophthalmoscope.

HAEMATOLOGY: Yes, blood for clinical pathology testing was collected in randomized order by retro-orbital bleeding.
- Time schedule for collection of blood: on day 94 of the study
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked: white blood cells, red blood cells, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, differential blood count, thromboplastin time

CLINICAL CHEMISTRY: Yes, blood for clinical pathology testing was collected in randomized order by retro-orbital bleeding.
- Time schedule for collection of blood: on day 94 of the study
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked : (sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

At the end of the 90-day exposure period all animals were necropsied and assessed by gross pathology postmortem.
The body weight and the weight of the lungs, liver, kidneys, adrenal glands and testes were determined.
Organs or tissues required by guidelines to be tested as well as all gross lesions were fixed in a 4% formaldehyde solution.
Histological examination and assessment of findings were carried out after histotechnical processing and staining with haematoxylin and eosin.

Other examinations:

For determination of cyanide-insensitive palmitoyl-CoA oxidation (Lazarow, 1981), which was carried out in liver homogenates taken after the animals had been killed, an automatic enzyme analyser (ACP 5040, Eppendorf, Hamburg, Germany) was used. Protein concentrations in liver homogenates were determined (Lowry et al., 1951).

Statistics:

Mean values and standard deviation were calculated for body weight and body weight change, haematological and clinical biochemistry parameters as well as for absolute and relative organ weights. The organ weights were statistically evaluated using the Dunnett's test (Dunnett, 1955 and 1964) for comparison of the exposure groups with the control group. The analysis of variance (Cochran, 1957) with subsequent Dunnett's test (Dunnett, 1955 and 1964) was used to compare body weight, body weight change as well as haematological and clinical biochemistry data of the treatment groups with those of the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects

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

Description (incidence and severity):

non-adverse effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs related to the treatment with EH were observed throughout the study period. No deaths were recorded during the study.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of the female animals of the 40 ppm and 120 ppm exposure groups was decreased in comparison to the control group on day 37. The males of the 15 ppm exposure group showed a statistically significantly higher increase in body weight on day 93 compared to the control group. The differences in body weight/ body weight gain were incidental not dose-related and therefore of no toxicological relevance.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmological examinations revealed no effects associated with the exposure of the animals to the chemical.

HAEMATOLOGY and CLINICAL CHEMISTRY
The haematological or clinical biochemistry investigations showed no changes related to exposure to the chemical (data not shown). The only differences observed were in the values for bilirubin in males exposed to 120 ppm (4.07 mmol/litre compared with 2.99 mmol/litre in the controls) and for glucose in females exposed to 15 ppm (6.98 mmol/litre compared with 7.81 mmol/litre in the controls). Both findings, however, are considered to be of no toxicological significance, since they occurred only in one sex. The changes in the values for bilirubin were only registered in males, for glucose only in females. Additionally, with regard to the value for glucose, no relationship to the administered substance concentration was observed.

ORGAN WEIGHTS
No exposure-related organ weight changes were observed (data not shown).

GROSS PATHOLOGY
There were no macroscopic findings that could be attributed to the treatment with the chemical.

HISTOPATHOLOGY: NON-NEOPLASTIC
All the histomorphological findings were considered to have occurred incidentally and were not associated with the exposure to EH.

Dose descriptor:

NOAEC

Effect level:

120 ppm

Based on:

test mat.

Remarks:

corresponding to the vapour saturation

Sex:

male/female

Basis for effect level:

other: No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time, necropsy and histological examination.

Dose descriptor:

NOAEC

Effect level:

638.4 mg/m³ air

Based on:

test mat.

Remarks:

corresponding to the vapour saturation

Sex:

male/female

Basis for effect level:

other: No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time, necropsy and histological examination.

Critical effects observed:

not specified

Cyanide-insensitive palmitoyl-CoA oxidation, a marker for peroxisome proliferation, was found elevated in a subchronic study in Fischer 344 rats after gavage application of 500 mg/kg but not under the conditions of this 90-day subchronic inhalation study.

Executive summary:

A 90-day subchronic inhalation toxicity study was performed on Wistar rats with 2-ethylhexan-1-ol (CAS No. 104-76-7) in accordance to OECD guideline 413 (Klimisch, 1998). 10 males and 10 females per group were exposed to 2-ethylhexanol vapours at concentrations of 15, 40 and 120 ppm (the latter corresponding to the vapour saturation at 20ºC) 6 hours/day on working days for 90 days. The respective controls inhaled clean air under the same conditions. No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time. There were no findings related to the treatment with 2-ethylhexanol either at necropsy or at histological examination. The highest concentration tested under these conditions (120 ppm) was found to be the NOAEL for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 2) study from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substances, the source substance being a product of the hydrolysis of the target substance (refer to endpoint discussion for further details).

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 grouping of substances and read-across

There are no data available on toxicity after repeated exposure of Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters (CAS 68334-05-4). In order to fulfil the standard information requirements set out in Annex X, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances and/or common chemical precursors or similar hydrolysis/breakdown products (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity and/or common chemical precursors or similar hydrolysis/breakdown products.

The read-across is either based structural similarity or on the metabolism of Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters (CAS 68334-05-4), in particular on the fact that the substance undergoes enzymatic ester hydrolysis resulting in the formation of Fatty acids, C18-unsatd., dimers and 2-ethylhexanol. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of repeated dose toxicity

CAS	Chemical name	Molecular weight	Repeated dose toxicity – oral	Repeated dose toxicity – inhalation	Repeated dose toxicity – dermal
68334-05-4 (a)	Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters	673.12	RA: CAS 68783-41-5 RA: CAS 104-76-7	RA: CAS 104-76-7	--
61788-89-4	Fatty acids, C18- unsatd., dimers	564.92	Experimental result: subchronic, rat NOAEL 741mg/kg bw/day	--	--
104-76-7 (b)	2-ethylhexanol	130.23	Experimental result: subchronic, rat NOAEL: 125 mg/kg bw/day	Experimental result: NOAEC = 638.4 mg/m³ air	--

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of structural similarity and common chemical precursors and common hydrolysis/breakdown products. The available endpoint information is used to predict the same endpoints for Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters (CAS 68334-05-4). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

Repeated dose toxicity - oral

CAS 61788-89-4

An oral subchronic repeated dose study was performed with the read across substance Fatty acids, C18-unsatd., dimers (CAS: 61788-89-4) as 13 week feeding study in rats according to OECD guideline 408 (Spurgeon, 1993). 20 Sprague-Dawley rats/sex/dose were fed concentrations of the test substance of 0.1, 1, 5% (w/w) corresponding to ca. 0, 74.1, 740.9, 3591.2 mg/kg bw/day for males and ca. 0, 90.5, 854.9, 4085.5 mg/kg bw/day for females (average dose weeks 0-13) daily in the diet. There were no treatment related clinical signs, mortality or body weight changes in any dose group. At the lowest dose level, 0.1 % (w/w) in diet, aggregation of macrophages, some of which were pigmented, was seen on microscopic examination of the mesenteric lymph node and the spleen. In the higher dose levels of 1.0 and 5.0% the same effects on lymph nodes and additionally on spleen, liver, adrenals and thyroids were reported. The NOAEL was set to 741 mg/kg bw/day. This would correspond to a NAEL for Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters of 889 mg/kg bw/d calculating with a molecular ratio factor of 1.2 (MW of the read across substance fatty acids, C18 -unsatd., dimers: 560.92 g/mol).

CAS 107-76-7

The subchronic oral toxicity of 2-ethylhexanol was investigated in Fischer 344 rats in a study performed similar to OECD guideline 408 (Astill, 1996). Groups of 10 male and 10 female rats received the test substance diluted in Cremophor EL once daily on 5 days/week at dose levels of 25, 125, 250, and 500 mg/kg bw/day for a period of 90 days. A similar constituted group of animals was administered the vehicle alone and served as controls. During the study period, no mortalities and no clinical signs of toxicity were observed in rats at any treatment level. At study termination, body weight gain was decreased in male (-6%) and female (-7%) rats compared to controls. No differences in food consumption were noted between treated animals and controls. Clinical chemistry revealed a 30 and 36% decrease in serum ALT activities in females treated with 250 and 500 mg/kg bw/day, respectively. At 500 mg/kg bw/day, a 16% decrease in serum cholesterol concentration in females and 13% decreases in total protein and albumin concentrations in males were observed. Furthermore, the reticulocyte numbers in male and female rats was increased (+25%) at 500 mg/kg bw/day. Moderate, but statistically significant increases in relative organ weights compared to controls were noted in the brain, kidneys, liver, stomach, and testes of male and female rats at 250 and 500 mg/kg bw/day. At necropsy, 2/10 males and 4/10 females exhibited single or multiple slightly elevated foci in the forestomach at 500 mg/kg bw/day. No other gross pathological findings were observed in any of the animals. Histopathological examination revealed dose-related findings in rats, which were limited to the forestomach and liver at 500 mg/kg bw/day. In the forestomach, generalised acanthosis of the 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 was observed. However, the effects on forestomach were likely to be attributed to gavage administration of the test substance. In the liver, hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal beta-cell hyperplasia in 3/10 female rats were noted.

Based on the results of this study, the NOAEL for 2-ethylhexanol in male and female Fischer 344 rats was established at 125 mg/kg bw/day. The molecular weight ratio of 2 -ethylhexanol (MW 130.23 g/mol) and Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters (MW 673.12 g/mol) is 5.17 (673.12/130.23). Thus, based on this molecular ratio factor the NAEL for Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters (CAS No. 68334 -05 -4) was calculated to be 646 mg/kg bw/d (125 mg/kg bw/d x 5.17).

 

Repeated dose toxicity - inhalation

CAS 104-76-7

A 90-day subchronic inhalation toxicity study was performed on Wistar rats with 2-ethylhexan-1-ol (CAS No. 104-76-7) in accordance to OECD guideline 413 (Klimisch, 1998). 10 males and 10 females per group were exposed to 2-ethylhexanol vapours at concentrations of 15, 40 and 120 ppm (the latter corresponding to the vapour saturation at 20ºC) 6 hours/day on working days for 90 days. The respective controls inhaled clean air under the same conditions. No substance-related adverse effects were observed for body weight, body weight gain, mortality, organ weights, ophthalmoscopy, clinical biochemistry and haematological parameters including clotting time. There were no findings related to the treatment with 2-ethylhexanol either at necropsy or at histological examination. The highest concentration tested under these conditions (120 ppm equivalent to 638.4 mg/m3) was found to be the NOAEC for male and female rats.

 

Repeated dose toxicity - dermal

No studies are available for the dermal route of exposure. Overall, the calculated low dermal absorption potential, the low water solubility, the molecular weight (>100), and the fact that the substance is not irritating to skin implies that dermal uptake of Fatty acids, C18 unsatd., dimers, 2-ethylhexyl esters in humans is considered as very limited.

Conclusions for repeated dose toxicity

No studies investigating repeated dose toxicity are available for Fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters. Oral, dermal or inhalative absorption of the registered substance is deemed unlikely due to its high MW and physico-chemical properties. As hydrolysis is possible to a limited extend, it will most likely be the cleavage products that are absorbed, thus a worst case approach considering the repeated dose toxicity of common and similar hydrolysis products was performed. None of the potential hydrolysis products (Fatty acids, C18-unsaturated, dimers, hydrogenated (CAS 68783-41-5), 2-ethylhexanol (CAS 104-76-7)) poses a risk for human health effects.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from structural surrogates (hydrolysis products). All available studies are adequate and reliable based on the common hydrolysis products between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from structural analogues/surrogates. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substances and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Hazard assessment is conducted by means of read-across from structural analogues/surrogates. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substances and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

The subchronic oral NOAEL for the toxicologically most critical metabolite 2 -ethylhexanol was found to be 125 mg/kg bw/d. Based on the molecular ratio this corresponds to a NOAEL of 646 mg/kg bw/d for Fatty acids, C18 -unsatd., dimers, 2 -ethylhexyl esters.

Thus according to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for repeated dose toxicity, fatty acids, C18-unsatd., dimers, 2-ethylhexyl esters have not to be classified.