2-ethylhexanal

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-detailled and very complete study conducted under GLP according to OECD guideline 414 .

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanal

EC Number:

204-596-5

EC Name:

2-ethylhexanal

Cas Number:

123-05-7

Molecular formula:

C8H16O

IUPAC Name:

2-ethylhexanal

Details on test material:

- Name of test material (as cited in study report): 2-ethylhexanal
- Physical state: clear colourless liquid
- Analytical purity: 98 %
- Impurities (identity and concentrations): not reported
- Purity test date: analysis of 2-ethylhexanal using Gas Liquid Chromatography in formulation with corn oil (see vehicle for details on exposure/vehicle)
- Lot/batch No.: Batch No. 9147-105, Lot No.000123057
- Stability under test conditions: yes, characterization of stability was tested prior to and after the study; the stability in suspension was checked in a parallel study
- Storage condition of test material: refrigerator (approximately 4°C) under nitrogen gas

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Charles River UK Limited, Margate, Kent, England
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 216-261 g
- Fasting period before study: no
- Housing: barried, limited-access rodent facility, caging in TR18 cages (Arrowmight Biosciences, Hereford, england and RB3 modified cages, North Kent Plastic Cages Ltd., Erith, Kent, England)
- Diet: ad libitum, source: SDS laboratory animal diet No.1, Supplier: Special Diets Services Limited, Wtihma, Essex, England
- Water: ad libitum (water quality governed by the Department of Environmental regulations)
- Acclimation period: 5 days prior to study begin


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C (daily recorded)
- Humidity (%): 40-70 % (daily recorded)
- Air changes (per hr): filtered air was passed into the room with positive pressure without recirculation; at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 2-ethylhexanal was prepared weekly as a suspension in corn oil for oral gavage, procedure: required quantity was weighed, addition of corn oil in immediately and mixing until homogenous; storage at 4°C, ca. 1h prior to use at RT; re-mixing with a magnetic stirrer before and during dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported, probably due to low solubility (10mg/L at 20°C)
- Concentration in vehicle: 0, 20, 60, 160 mg/ml (5ml total volume)
- Amount of vehicle (if gavage): 5ml/kg bw/day
- Purity: analysis of 2-ethylhexanal in corn oil formulation with Gas Liquid Chromatography (GCS), Huntingdon Life Sciences (HP 5890 Series GC System and HP Chemstation), determined for a preliminary study BGH061/9800121 (identical Batch No. 9147-105 as used in BGH062/993418)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

achieved concentrations of 2-ethylhexanal were tested using Gas Liquid Chromatography
- homogeneity and re-suspendibility of 2EHN were analyzed: proove of momogenous suspension
- Mean concentration of tested concentrations (prepared for dosing during first and last week): range 96.3 - 104% of nominal concentrations (see doses/concentrations for details on doses used)

Details on mating procedure:

- Impregnation procedure: cohousing
- M/F ratio per cage: 1M/1F per cage
- Length of cohabitation: not reported in detail, check for successful pairing each morning after pairing
- Further matings after two unsuccessful attempts: not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug (from at least 3 copulations) and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

from day 6 to 19 of gestation

Frequency of treatment:

once daily

Duration of test:

13 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 300, 800 mg/kg bw/day
Basis:
nominal conc.
oral gavage, vehicle: corn oil

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 100.0, 300.9, 797.6 mg/kg bw/day
Basis:
analytical conc.
(analysis during first week of the study)

No. of animals per sex per dose:

25 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: yes, based on preliminary studies

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weighing on days 0, 3 and 6 to 20 inclusive after mating

FOOD CONSUMPTION: Yes
- Time schedule: Days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: reproductive tract (complete with ovaries)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of live fetuses in each uterine horn

Fetal examinations:

- External examinations: Yes: [all per litter ] for body weight, sex and any external abnormalities
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter] for neck, thoracic and and abdominal cavitis

Statistics:

- Statistic test, employing analysis of variance followed by an inter-group comparison with the control were performed on the following parameters: body weight (bw), bw change adjusted for gravid uterine weight, food consumption, litter data, litter weight, fetal weight and placental weight
- Parametric tests (Snedecor and Cochran) followed by Williams´test and non-parametric test followed by Shirley´s test, Fisher´s exact test (> 75% same values)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
All rats survived

CLINICAL SIGNS
Numerous rats displayed a brown to yellowish staining of their body surface, probably due to the corn oil used as vehicle. In all treated groups, transitory salivation was seen after dosing. The number of rats displaying salivation increased with the dosage, from 4 of 25 animals affected at 100.0 mg/kg bw/day to 25 animals at 797.6 mg/kg bw/day.
At 797.6 mg/kg bw/day, further effects were seen, including piloerection and reduced activity that was some times accompanied by partly closing of the eyelids and hunched posture.
BODY WEIGHT
Bodyweight changes within the 100.0 and the 300.9 mg/kg bw/day groups were similar to controls. In contrast, at 797.6 mg/kg bw/day, bodyweight changes were significantly reduced compared to controls (-23 %). This results in lower body weight at the end of the study (-8 %)

FOOD CONSUMPTION
The mean group food consumption at 100.0 and 300.9 mg/kg bw/day was to control. At 797.6 mg/kg bw/day, the mean food consumption was reduced.

NECROPSY
Necropsy revealed no gross treatment-related abnormalities.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

300.9 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

300.9 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
REPRODUCTIVE PARAMETERS:
-In all groups, the females have live fetuses at day 20 of gestation. The mean number of corpora lutea, inplantations, live fetuses and resorptions were unaffected by the treatment.

FETUSES
- At 300.9 and 797.6 mg/kg bw/day, the placenta was found to display abnormalities including swelling, enlargement, presence of clotted blood around, pale coloration.
- The placental weight was also significantly reduced at 797.6 mg/kg bw/day.
- The fetal weight also was affected at 797.6 mg/kg bw/day.
- The visceral (soft tissues) and skeletal examination of the fetuses revealed no treatment-related changes at 100.0 mg/kg bw/day. In contrast, at 300.9 mg/kg bw/day, a slight increase in number of fetuses displaying rudimentary/absent renal papillae, dilated ureter and displaced testes compared to controls was observed. Because of the fact that the incidence of the first two abnormalities (renal papillae and ureter) was within the range of background control data, the authors considered these changes not to be treatment-related. The toxicological significance of the displaced testes was unclear. Furthermore and still at 300.9 mg/kg bw/day, the authors reported an increased incidence of fetuses with incomplete ossification of the 5th/6th sternebrae and of the sacrocaudal vertebral arches.
- At 797.6 mg/kg bw/day, visceral as well as skeletal abnormalities were numerous and this time clearly treatment-related. These abnormalities can be summarized as follows:
Visceral abnormalities: dilated ventricles in the brain, absent or rudimentary thyroid, partially undescended thymus, cardiovascular abnormalities, rudimentary/absent renal papillae, left umbilical artery, subcutaneous edema.
Skeletal abnormalities: irregularly ossified ribs, tail abnormalities related to abnormalities within the termination of the vertebral column, vertebral configuration abnormalities,thoracic and lumbar vertebral abnormalities, incomplete ossification of cranial centres, cervical and sacrocaudal vertebral arches, pelvic bones, metacarpals, metatarsals and sternebrae.
- cleft palate occured in a single case at highest dose

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Summary table of important results on maternal and fetal findings:

Group: Dosage: (mg/k bw/day analytical concentration) Volume: (5 ml/kg)	1	2	3	4
	0	100.0	300.9	797.6
Adult females
Females with sperm	25	25	25	25
Pregnant females	25	25	25	25
Evaluated pregnant females	25	25	25	25
Litters - group mean values
Corpora lutea	15.6	15.4	16.1	16.3
Implantations	14.2	14.1	15.0	14.8
Live fetuses	13.1	13.4	14.4	13.3
Resorptions	1.2	0.7	0.6	1.5
Weight of fetuses (g)        Males:                                           Females:	3.81	3.86	3.72	2.53
	3.64	3.67	3.50	2.35
Sex ratios of fetuses (% Male):	50.7	50.7	46.6	46.4
IMPORTANT MATERNAL FINDINGS
Bodyweight change Days 6 to 20 of gestation (g)	126	130	133	96
Gravid uterine weight (g)	77	80	83	61
IMPORTANT FETAL PATHOLOGY FINDINGS
- At 100.0 mg/kg bw/day: there were no fetal abnormalities. - At 300.9 mg/kg bw/day: slight fetal immaturity. - At 797.6 mg/kg bw/day increased incidence of fetuses with: Visceral findings: dilated brain ventricles, absent/rudimentary thyroid, partially undescended thymus, cardiovascular abnormalities, rudimentary/absent renal papillae, left umbilical artery, subcutaneous oedema. Skeletal findings: Thickened/kinked/irregularly ossified ribs, termination of vertebral column (generally associated with tail abnormalities observed at necropsy), vertebral configuration abnormalities (including cervical ribs, complete 14th ribs, additional lumbar ribs, additional thoracolumbar vertebrae and offset pelvic girdle), thoracic/lumbar vertebral abnormalities (including incomplete ossification and structural categories), incomplete ossification of cranial centres (including incomplete ossification of basisphenoid, basioccipital and palatine shelves), cervical and sacrocaudal vertebral arches, pelvic bones, metacarpals/metatarsals and stemebrae (5 th/6 th and others).

Applicant's summary and conclusion

Executive summary:

2-Ethylhexanal was administered by daily oral gavage to rats from day 6 to 19 of their pregnancy, at dosages up to 797.6 mg/kg bw/day, There was no obvious adverse maternal toxicity at 100.0 or 300.9 mg/kg bw/ day. In contrast, at 797.6 mg/kg bw/day there was clear evidence of maternal toxicity. (piloerection and underactivity, eyelids partly closed and hunched posture; low food consumption and reduction of overall absolute and adjusted bodyweight gain).

Embryo-fetal survival was unaffected by treatment with 2-Ethylhexanal. At 300.9 mg/kg bw/day, fetal weights were lower and there was delayed ossification. Although the relationship of these findings to treatment is uncertain, they are considered to be transient in nature, rather than representing permanent structural changes. Therefore the slight fluctuations in fetal weights and some ossification parameters are not considered

to be of toxicological relevance during later development.At 797.6 mg/kg bw/day fetal and placental weights were reduced and placental abnormalities were observed in a few litters, e.g. pale placenta (3 litters; about 10% of the placenta overall). Fetal pathology revealed visceral and skeletal abnormalities at 797.6 mg/kg bw/day (visceral: dilated brain ventricles, absent/rudimentary thyroid gland, cardiovascular abnormalities and rudimentary/absent renal papillae; skeleton: reduction in ossification, rib and vertebral abnormalities and changes in configuration not related to fetal immaturity). Fetal immaturity was noted universally at 797.6 mg/kg bw/day and, to a lesser extent, at 300.9 mg/kg bw/day.

In conclusion, there was no evidence of adverse maternal or embryo-fetal toxicity at 100.0 mg/kg bw/day. Similarly, at 300.9 mg/kg bw/day no obvious adverse maternal toxicity was seen but there was some delayed ossification. Effect is considered to be transient in nature, rather than representing permanent structural changes and and of no toxicological relevance during later development . At 797.6 mg/kg bw/day, maternal toxicity was clearly evident and 2-Ethylhexanal is toxic to the developing embryo and fetus. The NOAEL for both, maternal toxicity and developmental toxicity is considered to be 300.9 mg/kg bw/day.