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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
The Developmental Toxicity of 2-Ethylhexanoic Acid in Wistar Rats
Author:
SIRPA PENNANEN, KAI TUOVINEN, HANNELE HUUSKONEN, AND HANNU KOMULAINEN
Year:
1992
Bibliographic source:
FUNDAMENTAL AND APPLIED TOXICOLOGY 19, 505-511 (1992)
Report Date:
1992

Materials and methods

Principles of method if other than guideline:
20-21 pregnant females/dose were exposed to 2-EHA in their drinking water at doses of 100, 300, or 600 mg/kg/day on Days 6-19 of gestation. Control animals received vehicle water. The fetuses were examined (on Gestational Day 20) for external, visceral, and skeletal malformations and variations.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
2-ethylhexanoic acid (99.5%) was obtained from Merck (Darmstadt, Germany).

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Animals and animal husbandry:
Virgin female Han: Wistar rats (National Laboratory Animal Center, University of Kuopio, Finland) weighing 200-220 g (12- to 14-weeks old) and male rats of the same strain 360-380 g (16-weeks old) were used for this study. Rats were housed in stainless-steel wire mesh cages and kept under a photoperiodic cycle of 12 hr light/12 hr dark in an air-conditioned animal room. Temperature and relative humidity of the room were maintained at 21 ± 1°C and 55-65%, respectively. Animals were housed three per cage before mating and individually during gestation. The animals received commercial rat chow (R3-EWOS, Sodertalje, Sweden) and tap water ad libitum except 2-EHA solution during the experiment.

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
Mated females were exposed to 2-EHA on Days 6-19 of gestation via the drinking water at doses of 100, 300, and 600 mg/kg 2-EHA. 2-EHA was administered as a sodium salt by mixing 2-EHA and NaOH equimolariy in drinking water. Control animals received deionized water. Water consumption was recorded for the whole exposure period and the doses were corrected individually according to the most recent body weight taken on Days 0, 6, 13, and 20.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Female rats were paired overnight with males and the vaginal smears were checked the next morning for the presence of sperm. Sperm-positive female rats (109 females in total) were randomly assigned into a control group and three treatment groups by the stratified body weight procedure on Day 0 of gestation (sperm-positive day). The final group size was 20 or 21 dams. The females were identified by tattoos and housed individually.
Duration of treatment / exposure:
Mated females were exposed to 2-EHA on Days 6-19 of gestation.
Frequency of treatment:
Daily via drinking water.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 other: mg/kg/day
Dose / conc.:
300 other: mg/kg/day
Dose / conc.:
600 other: mg/kg/day
No. of animals per sex per dose:
20-21 pregnant females/dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
The animals were observed daily for clinical signs. Food consumption in the same gestational stage was recorded per cage during Days 13 to 16 of pregnancy.
Ovaries and uterine content:
On Day 20 of pregnancy, the dams were euthanized with a mixture of carbon dioxide and oxygen and the visceral cavity was cut open. The uterine contents and ovaries were examined for the number of corpora lutea, weight of uterus with its contents, number of implantation sites, sex and number of live and dead fetuses, number of early and late resorptions, individual fetal weights, external abnormalities, and placental weights.

The empty uteri were placed into a solution of ammonium sulfide to visualize hemorrhagic alterations in implantation sites of very early resorptions (Salewsky, 1964). A limited necropsy focusing on the thoracic and abdominal cavities was performed for each dam.
Fetal examinations:
Every second living fetus was fixed in ethanol and cleared in potassium hydroxide for double staining of cartilage and bone with Alcian blue and Alizarin red S (Inouye, 1976; Kimmel and Trammel, 1981) to detect skeletal anomalies. All fetuses with clubfoot were subjected to analysis of skeletal anomalies. Other fetuses were fixed in Bouin's solution and subjected to free-hand sectioning in order to detect visceral anomalies (Wilson, 1965).
Abnormalities detected in fetuses were classified as variations or malformations. Variations were defined as common structural changes and malformations as permanent abnormal structural (visceral or skeletal) changes (Black and Marks, 1986).
Renal pelvic dilatation and dilatation of the fetal ureters were assessed using a slightly modified grading method of Kavlock etal.(\ 987). Increasing scores of 1 to 4 for pelvic dilatation and from 1 to 5 for ureter dilatation were used. Only when scored >3 were dilatations considered abnormal. For each litter, preimplantation loss and postimplantation loss were calculated as a percentage, according to the formulas:
Preimplantation loss: (a - b)/a X 100
Postimplantation loss: (b - c)/b X 100
where "a" is number of corpora lutea, "b" is total number of implantations, and "c" is number of live fetuses.
We define the terms "embryotoxicity," "fetotoxicity," "teratogenicity," and "maternal toxicity" as described in Black and Marks (1986) and "developmental toxicity" to mean death, structural abnormality, altered growth, or functional deficiency during development (EPA, 1991).
Statistics:
Maternal, fetal, and placental weights, implantations, and living fetuses per litter were analyzed by one-way analysis of variance (ANOVA). Comparisons of significant group effects were conducted using the Fisher PLSD test. The fetal body weight data were also evaluated using covariate analysis with litter size and sex ratio as covariates but the test was not significant. Dose-response relationships for maternal and fetal body weights and affected fetuses and fetuses with skeletal or visceral malformations (group mean of litter percentages) were analyzed by the Pearson correlation test. Pre- and postimplantation losses and group mean values of litter percentages of skeletal and visceral anomalies were analyzed by the Mann-Whitney U nonparametric test. Other data were analyzed by the x^2 test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
The doses of 2-EHA employed did not cause visible maternal toxicity (clinical signs or behavioral changes).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the dams at 600 mg/kg decreased slightly (around 10%) from Day 13 onward. At termination the mean body weight of dams was 11 % (p < 0.001) lower at 600 mg/kg. The weight of the gravid uterus did not differ significantly between groups, but the corrected maternal body weight gain was reduced (53.8%, p < 0.001) at 600 mg/kg compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (g/kg body wt) did not differ significantly between groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At the 600 mg/kg dose level, the consumption of 2-EHA containing drinking water also exhibited some decrease from Day 6 onward, being maximally 20% less than in controls.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weight of the gravid uterus did not differ significantly between groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were noted upon necropsy in the organs of the dams.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The placental weight was slightly reduced (10.2%, p < 0.001) in both 300 and 600 mg/kg dose groups.

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted in pre- and postimplantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
2-EHA did not cause resorptions.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
2-EHA affected neither the number of implantations nor that of living fetuses.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy rate was slightly but, not significantly, decreased in the 300 and 600 mg/kg dose groups as compared to the control group (67 and 67% vs 84%).
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted in the number of corpora lutea.

Effect levels (maternal animals)

Key result
Remarks on result:
other: Maternal NOEL/NOAEL values were not stated in this study.

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean fetal body weight/litter showed a marginal decrease in males (5.6%, p < 0.001) and in females (8.6%, p < 0.001) at 600 mg/kg. The mean body weight of female fetuses was slightly decreased (5.7%, p < 0.001) also at 300 mg/kg.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All fetuses in treated and control groups were alive.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted in sex ratio.
External malformations:
not specified
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Clubfoot was the most severe skeletal malformation. It occurred at all dose levels, more notably at 300 and 600 mg/kg. Other skeletal malformations included abnormal cartilage in ankle (acampsia, strongly cartilagenous ankle, no flexure of the tarsal joints), absence of fibula, polydactyly, flabby legs (external, slightly paralyzed), scoliosis, lordosis, and extra thoracic ribs. With the exception of polydactyly and extra thoracic ribs, they did not occur spontaneously in control animals. Flabby legs, mild scoliosis, and lordosis occurred even at 100 mg/kg.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Only a few visceral malformations were found altogether but all cases were in 2-EHA-treated animals. There was one hydronephrosis at the dose level of 600 mg/kg. Kidney hypoplacia and rudimentary adrenal were also observed in the 300 and 100 mg/kg group respectively. A convoluted retina was also observed at the dose level of 600 mg/kg.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The incidence of several skeletal variations was increased in 2-EHA-treated animals, including the 100 mg/kg dose group. The frequency was highest at 600 mg/kg. The reduced ulna and lumbar ossification as well as the cases of twisted hind legs are in concordance with the more drastic malformations in feet.
Among visceral variations, curved or dilated ureter, congestion in the kidney cortex and displacement of the kidney occurred only in 2-EHA groups but pelvic dilatation was common in all animals. The incidence of slightly dilated brain ventricles was increased in the dose groups of 300 and 600 mg/kg. The frequency was higher in females than that in males (9 and 4 at 300 mg/kg, 15 and 9 at 600 mg/kg). The degree of dilatation of brain ventricles is known to reflect the developmental stage of the conceptus and the data fit well with the observation that the body weights of fetuses were slightly retarded at 600 mg/kg and in female fetuses also at 300 mg/kg.

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
100 other: mg/kg/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: eye
other: Dilation of brain ventricles
Description (incidence and severity):
2-EHA affected particularly skeletal development of the fetuses. The spectrum of changes ranged from skeletal variants to clubfoot, including a few cases of retarded lumbar and ulna ossification. Clubfoot does not occur spontaneously in rats and it must be regarded as a definitive indicator of teratogenicity. Most of the skeletal variations occurred also in control animals but with a lower frequency. In the present study variations already increased at doses which did not cause visible maternal toxicity. The skeletal variations are known to reflect to some extent delays in the stage of normal development. Meanwhile, 2-EHA did not markedly affect development of visceral tissues. There was only one case of hydronephrosis at the highest dose level and other minor changes were scattered without dose dependence. All cases, however, occurred in 2-EHA-treated animals.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 other: mg/kg/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

2-EHA affected development of the embryos/fetuses at all dose levels. The number of affected fetuses (having skeletal or visceral malformation) increased in a dose-dependent manner (p < 0.001) being 4.9, 8.9, and 15.3% per litter in 2-EHA groups vs 2.4% in the control group.

Applicant's summary and conclusion

Conclusions:
The results indicate that 2-EHA is teratogenic, i.e., increases malformations in Wistar rats at doses which are apparently not maternally toxic, embryotoxic, nor frankly fetotoxic. There was a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg. The dose 300 mg/kg was slightly fetotoxic in females as indicated by the reduced body weight in female fetuses. The 600 mg/kg dose was slightly fetotoxic in both sexes and maternally toxic as judged by the reduction of the body weights of the fetuses and dams. With regard to teratogenicity, the lowest-observed-adverse-effect-level (LOAEL) was 100 mg/kg and the NOAEL (no-observed-adverse-effect level) was lower than the lowest dose administered.
Executive summary:

The developmental toxicity of 2-ethylhexanoic acid (2-EHA), a wood preservative and a mammalian metabolite of di-(2-ethylhexyl) phthalate was examined in Wistar rats (20-21 pregnant females/dose). Mated animals were exposed to 2-EHA in their drinking water at doses of 100, 300, or 600 mg/kg/day on Days 6-19 of gestation. Control animals received vehicle water. The

fetuses were examined (on Gestational Day 20) for external, visceral, and skeletal malformations and variations.

2-EHA was marginally toxic to the dams at 600 mg/kg, but not at lower doses, since the mean near term body weight was reduced by 11%. This dose level was also slightly fetotoxic as indicated by a 5 to 8% decrease in the mean fetal body weight both in males and females. No treatment-related effects were observed in the number of implantations or live fetuses. At doses of 100 mg/kg and above, 2-EHA caused skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected. The number of affected fetuses increased in a dose-dependent way (4.9,8.9, and 15.3% of treated offspring at 100, 300, and 600 mg/kg/day, respectively, vs 2.4% control). See attachment Pennanen 1992 - Table 2 and Table 3.

These results indicate that 2-EHA is teratogenic in rats already at doses which are not yet maternally toxic. The skeleton appears to be the main target of 2-EHA in developing rats. With regard to teratogenicity, the lowest-observed-adverse-effect-level (LOAEL) was 100 mg/kg and the NOAEL (no-observed-adverse-effect level) was lower than the lowest dose administered.