2-ethylhexyl 3,5,5-trimethylhexanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Remarks:

supporting data on metabolite 2-ethyl-1-hexanol

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: detailed information fulfil scientific principles, well documented with the GLP guidelines, test conducted using a metabolite

Data source

Reference

Reference Type:

publication

Title:

The Developmental Toxicity of 2-Ethylhexanol Applied Dermally to Pregnant Fischer 344 Rats

Author:

R. W. Tyl et al.

Year:

1992

Bibliographic source:

FUNDAMENTAL. AND APPLIED TOXICOLOGY 19, 176-185 ( 1992)

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexan-1-ol

EC Number:

203-234-3

EC Name:

2-ethylhexan-1-ol

Cas Number:

104-76-7

Molecular formula:

C8H18O

IUPAC Name:

2-ethylhexan-1-ol

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

Animals and dosages.
Animals were virgin F344 rats [CDF (R) F344 Crl./Br] supplied by Charles River Breeding Laboratories (Kingston, NY), On arrival, males weighed 175 - 200 g and were 70 days old, and females weighed 130 - 150 g and were 63 days old. Animals were deemed suitable for use after fecal sampling, histological testing of selected organs, and serum viral antibody testing examination. After a 2-week quarantine period animals were mated 1:1. Gestational Day 0 was dated from the appearance of a copulatory plug. Pregnant females were housed singly in stainless steel wire-mesh cages and kept on a 12-hr photoperiod at 68-73 °F and at 42-65% humidity. Animals were supplied with feed and water ad lib.

Administration / exposure

Route of administration:

other: occluded dermal application

Vehicle:

unchanged (no vehicle)

Details on exposure:

For dermal treatment the appropriate volume of test or reference compound was dispensed from a 1.0-cc syringe on to the clipped and shaved dorsal skin (ca. 1.5 in.2) between the scapuaev under a 2-in. gauze square. The application site was occluded with a Lycra-Spandex jacket with Velcro closures. A 1.5 X 2.5-in. polyethylene patch was attached at Ihe application site under the jacket. After a 6-hr exposure period the gauze and jacket were removed, and the application site was wiped gently with moist gauze and blotted dry.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance 2-EH had purity of 99.7% at the outset and at the end of an 84-day period by gas chromatographic analysis. It is thus stable over the treatment period.

Details on mating procedure:

After a 2-week quarantine period animals were mated 1:1. Gestational Day 0 was dated from the appearance of a copulatory plug. Pregnant females were housed singly in stainless steel wire-mesh cages.

Duration of treatment / exposure:

from gestation day 6 to 15

Frequency of treatment:

once daily

Duration of test:

21 days

No. of animals per sex per dose:

range-finding: 8
main study: 25

Control animals:

other: dermal reference compound 2-ME (2-methoxy ethanol) and a sham control of deionized water

Details on study design:

Studies were performed according to current US EPA Health Effects Guidelines and Good Laboratory Practice Regulations. In the range finding study four treatment levels of 2-EH, two levels of the dermal reference compound 2-ME and a sham control of deionized water were applied dermally. A gavage reference compound (VPA) and a naive (untreated) control were also used. In the main study there were three treatment levels of 2-EH, one of 2-ME (methoxy ethanol), and a deionized water control, all applied dermally. Eight plug-positive females were used per treatment level in the range-¿nding study, and 25 per level in the main study. Dermal test and reference compounds were applied undiluted; VPA was given at a concentration of 200 mg/ml in corn oil. Treatment volumes were based on the animal body weight on gd 6 and were not adjusted for subsequent weight changes. Treatment days were gds 6 through 15.

Examinations

Maternal examinations:

In-life observations. Body weights were recorded on gds 0, 6, 9, 12, 15, and 21. Food consumption was measured for each 3-day interval from gds 0 through 21. Observations were made at least once daily for clinical signs and skin irritation. Skin irritation was measured before and after each 6-hr treatment period. Irritation was scored according to the FHSA standard (1985).

Ovaries and uterine content:

Females which delivered early were terminated, examined grossly, and removed from the study. Surviving females in both studies were euthanized by CO2 asphyxiation on Day 21. Maternal body cavities were opened by midline thoracolaporotomy. Maternal uterine and liver weights (both studies) and spleen, adrenals, kidneys, and thymus weights (main study) were recorded. Corpora lutea and uterine implantation sites were counted, and ovaries, cervices, vaginas, and abdominal and thoracic cavities were examined grossly. Uteri were examined externally, removed, and dissected longitudinally to expose the contents.

Fetal examinations:

All live and dead fetuses and resorption sites were noted; uteri from nangravid females were tested tor early resorptions with ammonium sulfide solution (Salewski, 1964). All live fetuses were sexed, weighed, and examined for external malformations and for variations. After external examination approximately 50% of the live fetuses per litter from the main study were examined for visceral (thoracic and abdominal; Staples, 1974, modified) and craniofacial abnormalities (Wilson, 1965, 1973, modified; Van Hulsingha and Bennett, 1977). The remainder were examined for skeletal malformations and variations after evisceration, fixation in ethanol, and staining with alizarin red S (Crary, 1962; Pletzer and Schardein, 1966).

Statistics:

Analysis of data. The units of comparison were the pregnant rat or the litter. Quantitative continuous variables such as maternal body and organ weights were compared between 2-EH and sham control groups, between dermal reference and sham control groups, and between gavage reference and naive control groups. Levene's test for equal variances (Levene, I960), ANOVA, and t-tests with Bonferroni probabilities for pairwise comparisons were used. The pooled t-test was used when Levene's test indicated homogeneous variances and ANOVA was significant. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974) followed when necessary by the separate variance t-test.
Non-parametric data following laparohystereotomy were evaluated using the Kruskal-Wallis test followed by the Mann-Whitney test when appropriate (Sokal and Rohif, 1969). Incidence data were compared using Fisher’s exact test (Sokal and Rohif, 1969). The fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Indices:

percentage of live fetuses, sex ratio (% males)

Historical control data:

N/A

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: There was also an increase in the numbers of early and late resorptions and dead fetuses at 840 mg of 2-ME/kg/day; thus the percentage of live fetuses at necropsy at this dose level was reduced by over 50% compared with the sham control.

Details on maternal toxic effects:
No females died, aborted, or were removed from either study in any control or treated group. In the main study two females in the sham control and two in the low dose group delivered early, and their data are omitted. Pregnancy rates at laparotomy for sham controls and for 2-EH- and 2-ME-treated animals were 75 - 92%. All females in 2-EH-treated groups had a 100% viability. Females treated with 2-ME showed a treatment-related incidence of pregnant animals lacking viable implants, amounting to 40% of pregnant animals at 840 and 100% at 1260 mg/kg bw/day. There was a corresponding decrease in the number of 2-ME-treated animals with live fetuses at necropsy, amounting to 60% at 840 and 0% al 1260 mg/kg/day.
In summary, clinical findings for 2-EH-treated animals were limited to body weight changes, skin irritation, and nasal and ocular effects. Clinical findings with 2-ME were limited to body weight changes, food intake changes, and nasal and ocular effects.
Gestational weight changes (gds 0 through 21) for 2-EH-treated animals in both studies were not significantly different from sham controls. Reductions in weight gain with 2-EH treatment were seen in the range-finding study for gds 6 through 15 at 1680 and 2520 mg/kg bw/day. Weight gain was significantly reduced in the main study with 2-EH for gds 6 through 9 at 2520 mg/kg/day compared with the sham control; there was also a non-significant reduction at 840 mg/kg/day. Females treated with 1260 mg of 2-ME/kg bw/day in the range-finding study showed an overall reduction in weight gain from gds 0 through 21. This was seen as a marked reduction in weight immediately following the onset of treatment and a continued post-treatment reduction. In the main study, 2-ME at 840 mg/kg/day produced reduced weight gain on gds 6 through 9 compared with sham controls. 2-ME produced no effects on weight at 420 mg/kg bw/day.
Food consumption: There were no significant changes in food consumption at any treatment level of 2-EH in either study throughout gestation (data not given). Animals treated with 2-MH at 840 and 1260 mg/kg/day showed a 12% reduction in food intake on gds 6 through 9 compared with the respective sham controls. Food consumption by animals receiving VPA was reduced by 32% on gds 6 through 9 and by 23% on gds 9 through 12 in comparison with naive controls and returned to normal in the post-treatment period.
Clinical observations. Treatment-related effects attributable to 2-EH at the application site were exfoliation, encrustation, and erythema. There was no edema. Exfoliation and encrustation occurred tn both range-finding and main studies at all treatment levels of 2-EH. In the main study, there was no erythema or edema in sham controls. Erythema occurred during treatment with 2-EH at levels of 840 mg/kg/day and above. Irritation was essentially mild. Maximum mean treatment scores occurred on gd 10 at 1680 mg/kg/ day (0.4, range-finding study), on gd 11 al 2520 mg/kg bw/day (1.1, range-finding study), and on gd 14 at 1680 mg/kg bw/day (0.3, main study). There was no exacerbation by continued treatment. Erythema subsided imtnediateiy after the cessation of treatment. There were no exfoliation, encrustation, erythema, or edema at the application site with 2-ME. Nasal encrustation and ocular encrustation and discharge were seen mostly in the main study with 2-EH and 2-ME and in sham controls. Since these effects occurred in controls and mostly disappeared after treatment ceased they are attributed to handling stress.
Necropsy Findings
The only treatment-related findings for 2-EH-dosed animals at necropsy were residual exfoliation and crusting at the application site at mid and high treatment levels. There were no differences from controls for any treatment level of 2-EH, in maternal body weights, gravid uterine or corrected body weights, or in relative or absolute liver, thymus, spleen, adrenal and kidney weights. Thus at 1260 mg of 2-ME/kg bw/day, mean body and gravid uterine weights were significantly less than those of sham controls and were reduced non-significantly at the next lower treatment level. Necropsy findings for 2-ME-treated animals were not otherwise different from controls.
Gestational Parameters:
In both studies 2-EH was without adverse effect at any treatment level, compared with controls, on total and non-viable implants, early or late resorptions, live or dead fetuses, fetal sex ratio, and mean fetal body weights per litter. In contrast, 2-ME treatment caused a treatment-related increase in the numbers of non-viable implants. These were significantly increased at 840 mg/kg bw/day (main study) and at 1260 mg/kg bw/day (range-finding study) all implants were non-viable. There was also an increase in the numbers of early and late resorptions and dead fetuses at 840 mg of 2-ME/kg bw/day; thus the percentage of live fetuses at necropsy at this dose level was reduced by over 50% compared with the sham control. In addition, the mean body weight per litter of fetuses at this dose level was reduced by 38% compared with controls, although the number of fetuses was also reduced.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

840 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 520 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no external visceral, or skeletal malformations associated with any treatment level of 2-EH, In contrast, 840 mg of 2-ME/kg/day produced significantly increased incidences of two external malformations, i.e. whole body edema and protruding tongue. In addition, the same dose produced significantly increased incidences of two visceral malformations, i.e. missing innominate arteries and dilation of the lateral ventricles of the brain. In the range-finding study 2- EH and 2-ME fetuses were examined for external malformalformations only; none were found. Regarding to the incidence of variations by individual fetus or by category, there were no treatment-related increases in external, visceral, or skeletal variations at any treatment level of 2-EH. There were increases in the numbers of poorly ossified centrum 5 and in bilobed centrum 11 at 840 mg of 2-EH/kg/day only, not attributable to treatment with 2-EH. At 840 mg of 2-ME/kg/day there were increased incidences ofecchymosis of the head (an external variation), fetal atelectasis (a visceral variation), and 54 skeletal variations, relative to the sham control.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

2 520 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased incidences ofecchymosis of the head (an external variation), fetal atelectasis (a visceral variation), and 54 skeletal variations, relative to the sham control.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results given in this study, the NOAEL of test article for maternal toxicity is considered to be 840 mg/kg/day, while that of developmental toxicity is greater than 2520 mg/kg/day under the conditions of the paper.

Executive summary:

This paper is published to investigate the developmental toxicity potential of 2-ethylhexanol (2-EH). Undiluted test article was administered by occluded dermal application for 6 hr per day on Gestation Days 6 through 15 to pregnant Fischer 344 rats, in range-finding (R) and main (M) studies. Total animals were exposed at levels of 0, 420, 840, 1680 and 2520 mg/kg/day in (R) and 0, 252, 840 and 2520 mg/kg/day in (M) studies. Numbers of plug-positive females per group were (R) 8 and (M) 25. Maternal weight gain was reduced for 2-EH at 1680 (R) and 2520 (R and M studies) mg/kg bw/day. Exfoliation and encrustation were seen at the application site in both studies at 840, 1680 and 2520 mg/kg bw/d. Maternal liver, kidney, thymus, spleen, adrenal and uterine weights and gestational and fetal parameters were unaffected by treatment with 2-EH. There were no treatment-related increases in the incidence of individual or pooled external, visceral and skeletal malformations or variations following the application of 2-EH. The NOAELs for the maternal toxicity of 2-EH were 252 mg/kg bw/day based on skin irritation and 840 mg/kg bw/day based on systemic toxicity. The developmental toxicity NOAEL was at least 2520 mg/kg bw/day, with no teratogenicity.

It is concluded that 2-EH is not developmentally toxic by the dermal route in the Fischer 344 rat at and below treatment levels which produce matermal toxicity.