Fatty acids, C18-(unsaturated) dimers and their monoesters and diesters with 2-ethylhexanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

1990-05-18 to 1990-08-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study, tested with the source substance 2-EH (CAS No. 104-76-7)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): 2-ethylhexanol (2-EH)
- CAS No. of test material (as cited in study report): 104-76-7
- Analytical purity: >99%
- Lot/batch No.: 005018

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Weight at study initiation: 23.52-31.59 g
- Housing: Plug-positive females were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and Ab-Sorb-Dri® cage litter (Laboratory Products, Garfield, NJ
- Diet (e.g. ad libitum): Ground Purina Certified Rodent ChoW® (#5002) available ad libitum throughout gestation
- Water (e.g. ad libitum): deionized/filtered water were available ad libitum throughout gestation
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2
- Humidity (%): 48
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: food grade modified corn starch microcapsules

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh supplies of dosed feed were obtained from refrigerated stock on the mornings of gestational day 0, 3, 6, 9, 12 and 15

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis by gas chromatography prior to use verified the formulations to be within 99-108% of the theoretical concentrations. 2-ethylhexanol/feed mixes were determined to be stable throughout the period of use for each study replicate.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage:1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Verification of same strain and source of both sexes: yes

Duration of treatment / exposure:

continuously exposure to 2-ethylhexanol (0, 0.009, 0.03, or 0.09%) microencapsulated in the feed from gestational day 0-17

Frequency of treatment:

continuously ad libitum feed

Duration of test:

sacrifice at gestational day 17

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.009, 0.03 and 0.09%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 17, 59 and 191 mg/kg bw/d
Basis:
other: calculated consumption, based on gestational food consumption

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: di-(2-ethylhexyl)phthalate (DEHP9 was previously evaluated for potential developmental toxicity in timed-pregnant Swiss (CD-I) mice exposed via the diet throughout gestation (gestational day 0 to 17) (Tyl et al. 1988). An average dose of 44 mg/kg/day (0.025% DEHP in feed) was the maternal and embryo/fetal NOAEL. An increased incidence of malformations was observed at 91 mg/kg/day (0.05% DEHP in feed) in the absence of other indications of maternal and embryo/fetal toxicity. At 191 and 292 mg/kg/day (0.10% and 0.15% DEHP in feed), maternal toxicity (reduced weight gain during treatment and increased relative liver weight) was observed, as well as decreased fetal weight and an increased incidence of prenatal mortality and fetal malformations. Based upon these findings, additional studies were designed to characterize the developmental toxicity of DEHP's principal metabolites (MEHP and 2-ethylhexanol) at approximately equimolar doses and under comparable experimental conditions as those from the study of DEHP in mice (Tyl et al., 1988). Accordingly, the concentrations of MEHP in feed included 0% (control), 0.017%, 0.035%, 0.070%, and 0.140%, with the average daily intake of 0, 35, 73, 134, and 269 MEHP mg/kg/day, respectively (NTP, 1990), approximately equivalent on a molar basis to the dose levels of DEHP used by Tyl et al. (1988). The target concentrations of 2-EH in feed employed for this study included 0.00% (control), 0.009%, 0.030%, and 0.090%. The expected average daily intake of 2-EH at the proposed dietary concentrations were 0, 15, 52.5, and 157.5 mg/kg/day, respectively. The actual intake was 0, 17, 59 and 191 mg/kg/day. Therefore, the target dietary dose levels of 2-EH employed for this study were intended to encompass the range of intakes obtained with DEHP.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from gestational day 0-17

BODY WEIGHT: Yes
- Time schedule for examinations: on gestational days 0, 3, 6, 9, 12, 15, and 17

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: on gestational days 0, 3, 6, 9, 12, 15, and 17

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestational day 17
- Organs examined: The maternal body, liver, and intact uterus were weighed and ovarian corpora lutea were counted

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

Fetal examinations:

- Body weight: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

General Linear Models (GLM) procedures were applied for the analyses of variance (ANOVA) of maternal and fetal parameters (SAS Institute, 1989a,b; 1990 a,b,c). Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967) and Bartlett's test for homogeneity of variance was performed on all data to be analyzed by ANOVA (Winer, 1962). GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions. Dose x replicate interactions were nonsignificant (p>0.05) for all maternal and embryo/fetal endpoints analyzed by ANOVA, thus indicating that the results were consistent across replicates. When ANOVA revealed a significant (p<0.05) dose effect, Dunnett's Multiple Comparison Test (Dunnett, 1955; 1964) compared each 2-ethylhexanol-exposed group to the control group. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights and fetal body weight .
Nominal scale measures were analyzed by a χ² test for independence and by a test for linear trend on proportions. When a χ² test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of each 2-ethylhexanol group with the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No females died, delivered early or were removed from the study. Pregnancy rates were high and equivalent across all groups (93-96%); maternal weight change for the gestational (and treatment) period, gestational day 0-17, was unaffected, as was weight change corrected for gravid uterine weight; maternal organ weights and food consumption were also unaffected; Treatment related clinical signs of toxicity were limited to hyperactivity observed in one dam on gestational day 6, 9 and 12 at 0.090% and in one dam on gestational day 6 at 0.030%

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

191 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 effects of exposure on the number of ovarian corpora lutea, or of uterine implantation sites (resorptions, dead fetuses or live fetuses) per litter. Live litter size and fetal body weight per litter (all fetuses, males or females) were equivalent across all groups.
There were also no effects of treatment on the incidence of malformations (external, visceral, skeletal or total) or variations, whether expressed as number or percentage of fetuses per litter or of litters with one or more affected fetuses. Examination of individual fetal findings also indicated no specific malformations or variations with a dose-related incidence.

Effect levels (fetuses)

Remarks on result:

not measured/tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

2-ethylhexanol administered in the diet during gestation (gestational day 0-17) in CD1 mice at concentrations of 0, 0.13, 0.46, and 1.49 mmol/kg (corresponding to 0, 17, 59, and 191 mg/kg/day) resulted in no maternal or developmental toxicity.
In conclusion, 2-ethylhexanol plays essentially no role in the expression of di-(2-ethylhexyl)phthalate (DEHP)-induced maternal and developmental toxicity.

Executive summary:

Microencapsulated 2-ethylhexanol (2 -EH) (0%, 0.009%, 0.03%, or 0.09% in feed, corresponding to an average intake of 0, 17, 59, and 191 mg/kg/day) was provided on gestational days 0 to 17 ad libitum to timed-mated CO-1 mice (28/group). At sacrifice (gestational day 17), the number of ovarian corpora lutea and uterine implantation sites, including resorptions, and dead or live fetuses, were recorded. Live and dead fetuses were weighed. Live fetuses were sexed and examined for external, visceral and skeletal malformations and variations.

 

No dams died, delivered early or were removed from the study. Pregnancy rate was high and equivalent across all groups. There was no treatment-related maternal toxicity observed in this study.

 

There were no effects of exposure to dietary 2-EH on any gestational parameter. The number of corpora lutea, uterine implantation sites, pre- and postimplantation loss, sex ratio and live fetal body weight per litter were all equivalent across all groups. There were also no treatment-related changes in the incidence of individual, external, visceral, skeletal or total malformations or variations. In conclusion, there were no maternal or developmental toxic effects of 2-EH dietary exposure throughout gestation up to a concentration of 191 mg/kg/day.