Registration Dossier

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: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexanol
- 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 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 gd 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 (GC) prior to use verified the formulations to be within 99-108% of the theoretical concentrations. 2EH/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-EH (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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.009, 0.03 or 0.09%
Basis:
nominal in diet
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: DEHP was previously evaluated for potential developmental toxicity in timed-pregnant Swiss (CD-I) mice exposed via the diet throughout gestation (gd 0 to 17) (Tyl et oZ., 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 2-18 ethylhexanol) at approximately equimolar doses and under comparable experimental conditions as those from the study of DEHP in mice (Tyl et aZ., 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 aZ. (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; see results.) 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 gestation day 0-17

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

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: The maternal body, liver, and intact uterus were weighed

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:
- 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:
analyses of variance (ANOVA), when ANOVA revealed a significant (p<0.05) dose effect, Dunnett's Multiple Comparison Test (Dunnett, 1955; 1964) compared each 2-EH-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, General Linear Models (GLM), chi square test, when a chi square test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of each 2-EH 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, gd 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 gd 6, 9 and 12 at 0.090% and in one dam on gd 6 at 0.030%

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
191 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

In conclusion, 2-EH administered in the diet during gestation (gd 0-17) in CD1 mice at concentrations comparable to or exceeding those employed for DEHP and MEHP in the same study design, resulted in no maternal or developmental toxicity. It is therefore clear that 2-EH plays essentially no role in the expression of DEHP-induced maternal and developmental toxicity.

Applicant's summary and conclusion