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Key value for chemical safety assessment

Effects on fertility

Description of key information
no data
Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment
Principles of method if other than guideline:
Groups of male and female Wistar rats received 0, 100, 300 or 600 mg/kg/day of test material in their drinking water. Males were exposed for 10 weeks and females for 2 weeks prior to mating, both sexes during mating and females during gestation and lactation, and toxicity to reproduction was studied.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Laboratory Animal Center, University of Kuopio, Finland
- Age at study initiation: males: 5-6 weeks; females: 9-10 weeks)
- Weight at study initiation: males: 125 ± 25 g; females: 200 ± 20 g
- Housing: wire mesh cages, 3 animals/cage; 1 male and 1 female per cage during mating; 1 female/litter per cage during gestation/lactation.
- Diet (e.g. ad libitum): commercial rat chow (R3-EWOS, Sodertalje, Sweden), ad libitum except during 2-EHA exposure
- Water (e.g. ad libitum): ad libitum except during 2-EHA exposure
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1ºC
- Humidity (%): 55-65%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
other: drinking water supplemented with NaOH (equal amounts as 2-ethylhexanoic acid)
Details on exposure:
Rats were given the test substance in their drinking water as a sodium salt by mixing equivalent amounts of 2-ethylhexanoic acid and NaOH. Concentrations of 2-ethylhexanoic acid solution were adjusted on the basis of water consumption and body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Exposure period: Males were exposed for 10 weeks and females for 2 weeks prior to mating, both sexes during mating and females during gestation and lactation.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300, 600 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Positive control:
None
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes, weekly

FOOD CONSUMPTION: Yes, weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, water consumption was recorded for each cage for the whole exposure period and doses were corrected by adjusting the concentration of the 2-ethylhexanoic acid solution acccording to the most recent body weights and water consumption. When more than one animals was housed per cage, a mean body weight was used.


Oestrous cyclicity (parental animals):
Yes, vaginal smears were evaluated microscopically
Sperm parameters (parental animals):
Parameters examined in all male parental animals: testis weight, right epididymis weight, left epididymis: sperm density, motility and morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter using equal sex distribution whenever possible; excess pups were examined externally, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (postnatal days 0, 4, 7, 14, 21). Pups were examined every other day from postnatal day 1 onwards and the appearance of the following developmental parameters was recorded: pinna reflex, placing reaction, righting reflex in 5 sec, cliff avoidance in 5 sec, approach/avoidance, ipsilateral flexor reflex (hind toe), grip reflex in 5 sec, air righting, opening of eyes, teeth eruption, and hair growth.


Postmortem examinations (parental animals):
SACRIFICE
- Male animals and non-gravid females: All surviving animals at the end of the mating period (maximum 21 days).
- Maternal animals: All surviving animals on postnatal day 21

GROSS NECROPSY
- Gross necropsy consisted of examination of any pathological changes

HISTOPATHOLOGY / ORGAN WEIGHTS
- Males: organ weight: testis, right epididymis. Histopathology (5 randomly selected animals/group): testes, right epididymis, seminal vesicle, prostate, and coagulating gland
- Non-gravid females and maternal animals: organ weight: ovaries. Histopathology (all non-gravid females and 5 randomly selected maternal animals per group): ovaries, uterus, cervix uteri, vagina
Postmortem examinations (offspring):
On postnatal day 21, all pups were examined externally and euthanized without further examination.
Statistics:
Body weights (adult males, adult females, litters, pups), organ weights, food and water consumption, number of live pups in litter, male fertility data, and data on pup development were analyzed by one-way ANOVA. Comparisons of significant group effects were conducted using Fisher PLSD test of Scheffe's test. The observations on pups (litter percentages) were analyzed by Scheffe's test and the dose-response relationship by the Pearson correlation test.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased (reversible)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased (reversible)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
slight dilatation of the lumen in uterus and epithelial hyperplasia in vagina
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: slightly reduced
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
sperm qualitiy slightly affected
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
delay in fertility
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A 9-12% decrease in bodyweight was observed from gestation day 7 onwards in females of the high-dose group, which disappeared during lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Liquid consumption by pregnant females was slightly reduced at the high dose.

FERTILITY PARAMETERS (PARENTAL ANIMALS)
A dose-dependent delay in fertility was observed. All non-pregnant animals belonged to the 2-ethylhexanoic acid-treated groups. Six males at the high dose and three males at the mid dose copulated occasionally with females in diestrus while all control and the lowest dose group males copulated in estrus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm quality was slightly, but not uniformly dose-dependent, affected. The total number of spermatozoa in the cauda epididymis was 14% lower at the high dose level, but not statistically reduced. The proportion of motile spermatozoa had decreased by 37% at the low dose and by 22% at the high dose. The share of nonmotile spermatozoa was highest in the low-dose group. The number of animals with morphologically abnormal spermatozoa was increased, however not statistically significant, at the two highest dose levels. The most common abnormalities were agglutinations and abnormal heads of spermatozoa. The latter was observed in 13% and 21% of the animals of the mid- and high-dose groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Relative weights of the right epididymides were increased at the high dose.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In two of five dams of the mid- and high-dose group, a slight dilatation of the lumen in uterus and epithelial hyperplasia in vagina were observed.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Delay in fertility
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
lethargy, hematomas etc.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
tendency to post natal mortality
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
transiently decreased in high dose group
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings:
not examined
LITTER SIZE (OFFSPRING)
Average litter size was reduced by 16% at the high dose. Postnatal deaths tended to be more common in the 2-ethylhexanoic acid-treated groups, but this was not statistically significant.

CLINICAL SIGNS (OFFSPRING)
The frequency of lethargy, hematomas, and abnormally thin hair was higher at the two highest dose levels. Kinky tail showed a dose-dependent increase, and the frequency of abnormal legs (e.g. severe flabby legs) was higher in the 2-ethylhexanoic acid-treated animals. The latter animals were cannabalized by the dams soon after birth.

BODY WEIGHT (OFFSPRING)
Bodyweights were similar at birth, but decreased transiently at the high dose during lactation.

PHYSICAL DEVELOPMENT
Exposure to 2-ethylhexanoic acid delayed physical development of the pups. Ears raised later in mid- and high-dose groups, and eye opening, eruption of teeth, and hair growth occurred significantly later at the high dose level. The development of the grip and cliff avoidance reflexes were delayed, more clearly in males than females.

GROSS PATHOLOGY (OFFSPRING)
One male pup of the high dose had a mass in the left testis and the left epididymis was missing.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: lower litter size, lower body weights and delayed physical development
Reproductive effects observed:
not specified
Conclusions:
2-Ethylhexanoic acid caused delayed fertility and development in rats when applied to the parental generation via drinking water. Based on a delay in fertility the NOAEL(parental) was considered to be 300 mg/kg bw/d and based on lower litter size, body weight and delayed physical development the NOAEL(developmental) was set at 100 mg/kg bw/d.
Based on a read-across approach, the same NOAELs are taken into account in case of sodium 2-ethylhexanoate.
Executive summary:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

Groups of male and female Wistar rats received 0, 100, 300 or 600 mg/kg/day of test material in their drinking water. Males were exposed for 10 weeks and females for 2 weeks prior to mating, both sexes during mating and females during gestation and lactation, and toxicity to reproduction was studied.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

A 9-12% decrease in bodyweight was observed from gestation day 7 onwards in females of the high-dose group, which disappeared during lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

Liquid consumption by pregnant females was slightly reduced at the high dose.

FERTILITY PARAMETERS (PARENTAL ANIMALS)

A dose-dependent delay in fertility was observed. All non-pregnant animals belonged to the 2-ethylhexanoic acid-treated groups. Six males at the high dose and three males at the mid dose copulated occasionally with females in diestrus while all control and the lowest dose group males copulated in estrus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Sperm quality was slightly, but not uniformly dose-dependent, affected. The total number of spermatozoa in the cauda epididymis was 14% lower at the high dose level, but not statistically reduced. The proportion of motile spermatozoa had decreased by 37% at the low dose and by 22% at the high dose. The share of nonmotile spermatozoa was highest in the low-dose group. The number of animals with morphologically abnormal spermatozoa was increased, however not statistically significant, at the two highest dose levels. The most common abnormalities were agglutinations and abnormal heads of spermatozoa. The latter was observed in 13% and 21% of the animals of the mid- and high-dose groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Relative weights of the right epididymides were increased at the high dose.

HISTOPATHOLOGY (PARENTAL ANIMALS)

In two of five dams of the mid- and high-dose group, a slight dilatation of the lumen in uterus and epithelial hyperplasia in vagina were observed.

LITTER SIZE (OFFSPRING)

Average litter size was reduced by 16% at the high dose. Postnatal deaths tended to be more common in the 2-ethylhexanoic acid-treated groups, but this was not statistically significant.

CLINICAL SIGNS (OFFSPRING)

The frequency of lethargy, hematomas, and abnormally thin hair was higher at the two highest dose levels. Kinky tail showed a dose-dependent increase, and the frequency of abnormal legs (e.g. severe flabby legs) was higher in the 2-ethylhexanoic acid-treated animals. The latter animals were cannabalized by the dams soon after birth.

BODY WEIGHT (OFFSPRING)

Bodyweights were similar at birth, but decreased transiently at the high dose during lactation.

PHYSICAL DEVELOPMENT Exposure to 2-ethylhexanoic acid delayed physical development of the pups. Ears raised later in mid- and high-dose groups, and eye opening, eruption of teeth, and hair growth occurred significantly later at the high dose level. The development of the grip and cliff avoidance reflexes were delayed, more clearly in males than females.

GROSS PATHOLOGY (OFFSPRING)

One male pup of the high dose had a mass in the left testis and the left epididymis was missing.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable with restrictions
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

In an one generation reproductive toxicity study according to OECD 415 (Pennanen et al., 1993), male and female Wistar rats (24/sex/dose) were exposed to 2-ethylhexanoic acid as the sodium salt in the drinking water at calculated average doses of 0, 100, 300 and 600 mg/kg bw/day. Animals received the test substance in drinking water for 10 weeks (males) or 2 weeks (females) prior to mating, during mating, gestation, and 21 days following parturition. Body weights of males were not affected, but pregnant 600 mg/kg bw/day females had reduced body weights (9-12%) beginning on gestation day 7. Pregnant females in the 600 mg/kg bw/day group had significantly reduced water consumption (14%), and overall gestational weight gain at the 600 mg/kg bw/day dose was reduced (21%). Female body weights at all dose levels were similar to the control at sacrifice on postnatal day 21. 2-ethylhexanoic acid caused a delay in fertilization, as several treated animals (1, 2, and 4 animals respectively in the 100, 300 and 600 mg/kg bw/day groups) required three or four estrus cycles to conceive, compared to one or two cycles for all control animals. However, no effects on fertility were observed and high pregnancy rates were achieved: only three of 71 females exposed to 2-ethylhexanoic acid failed to conceive (2 in the low-dose group, 1 in the high-dose group), and the pregnancy index was 100% for controls. All pregnant females delivered litters. Litter size was reduced by 16% at 600 mg/kg bw/day relative to control (9.2 vs. 10.9), but individual animal data were not provided, and it is not known whether the decrease represents all or only selected dams. No statistically significant dose-related differences were seen between groups in the number of pups, sex ratio, numbers of stillbirths, postnatal deaths, or lactation index at age of weaning. No effects of treatment were noted at gross necropsy of the parental males or females (both non gravid and gravid). Relative, but not absolute, weight of the right epididymis was increased 17% at 600 mg/kg bw/day. Relative testes and ovary weights were not affected by treatment. No histologic changes were seen in the epididymis or testis. Microscopic pathology findings in dams were restricted to slight dilatation of the uterine lumen and vaginal epithelial hyperplasia in two of five animals at both 300 and 600 mg/kg bw/day. All other examined tissues were normal. A slight but non-statistically significant decrease (14%) in sperm count was noted in the high-dose group. Sperm quality was affected by exposure to 2-ethylhexanoic acid, but no statistically significant dose-related effects were observed e.g., the proportion of non motile spermatozoa was highest at 100 mg/kg bw/day. Numbers of animals with morphologically abnormal spermatozoa were increased at 300 and 600 mg/kg bw/day, but the incidence per animal was not provided and differences were not statistically significant. In 2-ethylhexanoic acid exposed pups, the frequency of lethargy, hematomas, and abnormally thin hair was increased at 300 and 600 mg/kg bw/day but only lethargy was statistically significant and then only for the 300 mg/kg bw/day group. The incidence of kinky tail was also statistically increased in the 300 and 600 mg/kg bw/day groups, but the percentage of pups with this abnormality was similar in both groups, and the effect was also seen in the control group. Pups with leg abnormalities were seen only in 2-ethylhexanoic acid-exposed groups but incidences were low, not dose-related, and not statistically significant. Both male and female pups in the 600 mg/kg bw/day group had reduced weight gains during lactation when compared with the other groups, but this difference was transient. Delayed physical development seen in the 600 mg/kg bw/day group of animals included later raising of ears, eye opening, eruption of teeth, and hair growth, and grip and cliff avoidance. Significant effects seen in pup development at lower dose levels were restricted to a delayed raising of ears at 300 mg/kg bw/day. Reflex responses were not affected. The NOAEL for reproductive and systemic toxicity was 300 mg/kg bw, the NOAEL(F1) was 100 mg/kg bw/d.

In an oral subchronic toxicity study (CMA, 1988; see chapter repeated dose toxicity), groups of ten male and female F344 rats were administered feed containing 0, 0.1, 0.5 or 1.5% (males: 0, 61, 303, 917 mg/kg bw; females: 0, 71, 360, 1068 mg/kg bw) 2-EHA for 91-93 consecutive days. Additional groups were assigned to satellite recovery groups and were fed diets containing either 0 or 1.5% 2-ethylehxanoic acid for 94 days and then were offered standard diets for 27-28 days. Body weights were reduced in non-recovery males and females in the 1.5% dietary group from the first week through study termination. By day 91, weights were reduced 8% in males and 10% in females. In recovery groups of animals, body weights at the 1.5% dietary level were reduced relative to control by 9% (males) and 8% (females) at the end of the treatment period. Weight differences were less following recovery, with weight reductions of only 5% in males and 3% in females. Absolute testes weights were similar to controls for all treatment levels of non-recovery males. Relative testes/body and testes/brain weights were higher for the 0.5 and 1.5% males. For the 1.5% recovery males, absolute testes weights were comparable to controls while relative testes/body and testes/brain weights were statistically significantly increased compared to controls. The differences in relative testes weights reflect slightly lower body weights rather than target organ effects on the testes. The absolute ovary weights and the relative ovary/body and ovary/brain weights for all treatment levels of non-recovery and recovery animals were similar to controls. There were no gross or histological changes in either the testes or ovaries that were treatment related. In addition, there were no gross or histologic changes in the uterus, vagina or fallopian tubes or gross changes in male accessory sex glands or epididymides. The NOAEL for reproductive toxicity was 1.5% 2-ethylhexanoic acid in the diet. The LOAEL for systemic toxicity was 1.5%.


Short description of key information:
The available one generation reproductive toxicity study and repeated dose study with rats suggest that 2-ethylhexanoic acid, the source substance of the read across approach, is not a reproductive toxicant. For read across justification please refer to the attached document, IUCLID Chapter 13.

Justification for selection of Effect on fertility via oral route:
Non-GLP, non-guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment

Effects on developmental toxicity

Description of key information
Several studies with rats have demonstrated developmental toxicity in response to oral exposure to 2-ethylhexanoic acid, the source substance of the read across approach. Adverse fetal effects included reduced body weight, skeletal malformations and variations. The NOAEL was determined to be 100 mg/kg bw/day. Mechanistic studies indicated that the developmental toxicity of 2-ethylhexanoic acid is at least partially related to disruption of zinc metabolism and distribution in the parental animals. Developmental effects in the one-generation reproduction study occurred at the same levels, also with a NOAEL of 100 mg/kg bw/day. For read across justification please refer to the attached document, IUCLID Chapter 13.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, with minor restrictions in design and/or reporting but otherwise adequate for assessment.
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at arrival: males: 70 days; females: 63 days
- Weight at study initiation: males: 175-200 g; females: 130-150 g
- Housing: stainless steel wire mesh cages
- Diet (e.g. ad libitum): Prolab Certified Ground Rodent Chow, RMH-3200; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 69-72
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test chemical was diluted in certified corn oil (Mazola, Best Foods, Inc., CPC International) and mixed by inversion prior to the onset of the study. GC analysis indicated that the test material was stable for at least 21 days when stored at room temperature and uniformly distributed in corn oil. A dose volume of 2 ml/kg bodyweight (concentration in vehicle: 0 - 250 mg/ml) was administered by gavage, based on the body weight of each animal on gestation day 6.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual concentrations of test material in corn oil were determined by GC analysis and were 50.4, 132.0 and 246.0 mg/ml for the 100, 250 and 500 mg/kg/day doses, respectively. Analysis values ranged from 92.5 to 109.7% of the nominal dosing concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Each male was used only once
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy (GD 0)
- Successfully mated females weighing 147-174 g were housed singly and were randomly assigned (stratified by body weight) to each experimental group.
Duration of treatment / exposure:
Gestation day 6 through 15
Frequency of treatment:
Daily
Duration of test:
Up to gestation day 21
Remarks:
Doses / Concentrations:
0, 100, 250 or 500 mg/kg bodyweight/day
Basis:
nominal in diet
No. of animals per sex per dose:
25 (females only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study
Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 6 (prior to dosing), 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Food consumption (g food/animal/day) was measured for the intervals GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 (CO2)
- Gross examination: gravid uterus, ovaries (including corpora lutea), cervix, vagina and abdominal and thoracic cavities.
- Organ weights: liver, uterus
- The liver was fixed for possible future examination
Ovaries and uterine content:
The uterus was externally examined for signs of hemorrhage, removed from the abdominal cavity and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites were noted and recorded. Uteri from females that appeared nongravid were placed in ammonium sulfide for detection of early resorptions.
Fetal examinations:
All live fetuses were weighed and sexed. All fetuses were examined for external malformations including cleft palate and variations. One half of the fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. These fetuses were decapitated and their heads were fixed for examination of craniofacial structures. Remaining fetuses were eviscerated, fixed and examined for skeletal malformations and variations. Decapitated fetuses were also processed for staining but were not examined.
Statistics:
The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables were intercompared for the dose groups by the Levene's test for equal variances, ANOVA, and t-tests with Bonferroni probabilities for pairwise comparisons. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by ANOVA for unequal variances followed, when necessary, by the separate variance t-test. Nonparametric data obtained following laparohysterectomy were treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: hypoactivity, ataxia, ocular discharge

Details on maternal toxic effects:
Clinical signs were observed at the high-dose level only and included hypoactivity, ataxia, audible respiration, ocular discharge and periocular encrustations. No effects on body weight and mortality were observed. Liver weight (absolute and relative) was increased in the high-dose group.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: reduced skeletal ossification

Details on embryotoxic / teratogenic effects:
Fetal body weights per litter were reduced in the high-dose group, but these findings may be confounded by the slightly larger mean litter size in this group. Reduced skeletal ossification was observed in the mid- and high-dose group. Dilation of the lateral ventricles of the brain with no tissue compression and extra (14th) thoracic centrum and arches were observed in all groups but were significantly increased at the high dose only.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Prgenant rats were dosed with 0, 100, 250 or 500 mg/kg bw/d ethylhexanoic acid from gestation day 6 to 15. Based on the observed effects the NOAEL(maternal) was considered to be 250 mg/kg bw/d and the NOAEL(developmental) was set at 100 mg/kg bw/d.
Based on a read-across approach, the same NOAELs are taken into account in case of sodium 2-ethylhexanoate.
Executive summary:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

Timed-pregnant Fischer 344 rats were exposed to 2 -ethylhexanoic acid by gavage on gestational day 6 through 15 at doses of 0, 100, 250 of 500 mg/kg bw/d in corn oil (25 plug-positive females/group). The does volume employed was 2 mL/kg bw based on the weight of each femal on gestation day 6. Clinical observations were taken daily and maternal body weights were taken on gestation day 0, 6, 12, 15, 18 and 21. Food consumption was measured for the intervals gestation days 0 -3, 3 -6, 6 -9, 9 -12, 12 -15, 15 -18 and 18 -21. At scheduled sacrifice on gestation day 21, the dams were evaluated for body weight, liver weight, gravid uterine weight and status of implantation sites. Maternal livers were retained in fixative for possible subsequent microscopic examination. Live fetuses were dissected from the uterus, counted, weighed, sexed and examined for external abnormalities. Approx. one-half of the live fetuses in each litter were examined for visceral malformations and variations. These fetuses were then decapitated and the heads feixed in Bouin`s solution; serial free hand sections of the heads were examined for soft tissue craniofacial malformations and variations. The remaining (intact) fetuses in each litter were eviscerated, fixed in alcohol, stained with alizerin red S and examined for skeletal malformations and variations.

There were no treatment-related maternal deaths. No dams aborted. One female, at 250 mg/kg bw/d, delivered early, and was removed from the study. A total of 21 -24 litters were examined in each dose group. There were no statistically significant differences among the groups for gestational maternal body weights or weight gain, or food consumption. Treatment-related clinical signs, observed only at 500 mg/kg bw/d, included hypoactivity, ataxia, and audible respiration; clinical signs with significantly increased incidence at 500 mg/kg bw/d included ocular discharge and periocular encrustation. At the gestation day 21 sacrifice there were no effects of treatment on maternal body weight or gestational weight gain, or on gravid uterine weight. Liver weights were significantly increased at 500 mg/kg bw/d. Gestational parameters, including number of ovarian corpora lutea, viable and non-viable implantations/litter, and sex ratio were unaffected by treatment. Fetal body weight/litter were significantly reduced at 500 mg/kg bw/d.

There was no significant increase in the incidence of malformations in any treatment groups relative to controls. There were no treatment-related differencesamong groups for individual external variations or for pooled external, visceral, skeletal or total variations. One visceral variation, dilated lateral ventricles on the brain with no tissue compression, exhibited an increased incidence at 500 mg/kg bw/d. Twenty-six fetal skeletal variations exhibited significantly different incidences at 500 mg/kg bw/d relative to those in controls; 19 of these were significant increases in incidence and seven were significant reductions in incidence (almost all of the reductions were in incidence of poorly ossified skeletal districts since the incidences of these same districts, unossified, increased). In addition, there were skeletal variations with increased incidences at 250 mg/kg bw/d, indicative of minimal fetotoxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restriction
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

In a developmental toxicity study (CMA, 1988) according to OECD 414, 2 -ethylhexanoic acid was administered via gavage to F344 rats from gestation day 6-15 at doses of 0, 100, 250 and 500 mg/kg bw. Maternal body weights, weight gain, and feed consumption were comparable among groups. Abnormal clinical signs were increased in the 500 mg/kg bw/day group only and included ocular discharge and ocular encrustation. Hypoactivity, ataxia, and audible respiration were also noted in animals from the 500 mg/kg bw/day group. Pregnancy rates in all groups were between 21/25 and 24/25, and all animals had viable fetuses. There were no statistically significant differences in terminal maternal body weight among groups but in the 500 mg/kg bw/day group, statistically significant increases were seen in absolute (9%) and relative (10%) liver weights. No embryotoxic effects were noted. Number of ovarian corpora lutea, total implants, pre- and postimplantation loss, and percent viable fetuses were comparable among groups. The mean fetal body weights (all fetuses, male or female) in litters from the 500 mg/kg bw/day dams were lower (8%) than that in the control group, but this may have been due to a higher average litter size in the 500 mg/kg bw/day group (9.3 vs. 8.4 in controls). There were no significant differences among groups in the incidence of total malformations, malformations by category, or individual malformations between groups. Sex ratios were equivalent. There were no significant differences among groups in the incidence of fetal external variations. One visceral variation, dilation of the lateral ventricle of the brain with no tissue compression, was significantly increased in fetuses of the 500 mg/kg bw/day dams. Increases were noted in both number of affected pups and percent of affected litters: 21/104 pups and 15/21 litters affected compared to the control group (3/100 pups and 2/23 litters). This effect was observed in 13.0, 20.8, 36.4 and 71.4% of litters (3, 7, 10 and 21 fetuses) in the 0, 100, 250 and 500 mg/kg bw/day groups, respectively but was only statistically significant at the high dose. There was a dose-related trend in the incidence of the related malformation, dilated lateral ventricles of the brain with tissue compression. This effect occurred in 8.7, 4.2, 4.5, and 28.6% of litters, respectively in the 0, 100, 250 and 500 mg/kg bw/day dose groups but the results were not statistically significant. A number of specific skeletal variations were observed in the 500 mg/kg bw/day group. These variations included extra thoracic centra and arches, which were significantly elevated when calculated on a litter basis, rudimentary ribs, extra ribs, and unilateral and bilateral bone islands in thoracic and lumbar arches. There was also an increase in the number of litters in the 250 mg/kg bw/day group with reduced ossification of the anterior arch of the atlas and proximal phalanges of the forelimb and hindlimb. However, total numbers of visceral and skeletal variations were not significantly altered by treatment. The NOAEL for maternal toxicity was 250 mg/kg bw and for developmental toxicity 100 mg/kg bw.

In a supporting developmental toxicity study (Pennanen et al., 1992) with Wistar rats, 2-ethylhexanoic acid was administered as a sodium salt in drinking water to groups of 20-21 animals at doses of 0, 100, 300 and 600 mg/kg bw from gestation day 6-19. The 600 mg/kg bw/day group showed a 20% reduction in water consumption. The mean body weight of dams in the 600 mg/kg bw/day group was slightly lower from Day 13 onward, and at the end of the study, they were 11% less than the control group. Maternal weight gain in the 600 mg/kg bw/day group reduced by 58%. No evidence of maternal toxicity was seen at the 300 or 100 mg/kg bw/day dose levels. The pregnancy rate was 84% in the control group and 67% in the 300 and 600 mg/kg bw/day groups, but these differences were unrelated to treatment, which was limited to Days 6-19 of gestation. No females aborted their litters. No differences were noted in gravid uterine weights. No embryotoxic effects were noted. There were no differences in the number of live fetuses, and total implants, pre- and post-implantation loss, and percent viable fetuses were comparable among groups. There were no differences among groups in the number of litters with resorptions. The mean fetal body weights were very slightly reduced at the 600 mg/kg bw/day and 300 mg/kg bw/day dose levels when compared to the control fetal body weight, but these minor differences were likely due to reduced maternal weights and weight gains. The differences in mean fetal weight were present in both male and female fetuses at the 600 mg/kg bw/day dose level, but only in females at the 300 mg/kg bw/day dose level. Paralleling reductions in weight gain, the mean placental weights were reduced 10% in the 600 and 300 mg/kg bw/day dams. The majority of the effects were skeletal malformations. Visceral malformations were rare; only five instances were noted and there was no relationship between their occurrence and administered dose of the test substance. Clubfoot occurred with means of 6.7 and 5.6 affected fetuses per litter at dose levels of 600 and 300 mg/kg bw/day, respectively. A mean of 0.8 affected fetuses per litter was seen at the 100 mg/kg bw/day dose level, but the difference from the control, in which clubfoot was not seen, was not significant. Other skeletal malformations observed in treated animals included abnormal ankle cartilage, absence of fibula, flabby legs, scoliosis, and lordosis, but none of these malformations was significantly increased in treated animals relative to control. Extrathoracic ribs were seen in all groups, including controls. Skeletal variations were seen in all dose groups, including controls. Significant increases in some variations were seen in the 600 mg/kg bw/day group. These included wavy ribs, non-ossified sternebrae, bipartite visceral centra, and reduced lumbar ossification. A statistically increased percentage of all fetuses having wavy ribs was also noted at the 100 and 300 mg/kg bw/day dose levels compared with the control group, but there was no dose-response relationship associated with the frequency of this variation. Similarly, reduced cranial ossification was seen in the 100 and 600 mg/kg bw/day groups, but this effect was not dose related, as there was no difference between occurrence in the 300 mg/kg bw/day group and the control. Among visceral variations, pelvic dilatation was seen at all dose levels, including the control. Other visceral variations observed in treated animals included congestion in the kidney cortex, kidney displacement, ureter dilatation and curved ureter, but none of these variations was statistically increased over control. Dilatation of brain ventricles was seen in all groups, including control, but was significantly increased only in the 600 mg/kg bw/day group. Based on the observed effects, the NOAEL for maternal toxicity was 300 mg/kg bw/day and the NOAEL for developmental toxicity was 100 mg/kg bw/day.

Results from Bui et al. confirmed that 2-ethylhexanoic acid induction of maternal metallothionien synthesis reduced distribution of zinc to the fetus and adversely affected fetal development. This study supports the hypotheses that 2-ethylhexanoic acid causes developmental toxicity indirectly and that developmental toxicity will only occur at dose levels that cause maternal liver toxicity and disrupt zinc metabolism and distribution. Based on these results, 2-ethylhexanoic acid is not likely to cause effects on fertility but is likely to be a developmental toxicant.


Justification for selection of Effect on developmental toxicity: via oral route:
GLP compliant, guideline study, with minor restrictions in design and/or reporting but otherwise adequate for assessment.

Toxicity to reproduction: other studies

Additional information

no further data mandatory

Justification for classification or non-classification

Results from mechanistic studies confirmed that induction of maternal metallothionien synthesis by 2 -ethylhexanoic acid reduced distribution of zinc to the fetus and adversely affected fetal development. This study supports the hypotheses that 2-ethylhexanoic acid causes developmental toxicity indirectly and that developmental toxicity will only occur at dose levels that cause maternal liver toxicity and disrupt zinc metabolism and distribution. Based on these results, 2-ethylhexanoic acid is not likely to cause effects on fertility but is likely to be a developmental toxicant. Classification according to EU Directive 67/548/EEC with Reproduction Category 3, R63: possible risk of harm to the unborn child is required (Annex I). Classficiation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 for Reproduction toxicity is: Hazard Category 2, with the Hazard statement H361d (suspected of damaging the unborn child) (Annex VI).

Based on a read-across approach, the same classification is considered to apply for sodium 2-ethylhexanoate.