Sodium 2-ethylhexanoate

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Effects of 2-Ethylhexanoic Acid on Reproduction and Postnatal Development in Wistar Rats

Author:

Pennanen S, Tuovinen K, Huuskonen H, Kosma V, Komulainen H

Year:

1993

Bibliographic source:

Fundamental and Applied Toxicology 21 (1993) 204-212

Report date:

1993

Materials and methods

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

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanoic acid

EC Number:

205-743-6

EC Name:

2-ethylhexanoic acid

Cas Number:

149-57-5

IUPAC Name:

2-ethylhexanoic acid

Details on test material:

Purity: 99.5%
Supplier: Merck (Darmstadt, FRD)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Delay in fertility

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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 levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.