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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
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
Justification for study design:
This one-generation study from peer-reviewed literature meets scientific principles and is judged adequate to fulfil the REACH information requirement for toxicity to reproduction

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 99.5%
Supplier: Merck (Darmstadt, FRD)

Test animals

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

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
other: drinking water supplemented with NaOH (equal amounts as 2-EHA)
Details on exposure:
Rats were given the test substance in their drinking water as a sodium salt by mixing equivalent amounts of 2-EHA and NaOH. Concentrations of 2-EHA 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-EHA 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 animals)

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-EHA-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

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-EHA-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-EHA-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-EHA 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:
In summary, 2-ethylhexanoic acid increased time to mating, inhibited implantation, and tended to decrease fertility in Wistar rats at 600 mg/kg. At this same dose level 2-EHA decreased pup weights during lactation and at and above 300 mg/kg delayed postnatal development of pups as noted in the reflex and physical parameters evaluated.
Executive summary:

Reproductive toxicity of 2-ethylhexanoic acid (2-EHA) was studied in Wistar rats. The animals (24 animals per sex per group) were given 2-EHA as a sodium salt in drinking water at daily doses of 100, 300, or 600 mg/kg. Control animals received plain water. Male rats were exposed to 2-EHA for 10 weeks and females for 2 weeks prior to mating, both sexes during the mating period and females during the entire gestation and lactation period. 2-EHA caused a slight but dose-dependent decrease in fertility, time to mating increased at 300 and 600 mg/kg and even total infertility ensued. 2-EHA slightly decreased sperm quality in males. The spermatozoa were significantly less motile at 100 and 600 mg/kg and abnormal sperm occurred more frequently at the two highest dose levels. The average litter size was reduced by 16% in the dose group receiving 600 mg/kg. The birth weights of the pups were unaffected but the body weight gain was transiently slower during lactation at 600 mg/kg. Several pups appeared abnormal (kinky tail, lethargic, slightly paralyzed legs) and the physical development assessed by several landmarks (opening of eyes, eruption of teeth, hair growth) and reflexes (grip reflex, cliff avoidance) was delayed at 300 and 600 mg/kg. In another experiment, a single dose of 600 mg/kg 2-EHA was given to pregnant females by gavage on Gestational Day 4, 5, 6, or 7 and the number of implantations were counted on Gestational Day 10. Administration on Day 6 decreased the number of implantations and caused resorptions. In conclusion, 2-ethylhexanoic acid caused impaired fertility in Wistar rats and delayed postnatal development of pups. The reduced fertility might result from disturbed implantation in uterus and the retarded development of pups from teratogenicity and pre- and postnatal toxic effects of 2-EHA.