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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Materials, methods and results well described/presented in text and tables

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Effects of 2-Ethylhexanoic acid on reproduction and postnatal development in Wistar rats
Author:
Pennanen, S. et al.
Year:
1993
Bibliographic source:
Fundamental and Applied Toxicology 21: 204 - 212.
Reference Type:
publication
Title:
TOXICOLOGICAL EVALUATION No. 275 2-ethylhexanoic acid 06/00
Author:
BG Chemie
Year:
2000
Bibliographic source:
BG Chemie InfoCenter TOXIKOLOGISCHE BEWERTUNGEN (www.bgchemie.de)

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
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.
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.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexanoic acid
EC Number:
205-743-6
EC Name:
2-ethylhexanoic acid
Cas Number:
149-57-5
Molecular formula:
C8H16O2
IUPAC Name:
2-ethylhexanoic acid
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexanoic acid (2-EHA)(obtained from Merck Darmstadt, FRD)
- Analytical purity: 99.5%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Han:Wistar rats
- Source: National Laboratory Animal Center, University of Kuopio, Finland
- Age at study initiation:
Experiment 1:
males: 5 - 6 weeks old; females: 9 - 10 weeks old;
Experiment 2:
males: 16 weeks old; females: 15 - 16 weeks old;
- Weight at study initiation:
Experiment 1:
males: 125 ± 25 g; females: 200 ± 20 g
Experiment 2:
females: 260 ± 10 g
- Housing: animals were housed in stainless-steel wire mesh cages; litters were caged with their dams for 3 weeks following birth.
- Diet (ad libitum, except during exposure to 2-EHA solution): commercial rat chow (R3-EWOS, Södertälje, Sweden)
- Water (ad libitum, except during exposure to 2-EHA solution): tap water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 1°C
- Relative humidity: 55 - 65%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: oral in drinking water (Experiment 1) or by gavage (Experiment 2)
Vehicle:
other: NaOH
Details on exposure:
Experiment 1:
The rats were given 2-EHA as a sodium salt in drinking water (by mixing equivalent amounts of 2-EHA and NaOH).
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 according to the most recent body weights and water consumption. When more than one animal was housed per cage, a mean body weight was used for dose calculation. During the mating period the body weight of female was used.

Experiment 2:
Females were given 2-EHA as a sodium salt (10 mL/kg).
Details on mating procedure:
Experiment 1:
- M/F ratio per cage: each male was cohabited with one female of the same treatment group
- Length of cohabitation: the female was separated from the male when the pregnancy was confirmed (oestrus cycle stopped in dioestrus).
- Proof of pregnancy: sperm in vaginal smear
- After successful mating each pregnant female was caged: at the end of the mating period (maximum 21 days) bred females were housed individually.

Experiment 2:
- Length of cohabitation: virgin female rats were paired overnight with males
- Proof of pregnancy: the vaginal smears were checked the next morning for the presence of sperm.
- After successful mating each pregnant female was caged: the females were housed individually
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Experiment 1.
Male rats were exposed to 2-EHA for 10 weeks (3 animals per cage) and females for 2 weeks prior to mating (3 animals per cage), both sexes during the mating period (1 male and female per cage) and females during the gestation (1 animal per cage) and lactation period (one litter per cage)
Experiment 2:
Mated females were given a single dose of the test substance in the morning of Day 4 (group A), Day 5 (group B), Day 6 (group C), Day 7 (group D) of gestation
Frequency of treatment:
Experiment 1:
daily
Experiment 2:
once
Details on study schedule:
not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 100, 300, or 600 mg/kg (Experiment 1)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
600 mg/kg (Experiment 2)
Basis:
actual ingested
No. of animals per sex per dose:
Experiment 1:
24 males / 24 females
Experiment 2:
Gestational day 4 (group A): 5 mated females
Gestational day 5 (group B): 6 mated females
Gestational day 6 (group C): 6 mated females
Gestational day 7 (group D): 12 mated females
Control animals:
other: concurrent no treatment (Experiment 1)... (see attached file)
Details on study design:
Experiment 1:
- Animal assignment: the animals were randomly assigned into a control group and three treatment groups by stratified body weight procedure.
Experiment 2:
- Animal assignment: sperm-positive female rats were randomly assigned into three groups by the stratified body weight procedure on Day 0 of gestation (sperm-positive day).
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
EXPERIMENT 1:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: clinical signs

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Food consumption were recorded weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: water consumption was recorded for each cage for the whole exposure period
Oestrous cyclicity (parental animals):
EXPERIMENT 1:
The females had a regular oestrus cycle for 2 weeks prior to mating and during the mating period. The oestrus cycle was checked by evaluation of vaginal smears microscopically after staining with 0.25% toluidine blue O.
Sperm parameters (parental animals):
EXPERIMENT 1:
- For evaluation of sperm density, motility, and morphology, the left cauda epididymis was dissected, weighed, and minced in 7 mL of phosphate-buffered saline with 0.1% glucose (pH 7.4, 37°C).
- The number of spermatozoa and their motility were determined within 30 minutes after dissection of the tissue.
- The qualitatively sperm morphology was determined.
- Testes and the right epididymis were weighed
Litter observations:
EXPERIMENT 1:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, the number of pups per litter was randomly reduced to 8 using equal sex distribution whenever possible. Culled pups were examined externally, euthanized, and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: the litters were examined for litter size, including stillbirths, and any gross anomalies. Pups were individually weighed on postnatal days 0, 4, 7, 14, and 21. Pups were examined every other day from Postnatal day 1 onwards and the appearance of the following developmental parameters were recorded: pinna reflex, placing reaction, righting reflex in 5 seconds, cliff avoidance in 5 seconds, approach/avoidance, ipsilateral flexor reflex (hind toe), grip reflex in 5 seconds, air righting, opening of eyes, teeth eruption, and hair growth (graded on a scale from 1 to 7 (Paget and Thomson, 1979)*.

*Reference:
- Paget, G.E., and Thomson, R. (1979). The evaluation of physical and functional 8behavioural) development in rodent neonates. In Standard operating Procedure in Pathology including Developmental Toxicology and Quality Assurance, pp. 447 - 458. MTP press Limited, Lancester.
Postmortem examinations (parental animals):
EXPERIMENT 1:
SACRIFICE / HISTOPATHOLOGY / ORGAN WEIGHTS
- Male animals and nonpregnant females: at the end of the mating period males and nonpregnant females were euthanised with mixture of carbon dioxide (70%) and oxygen (30%).
- In all nongravid females, ovaries, uterus, cervix uteri, and vagina were dissected and fixed in 10% neutral-buffered formalin for histopathological assessment. The ovaries were weighed before fixation. In males, the testes, the right epididymes, seminal vesicle, prostate, and coagulating gland were fixed in 10% neutral-buffered formalin. Histopathological assessment was donse in all nongravid females and five randomly selected males/group.
- On Postnatal Day 21, dams with litters were euthanized, and examined macroscopically for any pathological changes. Ovaries, uterus, cervix uteri, and vagina were dissected, ovaries weighed, and all these tissues fixed in 10% neutral-buffered formalin. Histopathology was done in five randomly selected animals/group.

EXPERIMENT 2:
SACRIFICE / HISTOPATHOLOGY / ORGAN WEIGHTS
On Day 10 of gestation, the dams were euthanized with a mixture of carbon dioxide (70%) and oxygen (30%) and the uterus was weighed, opened, and placed into a solution of ammonium sulfide to visualise haemorrhagic alterations in implantation sites of very early resorptions (Salewski, 1964)*.

*Reference:
- Salewski, E. (1964). Färbemethoden zum makroskopischen nachweis von inmplantations-stellen am uterus der ratte. Naunyn Schiedebergs Arch. Exp. Pathol. Pharmakol. 247, 367.
Postmortem examinations (offspring):
EXPERIMENT 1:
GROSS NECROPSY
On Postnatal Day 21, all pups were examined externally and euthanized without further examination.
Statistics:
Body weights (adult males, adults females, litters, pups), organ weights, food and water consumption, number of live pups in litter, male fertility data, and data on pup development were analysed by one-way analyses of variance. Comparisons of significant group effects were conducted using Fisher PLSD test or Scheffe's test. The observations on pups (litter percentage) were analysed by Scheffe's test and the dose-response relationship by the Pearson correlation test.
Reproductive indices:
Pregnancy index: number of pregnant females/number of mated females
Offspring viability indices:
Lactation index: number of pups live on postnatal day 21/postnatal day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

EXPERIMENT 1:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
2-EHA did not cause any visible clinical signs of toxicity or mortality in adult rats at any dose group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption (g/100 g body weight/day) did not differed significantly between groups prior to mating nor during the mating period.
- 2-EHA did not affect the body weights of males
- 9 to 12% decrease in the body weight of females at 600 mg/kg from gestational day 7 onwards. The gestational weight gain (gestational days 0 - 21) was statistically significantly lower (p < 0.01) at that dose. During lactation, the weight difference disappeared and the body weights of females at the time of sacrifice (postnatal day 21) were already similar.

WATER CONSUMPTION (PARENTAL ANIMALS)
- Liquid consumption (g/100 g body weight/day) did not differed significantly between groups prior to mating nor during the mating period.
- Liquid consumption by pregnant females was slightly reduced (14%, p < 0.05) at the highest dose level compared to that of the control group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- 2-EHA caused a dose-dependent delay in fertilization.
- Control animals conceived in the course of two oestrous cycles while for several 2-EHA-treated animals three or four cycles were needed.
- All nonpregnant animals belonged to the 2-EHA-treated groups.
- Six males at 600 mg/kg and three males at the 300 mg/kg dose group copulated occasionally with females in dioestrus while all control and the lowest dose group males copulated in oestrus.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- The relative weights of the right epididymides (epididymis/body weight) were increased by 12% (p < 0.05) at 600 mg/kg. Also the absolute weights of epididymides were increased in this dose but not statistically significant.
- The relative weights of testes were similar in the control and 2-EHA-treated groups.
- 2-EHA affected sperm quality slightly but the effect was not uniformly dose-dependent.
- Total number of spermazozoa in the causa epididymis was 14% lower at the highest dose (not statistically reduced)
- Proportion of motile spermatozoa decreased by 37% at 100 mg/kg and 22% at 600 mg/kg (p< 0.05)
- Share of nonmotile spermatozoa was highest in the 100 mg/kg group
- Number of animals with morphologically abnormal spermatozoa (most common: agglutinations and abnormal heads of spermatozoa) was increased at the two highest dose levels (not statistically significant)
- Abnormal heads included short heads, straight heads (heads without hook), and amorphous heads and they occurred in 13 and 21% of the animals at 300 and 600 mg/kg, respectively.
- Abnormal spermatozoa with looped and straight tails were also seen in the spermatozoa of the exposed animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- pregnancy index did not differ significantly in 2-EHA-treated groups from controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- The relative weights of ovaries were similar in the control and 2-EHA-treated groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- In the autopsy of the adult males, the nongravid females, and the dams the tissues appeared macroscopically normal.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- slight dilatation of the lumen in the uterus and epithelial hyperplasia in vagina were observed in two of five dams both at 300 and 600 mg/kg. Cervix uteri and ovaries were normal.
- sex organs of nongravid females (ovaries, uterus cervix, vagina) and males (epididymis, testes, seminal vesicle, prostate, coagulating gland) appeared histologically normal in all groups.

EXPERIMENT 2:
The number of implantations was lowest after administration of 2-EHA on gestational day 6 and there were resorptions in 80% (4/5) of the animals exceeding the historical control value of 0.6 resorptions per litter for this strain in the laboratory. There was total resorption in 40% (2/5) of the animals treated on gestational day 6. Effects were less severe after administration of 2-EHA on gestational day 7. Although there were resorptions, their incidence was smaller and total resorptions were absent.
The results suggest that 2-EHA disturbs most the stages of implantation timed on gestational day 6.

Please also refer to the results presented in "Attached background material" below.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the results that the test substance increased time to mating, inhibited implantation, and tended to decrease fertility in the rats at 600 mg/kg.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

EXPERIMENT 1:
VIABILITY (OFFSPRING)
- The number of stillbirths did not differ between groups while postnatal deaths tended to be more common in 2-EHA-treated animals (changes in postnatal deaths not dose related)

CLINICAL SIGNS (OFFSPRING)
- In the live 2-EHA-exposed pups, the frequency of lethargy, hematomas, and abnormally thin hair was higher at the two highest dose levels
- Kinky tail showed a dose-dependent increase (p < 0.001)
- Frequency of abnormal legs was higher in 2-EHA-treated animals the pups with leg anomalia were cannibalized by the dams soon after birth.
- 2-EHA delayed physical development of the pups
- Ears raised later in the dose groups 300 and 600 mg/kg
- Eye opening, eruption of teeth, and hair growth occurred significantly later (p < 0.01) at 600 mg/kg compared to control.
Development of the grip and cliff avoidance reflexes was delayed, more clearly in males than in females.
- Pinna reflex, placing reaction, approach/avoidance, and ipsilateral flexor reflex were not affected.

BODY WEIGHT (OFFSPRING)
- body weights of the pups were similar soon after birth but decreased transiently at 600 mg/kg during lactation.

GROSS PATHOLOGY (OFFSPRING)
- one male pup of the highest dose group had a mass in the left testis and left epididymis was missing.

OTHER FINDINGS (OFFSPRING)
- The average litter size was reduced by 16% (p < 0.05) at 600 mg/kg.
- At the age of weaning the lactation index did not differ significantly between groups

Please also refer to the results presented in "Attached background material" below.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on: lethargy, hematomas, and abnormally thin hair was well as kinky tail, reduced litter size

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (P-generation): 300 mg/kg bw/d (nominal)
According to the authors, 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.