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Key value for chemical safety assessment

Effects on fertility

Description of key information

In a published oral reproductive toxicity study using wistar rats, 2 -ethylhexanoic acid 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.

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.

The results of a published oral developmental toxicity / teratogenicity study in wistar rats indicate that 2-EHA is teratogenic, i.e., increases malformations in Wistar rats at doses which are apparently not maternally toxic, embryotoxic, or fetotoxic. There was a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg. 300 mg/kg was slightly fetotoxic in females as indicated by the reduced body weight in female fetuses.

2-EHA affected particularly skeletal development of the fetuses. The spectrum of changes ranged from skeletal variants to clubfoot, including a few cases of retarded lumbar and ulna ossification.

At doses of 100 mg/kg and above, 2-EHA caused dose-dependent skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected.

These results indicate that 2-EHA is teratogenic in rats already at doses which are not yet maternally toxic. The skeleton appears to be the main target of 2-EHA in developing rats.

On this basis a classification or Repro 2 is assigned to 2-hydroxypropyl)trimethylammonium 2-ethylhexanoate.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
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
Specific details on test material used for the study:
Purity: 99.5%
Supplier: Merck (Darmstadt, FRD)
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 with 2-EHA sodium salt
Details on exposure:
Rats were exposed via drinking water as a sodium salt 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:
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 via drinking water
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Positive control:
No
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:
- 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.

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):
At Sacrtifice
- 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

At Gross Necropsy
- Gross necropsy consisted of examination of any pathological changes

Organ Weights / Histopathology
- 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.
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.
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.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Delay in fertility
Details on results:
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.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lower litter size, lower body weights and delayed physical development
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: delayed postnatal development of pups
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
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:

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Purity 99.5% (Merck)
Species:
rat
Strain:
Wistar
Route of administration:
oral: drinking water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Day 6-19 of gestation
Frequency of treatment:
Daily via drinking water
Duration of test:
20 days to necropsy
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20-21 pregnant Females
Control animals:
yes, concurrent no treatment
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity - decreased body weight (10%) and water consumption (20%)
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: forelimb
skeletal: hindlimb
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Results of this study indicate that 2-EHA is teratogenic, showing a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg
Executive summary:

The results indicate that 2-EHA is teratogenic, i.e., increases malformations in Wistar rats at doses which are apparently not maternally toxic, embryotoxic, or fetotoxic. There was a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg. 300 mg/kg was slightly fetotoxic in females as indicated by the reduced body weight in female fetuses.

2-EHA affected particularly skeletal development of the fetuses. The spectrum of changes ranged from skeletal variants to clubfoot, including a few cases of retarded lumbar and ulna ossification.

At doses of 100 mg/kg and above, 2-EHA caused dose-dependent skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected.

These results indicate that 2-EHA is teratogenic in rats already at doses which are not yet maternally toxic. The skeleton appears to be the main target of 2-EHA in developing rats

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed

Justification for classification or non-classification

In a published oral reproductive toxicity study using wistar rats, 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.

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.

The results of a published oral developmental toxicity / teratogenicity study in wistar rats indicate that 2-EHA is teratogenic, i.e., increases malformations in Wistar rats at doses which are apparently not maternally toxic, embryotoxic, or fetotoxic. There was a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg. 300 mg/kg was slightly fetotoxic in females as indicated by the reduced body weight in female fetuses.

2-EHA affected particularly skeletal development of the fetuses. The spectrum of changes ranged from skeletal variants to clubfoot, including a few cases of retarded lumbar and ulna ossification.

At doses of 100 mg/kg and above, 2-EHA caused dose-dependent skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected.

These results indicate that 2-EHA is teratogenic in rats already at doses which are not yet maternally toxic. The skeleton appears to be the main target of 2-EHA in developing rats.

On this basis a classification or Repro 2 is assigned to 2-hydroxypropyl)trimethylammonium 2-ethylhexanoate.