Tin bis(2-ethylhexanoate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Stannous chloride and 2-ethylhexanoic acid [hydrolysis products of tin bis(2 -ethylhexanoate)] have been tested in 90 day toxicity studies in rats and mice and there were no effects noted on reproductive organs. 

Reproductive and developmental toxicity studies are not available for tin bis(2 -ethylhexanoate). Tin bis(2-ethylhexanoate) has not been evaluated for potential adverse effects in OECD 422 screening studies.

Rats were exposed to 2-ethylhexanoic acid in a one generation study, via drinking water,at 0, 100, 300, or 600 mg/kg bw/day. No mortality was observed. The relative epididymal weights in high-dose males were significantly increased, but no histologic changes were noted. A slight, but not statistically significant, increase in the number of abnormal sperm was reported in the two highest dose groups; however, the incidence per animal was not provided. A dose dependent delay in fertility was observed,and the mean litter size in high-dose females was significantly reduced. The mean pup weights in the high-dose group were significantly lower on postnatal day 7 and 14.

Development of the eyes, teeth and hair appeared to be slightly later in the pups from the high-dose groups; the significance of this finding is unclear since no data were presented on the length of gestation in treated and control dams. The high-dose of 600 mg/kg bw/day significantly reduced overall water consumption and body weights in female animals. The NOEL for reproductive effects in parental animals was 300 mg/kg-bw/day; this effect occurred in the presence of maternal toxicity. The NOEL for F1 offspring was 100 mg/kg-bw/day.

Short description of key information:
Data are considered to be fulfilled by studies on hydrolysis products. No effects were noted on reproductive organs.
Rats were exposed to 2-ethylhexanoic acid in a one generation study. EHA induced increased epididymal weights, but no histological changes were observed. A dose-dependent delay in fertility was observed, and the mean litter size in high-dose females was significantly reduced. The parental NOAEL was 300 mg/kg bw/day; this effect occurred in the presence of maternal toxicity.

Link to relevant study records

Reference

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:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

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

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

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

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-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.

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.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Delay in fertility

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

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:

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rat

Effects on developmental toxicity

Description of key information

Data are considered to be fulfilled by studies on the hydrolysis products of tin bis(2-ethylhexanoate), tin(II) and EHA:

 

In a reliable prenatal development study (Pennanen, 1992) in Wistar rats 100, 300 and 600 mg/kg of EHA was administered via drinking water to pregnant dams from gestational days 6 to 19. On gestational day 20, the fetuses were examined for external, visceral, and skeletal malformations and variations. Skeletal malformations (clubfoot, absence of fibula, polydactyly) were observed in all treatment groups, with the incidence increasing in a dose-dependent way. Also visceral malformations were noted at all dose levels, however, severity and incidence was less pronounced than the skeletal effects. At the highest dose tested (600 mg/kg), a 5 to 8% decrease in the mean fetal body weight both in males and females was noted in presence of a near-term body weight decrease of dams of 11 %. Due to the skeletal malformations at 100 mg/kg, a NOAEL could not be established. Benchmark dose analysis provided a lower confidence limit (BMDL) of 35 mg/kg, based on total skeletal malformations per litter.

 

In a supporting prenatal development study (Hendrickx, 1993), slight fetotoxicity (reduced ossification and increased incidence of skeletal variations) was noted in the offspring of rats exposed to gavage doses of EHA in oil of 250 mg/kg/d and above (NOEL = 100 mg/kg/d) and also in the offspring of rats exposed to EHA in the drinking water at an equivalent doses of 300 mg/kg/d and above (NOEL= 100 mg/kg/d).

 

Stannous chloride and EHA [hydrolysis products of tin bis(2-ethylhexanoate)] have been tested in 90-day toxicity studies in rats and mice and there were no effects noted on reproductive organs. Further, stannous chloride has been evaluated for potential adverse developmental effects in studies in four species (US FDA 71 -33). No adverse effects on development were found at doses up to and including 50 mg/kg/d during gestation in these studies.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Principles of method if other than guideline:

20-21 pregnant females/dose were exposed to 2-EHA in their drinking water at doses of 100, 300, or 600 mg/kg/day on Days 6-19 of gestation. Control animals received vehicle water. The fetuses were examined (on Gestational Day 20) for external, visceral, and skeletal malformations and variations.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

2-ethylhexanoic acid (99.5%) was obtained from Merck (Darmstadt, Germany).

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals and animal husbandry:
Virgin female Han: Wistar rats (National Laboratory Animal Center, University of Kuopio, Finland) weighing 200-220 g (12- to 14-weeks old) and male rats of the same strain 360-380 g (16-weeks old) were used for this study. Rats were housed in stainless-steel wire mesh cages and kept under a photoperiodic cycle of 12 hr light/12 hr dark in an air-conditioned animal room. Temperature and relative humidity of the room were maintained at 21 ± 1°C and 55-65%, respectively. Animals were housed three per cage before mating and individually during gestation. The animals received commercial rat chow (R3-EWOS, Sodertalje, Sweden) and tap water ad libitum except 2-EHA solution during the experiment.

Route of administration:

oral: drinking water

Details on exposure:

Mated females were exposed to 2-EHA on Days 6-19 of gestation via the drinking water at doses of 100, 300, and 600 mg/kg 2-EHA. 2-EHA was administered as a sodium salt by mixing 2-EHA and NaOH equimolariy in drinking water. Control animals received deionized water. Water consumption was recorded for the whole exposure period and the doses were corrected individually according to the most recent body weight taken on Days 0, 6, 13, and 20.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Female rats were paired overnight with males and the vaginal smears were checked the next morning for the presence of sperm. Sperm-positive female rats (109 females in total) were randomly assigned into a control group and three treatment groups by the stratified body weight procedure on Day 0 of gestation (sperm-positive day). The final group size was 20 or 21 dams. The females were identified by tattoos and housed individually.

Duration of treatment / exposure:

Mated females were exposed to 2-EHA on Days 6-19 of gestation.

Frequency of treatment:

Daily via drinking water.

Duration of test:

20 days

Dose / conc.:

100 other: mg/kg/day

Dose / conc.:

300 other: mg/kg/day

Dose / conc.:

600 other: mg/kg/day

No. of animals per sex per dose:

20-21 pregnant females/dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

The animals were observed daily for clinical signs. Food consumption in the same gestational stage was recorded per cage during Days 13 to 16 of pregnancy.

Ovaries and uterine content:

On Day 20 of pregnancy, the dams were euthanized with a mixture of carbon dioxide and oxygen and the visceral cavity was cut open. The uterine contents and ovaries were examined for the number of corpora lutea, weight of uterus with its contents, number of implantation sites, sex and number of live and dead fetuses, number of early and late resorptions, individual fetal weights, external abnormalities, and placental weights.

The empty uteri were placed into a solution of ammonium sulfide to visualize hemorrhagic alterations in implantation sites of very early resorptions (Salewsky, 1964). A limited necropsy focusing on the thoracic and abdominal cavities was performed for each dam.

Fetal examinations:

Every second living fetus was fixed in ethanol and cleared in potassium hydroxide for double staining of cartilage and bone with Alcian blue and Alizarin red S (Inouye, 1976; Kimmel and Trammel, 1981) to detect skeletal anomalies. All fetuses with clubfoot were subjected to analysis of skeletal anomalies. Other fetuses were fixed in Bouin's solution and subjected to free-hand sectioning in order to detect visceral anomalies (Wilson, 1965).
Abnormalities detected in fetuses were classified as variations or malformations. Variations were defined as common structural changes and malformations as permanent abnormal structural (visceral or skeletal) changes (Black and Marks, 1986).
Renal pelvic dilatation and dilatation of the fetal ureters were assessed using a slightly modified grading method of Kavlock etal.(\ 987). Increasing scores of 1 to 4 for pelvic dilatation and from 1 to 5 for ureter dilatation were used. Only when scored >3 were dilatations considered abnormal. For each litter, preimplantation loss and postimplantation loss were calculated as a percentage, according to the formulas:
Preimplantation loss: (a - b)/a X 100
Postimplantation loss: (b - c)/b X 100
where "a" is number of corpora lutea, "b" is total number of implantations, and "c" is number of live fetuses.
We define the terms "embryotoxicity," "fetotoxicity," "teratogenicity," and "maternal toxicity" as described in Black and Marks (1986) and "developmental toxicity" to mean death, structural abnormality, altered growth, or functional deficiency during development (EPA, 1991).

Statistics:

Maternal, fetal, and placental weights, implantations, and living fetuses per litter were analyzed by one-way analysis of variance (ANOVA). Comparisons of significant group effects were conducted using the Fisher PLSD test. The fetal body weight data were also evaluated using covariate analysis with litter size and sex ratio as covariates but the test was not significant. Dose-response relationships for maternal and fetal body weights and affected fetuses and fetuses with skeletal or visceral malformations (group mean of litter percentages) were analyzed by the Pearson correlation test. Pre- and postimplantation losses and group mean values of litter percentages of skeletal and visceral anomalies were analyzed by the Mann-Whitney U nonparametric test. Other data were analyzed by the x^2 test.

Clinical signs:

no effects observed

Description (incidence and severity):

The doses of 2-EHA employed did not cause visible maternal toxicity (clinical signs or behavioral changes).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of the dams at 600 mg/kg decreased slightly (around 10%) from Day 13 onward. At termination the mean body weight of dams was 11 % (p < 0.001) lower at 600 mg/kg. The weight of the gravid uterus did not differ significantly between groups, but the corrected maternal body weight gain was reduced (53.8%, p < 0.001) at 600 mg/kg compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption (g/kg body wt) did not differ significantly between groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

At the 600 mg/kg dose level, the consumption of 2-EHA containing drinking water also exhibited some decrease from Day 6 onward, being maximally 20% less than in controls.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weight of the gravid uterus did not differ significantly between groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes were noted upon necropsy in the organs of the dams.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The placental weight was slightly reduced (10.2%, p < 0.001) in both 300 and 600 mg/kg dose groups.

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in pre- and postimplantation loss.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

2-EHA did not cause resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

2-EHA affected neither the number of implantations nor that of living fetuses.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate was slightly but, not significantly, decreased in the 300 and 600 mg/kg dose groups as compared to the control group (67 and 67% vs 84%).

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in the number of corpora lutea.

Key result

Remarks on result:

other: Maternal NOEL/NOAEL values were not stated in this study.

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean fetal body weight/litter showed a marginal decrease in males (5.6%, p < 0.001) and in females (8.6%, p < 0.001) at 600 mg/kg. The mean body weight of female fetuses was slightly decreased (5.7%, p < 0.001) also at 300 mg/kg.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

All fetuses in treated and control groups were alive.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant differences were noted in sex ratio.

External malformations:

not specified

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Clubfoot was the most severe skeletal malformation. It occurred at all dose levels, more notably at 300 and 600 mg/kg. Other skeletal malformations included abnormal cartilage in ankle (acampsia, strongly cartilagenous ankle, no flexure of the tarsal joints), absence of fibula, polydactyly, flabby legs (external, slightly paralyzed), scoliosis, lordosis, and extra thoracic ribs. With the exception of polydactyly and extra thoracic ribs, they did not occur spontaneously in control animals. Flabby legs, mild scoliosis, and lordosis occurred even at 100 mg/kg.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Only a few visceral malformations were found altogether but all cases were in 2-EHA-treated animals. There was one hydronephrosis at the dose level of 600 mg/kg. Kidney hypoplacia and rudimentary adrenal were also observed in the 300 and 100 mg/kg group respectively. A convoluted retina was also observed at the dose level of 600 mg/kg.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The incidence of several skeletal variations was increased in 2-EHA-treated animals, including the 100 mg/kg dose group. The frequency was highest at 600 mg/kg. The reduced ulna and lumbar ossification as well as the cases of twisted hind legs are in concordance with the more drastic malformations in feet.
Among visceral variations, curved or dilated ureter, congestion in the kidney cortex and displacement of the kidney occurred only in 2-EHA groups but pelvic dilatation was common in all animals. The incidence of slightly dilated brain ventricles was increased in the dose groups of 300 and 600 mg/kg. The frequency was higher in females than that in males (9 and 4 at 300 mg/kg, 15 and 9 at 600 mg/kg). The degree of dilatation of brain ventricles is known to reflect the developmental stage of the conceptus and the data fit well with the observation that the body weights of fetuses were slightly retarded at 600 mg/kg and in female fetuses also at 300 mg/kg.

Key result

Dose descriptor:

LOAEL

Effect level:

100 other: mg/kg/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: eye

other: Dilation of brain ventricles

Description (incidence and severity):

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. Clubfoot does not occur spontaneously in rats and it must be regarded as a definitive indicator of teratogenicity. Most of the skeletal variations occurred also in control animals but with a lower frequency. In the present study variations already increased at doses which did not cause visible maternal toxicity. The skeletal variations are known to reflect to some extent delays in the stage of normal development. Meanwhile, 2-EHA did not markedly affect development of visceral tissues. There was only one case of hydronephrosis at the highest dose level and other minor changes were scattered without dose dependence. All cases, however, occurred in 2-EHA-treated animals.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 other: mg/kg/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

2-EHA affected development of the embryos/fetuses at all dose levels. The number of affected fetuses (having skeletal or visceral malformation) increased in a dose-dependent manner (p < 0.001) being 4.9, 8.9, and 15.3% per litter in 2-EHA groups vs 2.4% in the control group.

Conclusions:

The results indicate that 2-EHA is teratogenic, i.e., increases malformations in Wistar rats at doses which are apparently not maternally toxic, embryotoxic, nor frankly fetotoxic. There was a dose-dependent increase in the frequency of skeletal malformations starting at a dose level of 100 mg/kg. The dose 300 mg/kg was slightly fetotoxic in females as indicated by the reduced body weight in female fetuses. The 600 mg/kg dose was slightly fetotoxic in both sexes and maternally toxic as judged by the reduction of the body weights of the fetuses and dams. With regard to teratogenicity, the lowest-observed-adverse-effect-level (LOAEL) was 100 mg/kg and the NOAEL (no-observed-adverse-effect level) was lower than the lowest dose administered.

Executive summary:

The developmental toxicity of 2-ethylhexanoic acid (2-EHA), a wood preservative and a mammalian metabolite of di-(2-ethylhexyl) phthalate was examined in Wistar rats (20-21 pregnant females/dose). Mated animals were exposed to 2-EHA in their drinking water at doses of 100, 300, or 600 mg/kg/day on Days 6-19 of gestation. Control animals received vehicle water. The

fetuses were examined (on Gestational Day 20) for external, visceral, and skeletal malformations and variations.

2-EHA was marginally toxic to the dams at 600 mg/kg, but not at lower doses, since the mean near term body weight was reduced by 11%. This dose level was also slightly fetotoxic as indicated by a 5 to 8% decrease in the mean fetal body weight both in males and females. No treatment-related effects were observed in the number of implantations or live fetuses. At doses of 100 mg/kg and above, 2-EHA caused skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected. The number of affected fetuses increased in a dose-dependent way (4.9,8.9, and 15.3% of treated offspring at 100, 300, and 600 mg/kg/day, respectively, vs 2.4% control). See attachment Pennanen 1992 - Table 2 and Table 3.

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. With regard to teratogenicity, the lowest-observed-adverse-effect-level (LOAEL) was 100 mg/kg and the NOAEL (no-observed-adverse-effect level) was lower than the lowest dose administered.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

2 -Ethylhexanoic acid has been tested in 90 day toxicity studies in rats and mice, and there were no effects noted on reproductive organs.

Skeletal and visceral malformations were noted in the offspring of rats exposed to gavage doses of EHA in drinking water of 100 mg/kg/d and above (LOAEL = 100 mg/kg/d). Based on this effect on development, a classification as toxic to reproduction Category 2 (repr. 2) is applied. This classification is already a harmonised classification, as it is derived from the harmonised classification of the hydrolysis product 2-ethyl hexanoic acid.