2,2,2,4,4,4,4-heptakis[(2-ethylhexanoyl)oxy]cyclodimolybdoxan-2-yl 2-ethylhexanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting 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.

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

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

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:

Based on the results of this study the parental NOAEL was 300 mg/kg/day and the F1 NOAEL was 100 mg/kg/day.

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 and 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 decrease sperm quality in males. The spermatozoa were significantly less mobile 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 600 mg/kg dose group. The birth weight 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 paralysed 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 conclusion, 2 -EHA caused impaired fertility in Wistar rats and delayed postnatal development of pups. The parental NOAEL was 300 mg/kg bw/day and the F1 NOAEL was 100 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
did not demonstrate an adverse effect on fertility, although there were some findings that indicated possible effects on the reproductive system, such as delay in mating and some apparent decrease in sperm parameters.

Justification for selection of Effect on fertility via oral route:
Supporting study of adequate quality for use in hazards assessment and risk characterisation for the purpose of supporting qualitative risk management with quantitative assessments to ensure adequate protection against the other significant health effects.

Effects on developmental toxicity

Description of key information

From the data presented it is noted that there were maternal effects in the rat at 500 mg/kg/day and above, and there were foetal findings at 250 mg/kg/day and above, plus increased wavy ribs at 100 mg/kg/day in the Pennanen study.  This difference may have reflected the longer dosing period in the Pennanen study.  Wavy ribs are a variation that is considered to regress in the post-natal period (Kast, 1994 ). 
The findings described in the fertility study (Pennanen, 1993) tend to support the conclusions of the developmental toxicity study of Pennanen.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

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 ay 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

Vehicle:

other: 2-EHA was administered as a sodium salt by mixing 2-EHA and NaOH equimolarly in drinking water.

Details on exposure:

Mated females were exposed to 2-EHA on Days 6-19 of gestation via drinking water at doses of 100, 300 and 600 mg/kg bw/day. Control animals received deionised 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.

Duration of treatment / exposure:

14-days (days 6-19 of gestation).

Frequency of treatment:

Daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0, 100, 300 and 600 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

20-21 dams per treatment group
Control: 21 dams
100 mg/kg/day: 21 dams
300 mg/kg/day: 20 dams
600 mg/kg/day: 20 dams

Control animals:

yes, concurrent no treatment

Maternal examinations:

- The animals were observed daily for clinical signs.
- Water consumption was recorded for the whole exposure period and the doses were corrected individually according to the most recent bodyweight taken on days 0, 6, 13 and 20
- Food consumption was recorded in the same gestational stage during days 13 to 16 of pregnancy.

Ovaries and uterine content:

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, external abnormalities, and placental weights.

Fetal examinations:

Individual fetal weights, skeletal anomalies and visceral anomalies.

Statistics:

Maternal, fetal, and placental weights, implantations, and living fetuses per litter were analysed by one-way analysis of variance (ANOVA). Comparisons of significant group effects were conducted using the Fisher PLSD test. The fetal bodyweight data were also evaluated using covariate analysis with litter size and sex ratio as covariates. 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 analysed by the Pearson correlation test. Pre- and postimplantation losses and group mean values of litter percentages of skeletal and visceral anomalies were analysed by the Mann-Whitney U nonparametric test. Other data were analysed by the X2 test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced maternal weight gain at 600 mg/kg/day

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Lower fetal weight at 300 and 600 mg/kg/day
Increased incidences of clubfoot at 300 and 600 mg/hg/day
Increased wavy ribs at all doses, and of extra thoracic ribs at 600 mg/kg/day
Reduced ossification at 600 mg/kg/day
Increased incidences of fetuses with dialation of the lateral ventricles at 600 mg/kg/day.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The maternal NOAEL was 300 mg/kg bw/day and the NOAEL for teratogenicity was 100 mg/kg bw/day. Maternal effects included slightly lower pregnancy rate and reduced body weights. Teratogenic effects included reduced fetal weights and skeletal variations.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

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

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
Supporting study of adequate quality for use in hazards assessment and risk characterisation for the purpose of supporting qualitative risk management with quantitative assessments to ensure adequate protection against the other significant health effects.

Justification for classification or non-classification

A Classification of GHS Category 2 is considered appropriate for reproductive toxicity (adverse effects on fertility and adverse effects on development of the offspring) of HCAT due to the effects of 2-ethylhexanoic acid.