2-ethylhexanoic acid

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Feb 2015 - 03 Dec 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 443

Deviations:

yes

Remarks:

the deviations are considered not to have affected the validity and integrity of the study.

GLP compliance:

yes

Limit test:

no

Justification for study design:

ECHA requirement

Test material

Test material information

Constituent 1

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

Details on test material:

- Name of test material (as cited in study report): 2-Ethylhexanoic acid
- Molecular formula (if other than submission substance): C8H16O2
- Molecular weight (if other than submission substance): 144.21
- Physical state: Liquid, colourless to yellow
- Analytical purity: 99.6%
- Lot/batch No.: 15667956P0
- Expiration date of the lot/batch: 25 July 2016
- Storage condition of test material: ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: (P) 9 weeks females; 10 weeks (males)
- Weight at study initiation: (P) Males: 373.39 - 375.26 g; Females: 200.68 - 203.17 g;
- Housing: During the premating period, the animals were, as far as possible, housed in groups of 4/sex. For mating, one male and one female
were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack.After allocation to the
cohorts at weaning on PN day 21, the F1 animals were housed in groups of 5/sex/cage, except for the animals of cohort 2B that were housed per
lot (animals born on the same date) per dosing group until sacrifice on approximately PN day 22.
- Diet: ad libitum; cereal-based rodent diet (VRF-1) SDS, Witham, England
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65%; due to technical reasons, in animal room 5.1.08 of Cohort 1B animals, the relative humidity was higher than 65% from
29 June 2015 to 6 July 2015
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The intake of the test item per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body
weight at the end of the pertaining period

DIET PREPARATION
- Rate of preparation of diet (frequency)::The diets were mixed in a mechanical blender. Fresh batches of the experimental diets were prepared once
per month and stored in a freezer (<-18°C) in plastic bags in portions sufficient for three or more days.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurs of two weeks were elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 (GD 0)
- After successful mating each pregnant female was caged : individually for the birth and rearing of their pups

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated method of analysis for detection of the test item in the carrier was used as was also applied in the preceding DRF study with the same test
item. The test item in the diets was analysed by Gas Chromatography – Flame Ionization Detection (GC-FID) after extraction from the diet.
As homogeneity and stability were already demonstrated under similar conditions in the DRF study, analysis of the experimental diets was limited to
confirmation of concentration.The analytical report concludes that the concentration of the test substance was not always close (i.e. 90-110%) to the intended concentration for all individual diets at all dose levels (concentration of the low-dose ranged from -21% to +15%, concentration of the mid-dose was -12% for one occasion and the concentration of the high-dose ranged from -11% to +18%). However, taking into consideration all the
content determinations in all the batches of diets prepared during the entire study, it should be concluded that the concentration of the test
substance in the diet was close to intended at all dose levels. Besides, the test substance was considered to be homogenously distributed in the diets used during the lactation periods.

Duration of treatment / exposure:

Male animals received the test item via the diet during a 2-week premating period, during mating and up to and including the day of sacrifice.
Female animals were fed the diets during a 2-week premating period, and during mating, gestation and lactation and up to and including the day ofsacrifice. Animals of Cohort 1B received the diets from weaning up to and including the day of sacrifice.
The test substance was administered to the animals at constant concentrations in the diet, which remained the same for each group during the study except during during the lactation periods. During the lactation periods of the F0-generation and Cohort 1B animals, the concentration of the test item in the diet of the females was reduced to 50%. This dietary adjustment was made to maintain the dams at the desired target doses during this
period of increased food intake. Animals of Cohort 1B received the diets from weaning up to and including the day of sacrifice.

Frequency of treatment:

ad libitum ( daily, 7 days per week)

Details on study schedule:

The study started with four groups of 28 males and 28 females each, namely one control group and 3 test groups receiving different concentrations of the test item in the diet. The dose levels were selected in consultation with the sponsor and were based on the results an OECD 422 toxicity
study in rats with the same test item.

At the end of the premating period of each generation, each female was caged with one male from the same dosing group. Animals were caged
together until mating occurs or two weeks were elapsed. Mating pairs were clearly identified. Every consecutive morning during the mating period,
vaginal smears were made for determination of the presence of sperm cells. The day on which sperm was detected in the vaginal sperm was
considered as gestation day 0 (GD 0). Upon evidence of copulation, the females were caged individually for the birth and rearing of their pups.
On postnatal day 4, litters of more than 10 pups were adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 5
males and 5 females per litter. If it was not possible to retain 5 pups from each sex in a litter, unequal number of males and females were retained.
At weaning of the F1-generation pups on postnatal day 21, 75 male and 75 female pups per group were selected and placed into cohorts.
The first cohort was assessed for reproductive performance, the second cohort focused on neurodevelopmental endpoints and the third cohort
was examined for developmental immune toxicity.
Based on equivocal results of the F0-generation of this study, a second mating of the animals of cohort 1B was triggered. Therefore, the animals ofcohort 1B were used for breeding of a second generation.

Additionally, an extra group of 6 male and 6 female pups of the control group were included in Cohort 3 as a positive control group for
KLH-specific IgM response.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 80, 250, 800 mg/kg bw/day
Basis:
other: anticipated dose of test item

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1231, 3845, 12308 mg/kg
Basis:
other: dietary level of test item

No. of animals per sex per dose:

The study started with four groups of 28 males and 28 females each, namely one control group and 3 test groups receiving different concentrations of the test item in the diet.
Cohort 1A: One male and/or one female pup per litter was selected (20/sex/group)
Cohort 1B: One male and/or one female pup per litter was selected (25/sex/group)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the sponsor and were based on the results of an OECD 422 toxicity
study in rats with the same test item

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of
toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays,
Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly, during weighing, all F0 animals were subjected to detailed clinical observations.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded at allocation and on the start of the treatment (day 0).
Males were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were weighed on
days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4, 7, 14 and 21 of lactation. In addition, all animals were weighed on their
scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food in the feeders was replaced twice per week with fresh food from the freezer. Except for during the mating period, the food consumption was measured per cage over the same periods as the body weight was measured. The results are expressed in g per animal per day and g per kg
body weight per day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
The intake of the test item per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body
weight at the end of the pertaining period.

Oestrous cyclicity (parental animals):

Vaginal smears to evaluate the estrus cycle length and normality were made daily from the start of the premating period until confirmation of mating. Smears were made, stained and examined in all females.
Mating and pregnancy evaluation
At the end of the gestation period (GD 21), all remaining females were examined twice daily for signs of parturition. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups. Any abnormalities (signs of dystocia, nesting behaviour, nursingbehaviour) was recorded. The dates of pairing, the dates of insemination and the date of parturition were recorded and the precoital interval and the duration of pregnancy was calculated.

Sperm parameters (parental animals):

From all male animals, epididymal sperm analysis and testicular sperm count was performed.
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all F0 males of each group. For this purpose, the cauda
epididymis was dissected, weighed and then minced in M199 medium containing 0.5% Bovine Serum Albumin. Sperm motility and, after sonification
and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for males of all groups, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the
control and the high-dose males were examined microscopically for morphology.
The testes to be analysed was thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma was
weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were
enumerated using the IVOSThe daily sperm production was calculated. Sperm counts was only be conducted on the control and the highdose males.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: litters of more than 10 pups were adjusted by eliminating extra pups by random selection to yield, as nearly as
possible, 5 males and 5 females per litter. If it was not possible to retain 5 pups from each sex in a litter, unequal numbers of males and females
were retained. The anogenital distance was measured in all F1-generation pups. In F2- generation pups of Cohort 1B, the anogenital distance was
measured on PN day 4 and also on PN day 0.

PARAMETERS EXAMINED
The following parameters were examined: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead
pups and grossly malformed pups were evaluated on days 0, 4, 7, 14 and 21 of lactation. The pups were individually observed for clinical signs and weighed on days 0, 4, 7, 14 and 21 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
On postnatal day 12 and postnatal day 20 or 21 all surviving male F1 pups were examined for the presence of nipples/ areolas.

GROSS EXAMINATION OF DEAD PUPS:
Grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund
state during the study and macroscopic abnormalities were recorded. Pups euthanized at culling were examined for gross anatomical changes andblood was collected (pooled per sex per litter) and stored in a freezer at ≤-18 oC for possible evaluation of T4 and TSH in serum. All pups were examined for gross anatomical changes.

Postmortem examinations (parental animals):

At scheduled necropsy, all surviving male and female parent animals were sacrificed.
A vaginal smear was taken for determination of the stage of the estrus cycle on the day of necropsy.
- Male F0 animals were sacrificed after the mating period after at least 10 weeks of exposure.
- Female F0 animals were sacrificed shortly after weaning (females that did not mate with their assigned male and all non-pregnant females were
sacrificed before weaning and at least one week after the end of the gestation period). A necropsy was also performed on animals that died
intercurrently (if not precluded by autolysis) or that had to killed because of they were in a moribund condition.
At scheduled necropsy, samples of the following tissues and organs of all parental animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epidydymides and ovaries which were preserved in Davidson’s solution:
Adrenal glands, Bone marrow (femur), Brain , Cecum, Colon, Duodenum, Epididymides and cauda epididymides, Esophagus, Eyes, Heart, Ileum,
Jejunum, Kidneys, Liver, Lungs, Lymph node axillary, Lymph node mesenteric , Mammary gland (♂and ♀) , Ovaries, Oviducts, Optic nerve, Pancreas,
Peyer’s patches, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles with coagulation glands, Skeletal muscle (tigh), Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testis, Thymus, Thyroid (including parathyroid), Trachea, Urinary bladder, Uterus with cervix, Vagina,
Vas deferens.

During necropsy, blood was taken from the abdominal aorta under CO2/O2 anaesthesia from 10 randomly selected animals/sex/group after
overnight fasting. EDTA was used as anticoagulant. In each plasma sample the following determinations were carried out.
haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils,
lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, activated partial thromboplastin time (APTT), red blood cell distribution width (RDW)
The following parameters were calculated:
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

Clinical chemistry
During necropsy, blood was taken from the abdominal aorta under CO2/O2 anaesthesia from 10 randomly selected animals/sex/group after
overnight fasting. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. In each plasma sample the following
determinations were carried out:
alkaline phosphatase activity (ALP) , bilirubin (total), aspartate aminotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity,
triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin
(calculated), potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate (PO4), glucose (fasting), bile acids.

Blood sampling for hormone determinations
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from 10 randomly selected animals/sex/group after overnight fasting
for determinations of TSH and T4 hormone levels in serum samples

Urinalysis
Before necropsy, 10 randomly selected animals/sex/group were deprived of water for 24 hours and of food during the last 16 hours of this period.
During the last 16 hours of the deprivation period, the rats were kept in stainless-steel metabolism cages (one rat per cage) and urine was collected inglass tubes. The following determinations were carried out:
Volume, Density, Appearance, Dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen)
Microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).

Terminal body weight
Terminal body weight was determined for all F0 animals prior to necropsy.

Postmortem examinations (offspring):

Neonatal pup pathology
Grossly malformed pups were sacrificed and examined. Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund
state during the study and macroscopic abnormalities were recorded.
Culled pup pathology
Pups euthanized at culling were examined for gross anatomical changes and blood was collected (pooled per sex per litter) and stored in a freezer at ≤-18 oC for possible evaluation of T4 and TSH in serum. All pups were examined for gross anatomical changes.
Necropsy of non-selected pups at weaning
Non-selected pups at weaning were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and blood was sampled from maximally 10 pups/sex/group and stored in a freezer at ≤-18oC for possible serum hormone determinations (evaluation of T4 and TSH).
The following organs of maximally 10 non-selected pups/sex/group at weaning) were weighed and/or will be preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde: Brain, Spleen, Thymus, Mammary tissus, Gross abnormalities.

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
During the post-mating phase of this study, two male animals of the high-dose group were sacrificed in a moribund condition.
Further daily cage-side clinical observations and the weekly detailed clinical observations atthe day of weighing during the premating, postmating, gestation and lactation periods showed common findings in rats of this strain and age or occurred as individual fortuitous findings. The
distribution of the findings was equally amongst the various groups or occurred in only one or a few animals. Therefore, these findings were not
considered to be related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the 14-day premating period, a slight, not statistically significant, trend towards a decreased body weights in high-dose male and high-dose
female animals was observed. During this period, body weight changes of high-dose male animals were
statistically significantly decreased from days 0-7 and 0-14 whereas body weight changes of
high-dose female animals were statistically significantly decreased from days 0-14. During the first two weeks after the mating period, body weights
of the male animals of the high-dose group were slightly, not statistically significantly decreased whereas on days 22, 29, 36 and 43 post mating,
body weights of the male animals of this group were statistically significantly decreased as compared to the control males (approximately 5% on post mating day 43) . Body weight changes of the male animals of the high-dose group were statistically significantly decreased from post mating days 22 to 29. During the gestation period, body weights of the pregnant females of the high-dose group were statistically significantly decreased on GD 7, 14 and 21 (approximately 7.5% on GD 21) and body weight changes of these females were statistically significantly decreased from GD 0-7, GD 14-21 and GD 0-21. Body weight changes of the pregnant females of the mid-dose group were statistically significantly decreased from GD 0-7. During the lactation period, body weights of nursing dams of the high-dose group were statistically significantly decreased on PN days 4 and 21 whereas from PN day 4-7 a statistically significant increased body weight change was observed in the mid-dose group. No statistically significant effects on body
weight changes were observed in the high-dose groups. Observed effects on body weights and body weight changes were most probably related to
lower food intake by the animals of the high-dose group.
Food consumption (expressed as g/animal/day) was statistically significantly decreased in males and females of the high-dose group during both
weeks of the premating period. During the post mating period, no statistically significantly effects on food consumption of male
animals was observed. Food consumption of the pregnant and nursing female animals of the high-dose group was statistically significantly
decreased during three weeks gestation period and from PN days 4-7 and PN days 14-21 , respectively. Furthermore, during the gestation period,
food consumption of the animals of the low- and mid-dose groups was statistically significantly decreased from gestation days 0-7. During the
lactation period, food consumption in the low-dose group was statistically significantly decreased from PN days 4-7.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean test item intake of male animals during the premating period ranged between 66-73, 208-224 and 646-671 mg/kg body weight/day for
the low-, mid- and high-dose groups, respectively.
The mean test item intake of female animals during the premating period ranged between 83- 90, 256-277 and 783-810 mg/kg body weight/day for the low-, mid- and high-dose groups, respectively.
The mean test item intake of male animals during the post mating phase ranged between 51- 59, 164-197 and 535-606 mg/kg body weight/day for
the low-, mid- and high-dose groups, respectively .
The mean test item intake of pregnant female animals during the gestation period ranged between 86-90, 258-275 and 820-874 mg/kg body
weight/day for the low-, mid- and high-dosegroups, respectively.
The mean test item intake of nursing female animals during the lactation period ranged between 68-141, 234-465 and 742-1367 mg/kg body
weight/day for the low-, mid- and highdose groups, respectively.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
In the high-dose one female (female 191) had an acyclic estrus cycle whereas 8 females had at least one estrus cycle of 5 days. Consequently, the
mean length of the longest cycles in the high-dose group was statistically significantly higher as compared to the control group (mean of 4 days in
the control group versus 4.3 days in the high-dose group). This was considered as a fortuitous finding, mainly due to a low value in the control
group, since the range of historical control data was 4.2-5.5 days (see Annex 19). There were no animals showing a prolonged estrus period. The
number of complete estrus cycles in the 15 days examination period was comparable amongst the groups.

REPRODUCTIVE FUNCTION:/PERFORMANCE:
In each group 28 females were placed with males for mating. Within 2 weeks, 28, 27, 28 and 27 females of the control, low-, mid- and high-dose group, respectively, were mated (female101 of the low-dose group and female 211 of the high-dose group were not mated). Two, 2, 1,
and 1 females of the control, low-, mid- and high-dose group, respectively, were mated butwere not pregnant. This resulted in 26, 25, 27 and 26
pregnant females in the control, low-, mid- and high-dose group, respectively. There were no differences in pre-coital time, male and female mating
indices and male- and female fertility indices. The duration of gestation was
slightly, but statistically significantly, longer in the high-dose group as compared to the control group (mean length of the gestation period in the
control group was 22.5 days versus 22.9 days in the high-dose group). Since the animals showing the longest gestation period of 24 days were in themid-dose group (females 75, 89 and 91), the slightly longer gestation period as observed in the high-dose group was considered not biologically
relevant. In addition, the duration of gestation as observed in the high-dose group (22.9 days) was still within the range of historical control data (21.4-23.0 days) (see Annex 19). All pregnant females gave birth to a litter. All pups of all dams in each group were born alive, consequently, the
gestation index was 100% and perinatal loss was 0% for all groups. The mean number of implantation sites was slightly, not statistically significantly lower in the low- and high-dose groups as compared to the control group. In addition, also the number of lost implantations and mean
post-implantation loss were not statistically significantly lower in the low- and high-dose groups than in the control group. These findings were not
considered as adverse effects of treatment since no dose-relationship was observed (effect on low-dose group was more pronounced than in
high-dose group and no effects in mid-dose group) and the values observed in the high-dose group were within the range of historical control data.
Consequently, the mean number of pups per litter was lower in the low- and high-dose groups,vreaching the level of statistical significance in the
high-dose group (mean number of pups delivered in the control- and high-dose groups was 12.0 and 10.0, respectively). Since no
dose-relationship was observed and since the lower number of pups observed in the highdose group was well within the range of historical control
data , this finding was considered as an fortuitous finding and not related to treatment. No statistically significant effects were observed on prenatal
loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Terminal body weights of male animals of the high-dose group were statistically significantly decreased. The absolute and relative weights of the liver of males and females of the high-dose group was statistically significantly increased. The relative weights of the kidneys and thyroids of male animals of the high-dose group were statistically significantly increased.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic analysis of the F0-generation revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the
background pathology of rats of this strain and age.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination revealed a treatment-related effect in the kidneys of high-dose male animals. In the F0-generation, minimal to moderate
accumulation of proteinaceous droplets in the tubuli was observed. In the male animals of the high-dose group of Cohort 1A, an increase in both the incidence and the severity of proteinaceous accumulation was observed in the tubuli. Similarly, an increase in both the incidence and the severity of
basophilic tubuli formation was observed in the high-dose group in comparison to the control group.

OTHER FINDINGS (PARENTAL ANIMALS)
Clinical chemistry
In male animals of the high-dose group, the activity of gamma glutamyl transferase (GGT) was statistically significantly increased. In female animals ofthe high-dose group, the concentration of bilirubin was statistically significantly decreased.

Urinalysis
In the urine of male animals, the score for amorphous material was statistically significantly higher in the mid- and high-dose groups as compared to the control group whereas the pH of the urine of male animals of the high-dose group was statistically significantly lower than of the urine of the
control male animals. In urine of the female animals, the score for crystals was statistically significantly lower in the low-dose group than in the
control group whereas no statistically significant differences were observed for this parameter in the mid- and high-dose groups.

Sperm analysis
No statistically significant effects were observed on epididymal sperm motility, epididymal sperm count (Table F0-12.2) and epididymal sperm
morphology Homogenisation resistant sperm count and daily testicular sperm production were comparable among the various groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: effects on body weights, food consumption, kidney and liver weights and kidney pathology

Results: F1 generation

Details on results (F1)

Litter data and sex
The mean number of pups delivered per litter and the mean number of pups/litter on PN day 4 (before culling) in the low- and high-dose groups was lower than in the control group, reaching the level of statistical significance only in the high-dose group. After culling, the mean number of live pups/litter at day 7, 14 and 21 was statistically significantly lower in the low- and high-dose groups as compared to the control group.The lower number ofpups was not considered as related to treatment. All pups were born alive, no stillborn pups were found. The number of pups that was lost
(killed and missing) between PN day 0-4 amounted 0, 3, 1 and 1 in the control, low-, mid- and high-dose groups, respectively. After day 4, no pups
were missing, consequently, the viability indices from PN day 0-4 and PN day 5-21 were comparable among the various groups.
No effects were observed on the sex ration on PN day 0 and PN day 21.
Pups clinical observations
The incidental observed clinical observations in male and female pups were considered to be not related to treatment.
Pups weights
No statistically significant differences were observed on pup weights among the various groups.
Anogenital distance
The absolute anogenital distance of F1-generation male and female pups of the high-dose group as measured on PN day 4 was statistically
significantly increased (approximately 9% for both sexes). However, after correction for pup weight, in female pups no effect was observed. In male
pups, the anogenital distance related to the cube root of pup weight was statistically significantly higher in the male pups of the mid- and high-dose groups (approximately 3 and 7%, respectively) as compared to the control group. This is considered a fortuitous finding that was not related to
treatment because in case of endocrine activity a decrease in anogenital distance would be expected. Nipple retention
No differences were observed on nipple retention of male animals among the various groups.
Sexual maturation of all selected pups
No statistically significant effects among the various groups were observed. All male pups scored positive for preputial separation whereas two
female pups of the control group and two pups of the mid-dose group scored negative for vaginal opening until sacrifice.
Macroscopic examination of pups Except for an pup of which the hipbone was accidentally broken and, consequently, was killed no macroscopic
observations were reported. One male pup of the low dose group was very small (32.2 grams on PN day 21; F and for that reason a necropsy was
performed at PN day 21. At macroscopic examination, the pup showed cerebellar hypoplasia.
Pup organ weight
No statistically significant effects were observed on pup terminal body weights nor on absolute and relative organs weights of brain, spleen and
thymus.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Results of Cohort 1A

Mortalities and clinical observations

Female animal 073-06 of the low-dose group of Cohort 1A was found dead on day 20 (at an age of 43 days). No clinical signs were observed before this animal was found dead. At autopsy of this animal, macroscopic analysis only revealed that the cecum was filled with air. Daily cage-side clinical observations and the weekly detailed clinical observations at the day of weighing showed only common findings in rats of this strain and age or occurred as individual fortuitous findings. The distribution of the findings was equally amongst the various groups or occurred in only one or a few animals. Therefore, these findings were not considered to be

related to treatment.

Body weight and body weight changes

Except for day 56, mean body weights of the male animals of the high-dose group of Cohort 1A were statistically significantly lower than the mean body weights of control animals (approximately 6%). On one occasion (day 42) mean body weights of the Cohort 1A male animals of the low-dose group were statistically significantly higher than the mean body weights of the control animals.

Mean body weight changes of the male animals of the low- and mid-dose groups were statistically significantly increased as compared to the control group from days 0-7, 21-28 and 35-42 (low-dose group) and from days 21-28 (mid-dose group), respectively. In the high-dose group, mean body weight changes of the male animals of Cohort 1A were statistically

significantly decreased as compared to controls from days 0-7 and 7-14.

Mean body weights of the female animals were comparable amongst the various groups. Statistically significant differences between mean body weight changes of female animals of the control group were observed in the mid-dose group from days 0-7 (increased body weight change) and in the low- and high-dose groups from days 35-42 (decreased body weight change).

Food consumption

In male animals of Cohort 1A, food consumption (expressed as g/animal/day) was statistically significantly increased in the low-dose group from day 14-21 and statistically significantly decreased in the high-dose group from day 0-7, 7-14, 14-21 and 28-35, respectively. No effects on food consumption were observed in female animals of Cohort 1A.

Intake of test item Cohort 1A

The mean test item intake of male animals ranged between 76-181, 238-560 and 752-1749 mg/kg body weight/day for the low-, mid- and high-dose groups, respectively. The mean test item intake of female animals ranged between 88-172, 267-529 and 830-1668 mg/kg body weight/day for the low-, mid- and high-dose groups, respectively.

Estrus cycle

Vaginal patency in the pups of the high-dose group of Cohort 1A only was slightly butm statistically significantly higher. Since no effect was observed on vaginal opening when pups of all Cohorts were included, the finding in Cohort 1A animals was considered to be a fortuitous finding. The first estrus stage after the onset of vaginal patency occurred between 0-4 days. The mean

period between vaginal opening and the first estrus stage was 0.7, 0.7, 1.1 and 0.4 days for the control, low-, mid- and high-dose groups, respectively. Estrus cycle evaluation for 2 weeks before sacrifice showed a statistically significantly higher mean cycle length of the female animals of the high-dose group (4.16 days in the control group versus 4.70 days in the high-dose group, respectively) and four animals of the highdose group showed a longer estrus period. Consequently, the number of complete estrus cycles in the 15 days evaluation period was statistically significantly decreased in the highdose group. Both the mean length of the longest cycle as well as the number of animals with a prolonged estrus period as observed in the high-dose group were within historical control ranges. For that reason, these findings were not considered as adverse effects of the test item.

Haematology

In male animals of Cohort 1A, mean corpuscular haemoglobin (MCH) was slightly (approximately 5%), but statistically significantly, lower in the high-dose group. No other statistically significant effects were observed on any of the red- and while blood cell

parameters measured in the male animals of Cohort 1A. In female animals of Cohort 1A, mean corpuscular volume (MCV) was slightly (approximately 3%), but statistically significantly, lower in the high dose group. Prothrombin time was statistically significantly longer in the female animals of the low- and mid-dose groups (approximately 9% and 8%, respectively) whereas the prolongation in the high-dose group (approximately 7%) did not reached the level of statistical significance. No other statistically significant effects were observed on any of the red- and while blood cell parameters measured in the female animals of Cohort 1A.

Clinical chemistry

Statistical evaluation showed lower total protein (approximately 5%), higher albumin toglobulin ratio (approximately 7%) and sodium values (approximately 2%) in males of the high dose group. Sodium values were also statistically significantly higher in males of the low dose group (approximately 3%). ALP was statistically significantly increased in females of the middose group (approximately 56%) whereas the difference in the high-dose group did not reach the level of statistical significance.

Hormone determinations

Exposure to 2-Ethylhexanoic acid via the diet did not affect the concentrations of TSH and T4 hormones in the sera of male and female animals of Cohort 1A.

Urinalysis

In the urine of the male animals of Cohort 1A, the score for epithelial cells was statistically significantly decreased in the mid-dose group, whereas no effect was observed in the highdose group. In the urine of the high-dose males, the pH and ketones were statistically significantly decreased. No other statistically significant effects were observed on any of the other urine parameters measured in the male and female animals of Cohort 1A.

Sperm analysis

No statistically significant effects were observed on epididymal sperm motility epididymal sperm count and epididymal sperm morphology. Homogenisation resistant sperm count and daily testicular sperm production were comparable among the various groups

Organ weights of Cohort 1A

In male animals, the absolute weight of the heart was statistically significantly higher in the low-dose group (approximately 7%) whereas no effects were observed in the mid- and high-dose groups. In male animals of the high-dose group, the relative weights of the heart, kidneys, liver and testes were statistically significantly increased (approximately 6%, 12%, 15% and 10%, respectively). In the low-dose group, and not in the mid- and high-dose groups, the relative weight of the cauda epididymides was statistically significant decreased (approximately 8%). In the female animals of the high-dose group, the absolute weights of the liver (approximately 10%) and the relatively weights of the kidneys and liver (approximately 7% and 15%, respectively) were statistically significantly increased.

Splenic lymphocyte subpopulation analysis

Exposure to 2-Ethylhexanoic acid via the diet did not affect the splenic lymphocyte composition of male and female animals of Cohort 1A.

Macroscopic examination of Cohort 1A animals

Macroscopic analysis at necropsy of the remaining animals revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Microscopic examination of Cohort 1A animals

Microscopic analysis of one animal which was found dead revealed no abnormalities of the caecum.

Microscopic analysis revealed a treatment-related effect in the kidneys of the male animals. In the mid and high-dose groups, respectively 14 and 15 out of 20 animals showed minimal to moderate accumulation of proteinaceous droplets in the tubuli. In comparison only 6 out of 20 low-dose animals and 3 out of 20 control animals showed minimal to mild accumulation of

proteinaceous droplets in the tubuli. In addition, minimal to mild basophilic tubuli were observed in 9 out of 20 animals of the high dose-group. In comparison respectively 2, 0 and 6 out of 20 animals of the control, low and mid-dose groups showed a minimal presence of basophilic tubuli. The remaining findings were considered unremarkable and part of the background pathology

of rats of this strain and age.

Differential ovarian follicle count

The absolute number of follicles in the high-dose group was lower than in the control group, however, the relative distribution of the follicles in each phase (small, growing, antral and corpora lutea) was comparable in the control and high-dose groups. Although the

development of small follicles into growing follicles was statistically significantly slower in highdose females than in control females animals, no effects were observed in the development of small follicles into antral follicles and into corpora lutea, indicating that treatment had no effect on the development of the follicles.

Results of Cohort 1B

Mortalities and clinical observations

During the premating period of Cohort 1B, 4 male animals showed iatrogenic encrustations and/or wounds due to implantation of the transponders. One female animal of the mid-dose group howed vaginal occlusion, and, consequently, was not mated during the

mating period. Further daily cage-side clinical observations and the weekly detailed clinical observations at the day of weighing during the premating, postmating, gestation and lactation periods showed common findings in rats of this strain and age or occurred as individual fortuitous findings. These findings were not considered to be related to treatment.

Body weight and body weight changes

During the major part of the premating and post-mating periods of Cohort 1B, body weights of the male animals of the high-dose group were statistically significantly decreased. During these periods, no effects on body weights of the female animals were observed Statistically significant effects on body weight changes of male animals were observed in the low-dose group. During the premating period, in female animals of the mid-dose group, body weight changes were statistically significantly decreased from days 35-42 and statistically significantly increased from days 42-49. Except from a statistically significant decreased body weight change of the female animals of the high-dose from gestation days 7-14, no effects were observed on body weights and body weight changes of the females animals during the gestation and lactation periods . Observed effects on body weights and body weight changes were most probably related to lower food intake by the animals of the high-dose group

Food consumption

Food consumption (expressed as g/animals/day) of the male animals of the high-dose group was statistically significantly decreased during almost the entire premating and postmating periods, whereas, during the premating period no effect on food consumption of female animals was observed . Food consumption of pregnant females was statistically significantly decreased from gestation days 0-7 (mid- and high-dose groups) and 7-14 (high-dose group) . During the lactation period, food consumption was comparable amongst the various groups.

Intake of test item

The mean test item intake of male animals during the premating period ranged between 66-176, 206-551 and 666-1745 mg/kg body weight/day for the low-, mid- and high-dose groups. The mean test item intake of female animals during the premating period ranged between 78- 168, 247-529 and 770-1668 mg/kg body weight/day for the low-, mid- and high-dose groups. The mean test item intake during the post mating phase ranged between 55-60, 172-184 and 74-591 mg/kg body weight/day for the low-, mid- and high-dose males. The mean test item intake during the gestation period ranged between 74-79, 223-248 and 732-744 mg/kg body weight/day for the low-, mid- and high-dose groups. The mean test item intake during the lactation period ranged between 71-137, 210-408 and 706-1414 mg/kg body weight/day for the low-, mid- and high-dose groups.

Mating and pregnancy evaluation Cohort 1B In each group, 25 females were placed with male animals for mating. Within 2 weeks, 25, 25, 24 and 25 females of the control, low-, mid- and high-dose group, respectively, were mated (female 121-06 of the mid-dose group was not mated and female 107-06 of the low-dose group was judged to be non-mated but this animal appeared to be pregnant). Animals 001-7 of the control group, 157-04 of the mid-dose group and 197-07 of the high-dose group were mated but were not pregnant. This resulted in 24, 25, 23 and 24 pregnant females in the control, low-, mid- and high-dose group, respectively. Pre-coital time, male- and female mating indices, male- and female fertility indices and duration of gestation was comparable amongst the groups. All pregnant females gave birth to a litter. In the control, low-, mid- and high-dose groups, 0, 2, 1, and 2 females delivered stillborn pups but no female delivered only stillborn pups. Consequently, the gestation index was 100% for all groups and the mean perinatal loss was 0, 0.6, 2.6 and 1.3% for the control, low-, mid- and high-dose group, respectively. The mean number of pups delivered per litter ranged from 10.5 in the high-dose group and 11.5 in the low-dose groups and was comparable amongst the groups. The incidence of stillborn pups was 0, 0.7, 1.2 and 1.2% in the control, low-, mid- and high-dose groups, respectively and, consequently, the incidence of live born pups was 100, 99.3, 98.8 and 98.8% in the control, low-, mid- and high-dose groups, respectively

Organ weights of Cohort 1B

In male animals of the mid-dose group, the absolute and relative weights of the testes were statistically significantly increased (approximately 9% and 7%, respectively). In male animals of the high-dose group, terminal body weight was statistically significantly decreased and the absolute weight of the kidneys (approximately 9%) and the relative weights of the liver (approximately 15%), kidneys (approximately 18%), testes (approximately 12%) and cauda epididymides (approximately 10%) were statistically significantly increased. In female animals, no effects on absolute organ weights were observed whereas the relative weights of the liver and kidneys of the female animals of the high-dose group were statistically significantly increased (approximately 10% and 8%). Macroscopic examination of Cohort 1B animals

Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Microscopic examination of Cohort 1B animals

The results of histopathological examination of the tissues and organs of the animals of Cohort 1A did not indicate a need for additional histopathological examination of the tissues and organs of the animals of Cohort 1B.

Litter and pup data F2-generation of Cohort 1B

Litter and sex data

The mean number of pups delivered and the mean number of live pups per litter at day 0, 4, 7, 14 and 21 was comparable amongst the various groups. The number of stillborn pups amounted 0, 2, 3, and 3 in the control, low-, mid- and high-dose groups, respectively. Only a limited number of pups were killed and or were missing between days 0-4 (4, 2, 3 and 2 in the control, low-, mid- and high-dose groups, respectively) and consequently, no effect was observed on the viability index from day 0 to 4. Furthermore, since only 1 pup of the low-dose and 1 pup of the mid-dose was missing between day 8-14 no effect was observed on the viability index from day 4-21. Sex ratios on day 0 and 21 were comparable amongst the various groups.

Pup clinical observations

The incidental observed clinical observations in male and female pups were considered to be not related to treatment.

Pup weights

Pup weights of male and female pups separately and/or combined were comparable amongst the groups on postnatal days 0, 4, 7 and 14 but on postnatal day 21 the mean weights of the pups of the high-dose group were statistically significantly increased as compared to the control group. The increased weight of the pups on postnatal day 21 is not considered as an adverse effects of treatment. Partly, it might be due to the slightly lower number of pups in the high-dose group as compared to the control group (mean number of pups in the high-dose group was 9.3 versus 9.8 in the control group).

Anogenital distance

The absolute anogenital distance and the anogenital distance related to the cube root of pup weight of F2-generation male and female pups as measured on postnatal day 0 and on postnatal day 4 were comparable amongst the various groups.

Macroscopic observation of pups

The macroscopic observations of culled pups and of pups that died during lactation and the macroscopic observations of pups killed at weaning did not show any remarkable and/or treatment related findings.

Applicant's summary and conclusion

Conclusions:

There were no effects of the test item on fertility and reproductive performance of F0- and F1-generation animals.
There were no effects of the test item on general and sexual developmental parameters.
There were no effects of the test item on (developmental) neurotoxicity parameters.
There were no effects of the test item on (developmental) immune toxicity parameters.
Based on the effects on body weights, food consumption, kidney and liver weights and kidney pathology in animals of the high-dose group, the
NOAEL for parental effects was placed at the mid-dose concentration of 3845 mg test item per kg diet.

Executive summary:

The reproductive toxicity of 2-ethylhexanoic acid was investigated in an extended one generation reproductive toxicity study according to OECD 443. The objective of this study was to provide data on the possible effects on reproductive performance of male and female Wistar rats and on the development of pups consequent to daily oral administration of various concentrations (0, 1231, 3845 and 12308 mg/kg diet) of the test item during a premating period of 2 weeks and during mating and gestation. During the lactation period, the concentration in the diets was halved. At weaning, pups were distributed to different cohorts and exposed to the same dose levels as their parents during growth into adulthood. The first cohort was assessed for reproductive performance, the second cohort focused on neurodevelopmental endpoints and the third cohort was examined for developmental immune toxicity. Based on equivocal results of the F0-generation of this study, a second mating of the animals of cohort 1B was triggered. Therefore, the animals of cohort 1B were used for breeding of a second generation.

During the F0-generation, two male animals of the high-dose were untimely sacrificed in a moribund condition. During the F1-generation, one female animal of the low-dose group of Cohort 1A and one male animal of the cyclosporine A positive control group of Cohort 3 were found dead. The macro- and micro-observations in these animals were not considered to be related to treatment. Furthermore, no treatment-related clinical signs were observed during the study. Body weight and food consumption of male and to a lesser extend of female animals of the high-dose group were slightly, but statistically significantly decreased in major parts of the F0- generation and F1-generation. During the second part of the lactation periods of the F0-generation and the F1-generation of Cohort 1B (most probably the pups start to eat) and in the first weeks of the F1-generation of each Cohort (food consumption is relatively high in young animals) the food consumption was relatively high. Consequently, test item intake during these period was higher than anticipated and reached a maximum value of 183, 669 and 1842 mg/kg body weight/day for the low- (F1- generation female animals of Cohort 2A and Cohort 3) and mid- (F1-generation male animals of Cohort 3) and high-dose (F1-generation male animals of Cohort 2A) groups, respectively. The statistically significant slight increases in both the absolute and relative weights of the liver and kidneys of males and females of both generations were considered to be related to treatment. Microscopic examination revealed a treatment-related effect in the kidneys of high-dose male animals of the F0-generation and of the male animals of Cohort 1A of the F1-generation. In the F0-generation, minimal to moderate accumulation of proteinaceous droplets in the tubuli was observed. In the male animals of the high-dose group of Cohort 1A, an increase in both the incidence and the severity of proteinaceous accumulation was observed in the tubuli. Similarly, an increase in both the incidence and the severity of basophilic tubuli formation was observed in the high-dose group in comparison to the control group.

Regarding fertility and reproductive performance parameters, no treatment-related effects were observed on estrus cycle in animals of the F0-generation and in animals of Cohort 1A of the F1-generation. Furthermore, no treatment-related effects were observed on fertility and reproductive performance of male and female animals of the F0-generation and of Cohort 1B of the F1-generation and no effects were observed on epididymal and testicular sperm parameters in animals of the F0-generation and of Cohort 1A of the F1-generation. No effects were observed on TSH and T4 analysis in animals of the F0-generation and of Cohort 1A of the F1-generation.

Regarding general and sexual developmental parameters, no treatment-related effects were observed on the incidences of liveborn and stillborn pups, pup viability indices, sex-ratio’s, pup weights and clinical and macroscopic observations in F1- generation pups and Cohort 1B F2-generation pups. No effects were observed on nipple retention in male F1-generation pups. No effects were observed on sexual maturation parameters (preputial separation and vaginal opening) in F1-generation pups and no effects were observed on organ weights of F1-generation pups. Furthermore, no treatment-related effects were observed on the development of the follicles from primordial small follicles into corpora lutea. The statistically significant effects on anogenital distance after correction for pup weights as observed in F1-generation male pups on postnatal day 4 were considered fortuitous findings that were not related to treatment. The anogenital distance in the male pups was higher than in control male pups whereas in case of endocrine related activity a decrease would be expected. Furthermore, no effects were observed on anogenital distance on postnatal days 0 and 4 in F2-generation pups.

Regarding neuro(developmental) parameters, Functional observation battery (FOB) and spontaneous motor activity analysis did not reveal any effect of the test item in animals of Cohort 2A of the F1-generation. The results of the auditory startle response did not indicate any neurotoxic potential of the test item in animals of Cohort 2A. No treatment-related adverse effects were observed on brain weight, brain length and brain width measurements in animals of Cohort 2A and Cohort 2B, respectively. No treatment-related differences were observed in thicknesses of 10 major regions of the brain regions as measured in male and female animals of the control and high-dose group. Macroscopic observation at sacrifice of animals of Cohort 2A and Cohort 2B did not reveal any treatment-related abnormalities. Microscopic observations of brains and neuronal tissues of animals of Cohort 2A and brains of animals of Cohort 2B revealed no treatment-related abnormalities.

Regarding immune(developmental) parameters, no effect was observed on the composition of the splenic lymphocyte subpopulation in animals of Cohort 1A of the F1-generation. In contrast to the positive control cyclosporine A, the test item had no effect on the KLHspecific IgM antibody levels in animals of Cohort 3 of the F1-generation.

In conclusion, the NOAEL for fertility and reproductive performance of F0- and F1-generation animals, general and sexual developmental parameters, (developmental) neurotoxicity and immune toxicity parameters was the highest dose tested (12308 mg/kg diet).

Based on the effects on body weights, food consumption, kidney and liver weights and kidney pathology in animals of the high-dose group, the NOAEL for parental effects was the mid-dose concentration of 3845 mg/kg diet.