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Diss Factsheets

Administrative data

Endpoint:
neurotoxicity: oral
Remarks:
other: Extended One Generation Reproductive Toxicity Study
Type of information:
experimental study
Adequacy of study:
weight of evidence
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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 443
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
2-ethylhexanoic acid
EC Number:
205-743-6
EC Name:
2-ethylhexanoic acid
Test material form:
other: liquid
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:
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 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.
Frequency of treatment:
ad libitum ( daily, 7 days per week)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 80, 250, 800 mg/kg bw/day
Basis:
other: anticipated dose of test item
Remarks:
Doses / Concentrations:
0, 1231, 3845, 12308 mg/kg
Basis:
other: dietary level of test item
No. of animals per sex per dose:
Cohort 2A: One male or one female pup per litter was selected (10/sex/group)
Cohort 2B: One male or one female pup per litter was selected (10/sex/group1)
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

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
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.

DETAILED CLINICAL OBSERVATIONS: Yes
Once weekly all animals were subjected to detailed clinical observations, e.g. during weighing. In addition, the animals of Cohort 2A were weekly
observed in more detail in an arena. Between postnatal days 63 – 75 arena testing formed part of the Functional Observational Battery tests. Signs
noted include but not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic
movements, stereotypies and bizarre behaviour.

BODY WEIGHT: Yes
Body weights of male and female animals in cohort 2A were recorded weekly. Additionally, body weight was recorded on the day of attainment of
vaginal patency or balano-preputial separation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The food in the feeders was refreshed twice per week. 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.
Intake of the test item (Cohort 2A)
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.

OTHER:
Sexual maturation (Cohort 2A)
The following landmarks for sexual maturation were recorded in F1 animals in cohort 2A:
- males: preputial separation from postnatal day 39 onwards
- females: vaginal opening from postnatal day 31 onwards
Daily checks were discontinued after 95% of the animals had reached sexual maturation. At the day the animals were scored positive for preputial
separation or vaginal opening, the body weight was measured.
Auditory startle response (Cohort 2A)
On postnatal day 24 (+/- 1 day) an auditory startle test was performed in all Cohort 2A animals. Each session consisted of 50 trials (5 blocks of 10 trials).
Neurobehavioural examinations performed and frequency:
Functional Observational Battery (FOB) and spontaneous motor activity (Cohort 2A)
FOB and motor activity testing were performed in all Cohort 2A animals approximately between postnatal days 63 - 75. The FOB used in our
laboratory was adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity#
Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization. Details on the conduct of observations included in this battery and operational definitions of the different scores for each item are given in the FOB-manual
entitled "Functional Observational Battery. Operational Definitions" (Lammers, 2000).
Unlike the daily, general clinical observations described in the previous section (intended to detect all abnormalities, signs of ill health or reactions to treatment), the FOB is a series of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional
integrity of the rat. The measures included according to functional domain are as follows:

Autonomic:l acrimation (R), salivation (R), pupil response to light (Q), palpebral closure (R), piloerection (Q), defecation (C), urination (C)
Neuromuscular:gait (D,R), mobility (R), forelimb and hindlimb gripstrength (I), landing foot splay (I), righting reflex (R)
Sensorimotor: response (R) to tail pinch, click, touch and approach of a visual object
Convulsive: clonic and tonic movements (D)
Excitability: ease of removal (R), handling reactivity (R), arousal (R), vocalizations (Q)
Activity: rearing (C), posture (D)
Physiological:body temperature (I)

Abbreviations: D=descriptive rank R=rank order data C=count data Q=quantal data I=interval or continuous data

At least one hour prior to the start of the observations, the animals were placed individually in macrolon cages in a waiting area in the examination room. First, measurements were carried out in the cage. The rat's posture, palpebral closure and the possible presence of clonic and tonic convulsions was recorded. Then the rat were removed from the cage and the ease of removal and handling was rated. Palpebral closure and any lacrimation or
salivation were also rated, and the presence or absence of piloerection and vocalizations was recorded. In addition, other signs, such as changes in
skin and fur, exophthalmus, crustiness around the eyes, bite marks on the tail or paws, missing toe nails or emaciation (shallow stomach,
protruding spinal vertebrae) was recorded. The rat was then placed in an open arena (77 l x 55 w x 7 h cm) and observed for 3 minutes. Rears (both
supported and unsupported) were counted. At the same time, gait characteristics were recorded and ranked, the ease with which the rat locomotes
was ranked, and arousal was assessed and recorded. Further, the occurrence of clonic and/or tonic convulsions, stereotypies and bizarre behaviour was recorded. At the end of the observation period, the number of faecal boluses and urine pools were recorded. Following this observation period,
reflex testing was conducted. Reflex testing consists of recording the rat's responses to the approach of a pencil, a touch of a pencil to the rump, a
click stimulus, tail pinch, and the constriction of the pupil to light. Aerial righting was rated next. Forelimb and hindlimb grip-strength were measuredThree valid determinations (from a maximum of five attempts) were taken for each grip-strength measure. The rectal temperature was taken with the
rat restrained by hand. Finally, the hindlimb feet was painted lightly and landing foot splay was measured.

Motor activity measurements (Cohort 2A)
Motor activity was assessed following FOB testing. Changes in spontaneous motor activity was assessed using an automated quantitative
microprocessor-based video image analysis system. Rats were placed individually in open roofed cages measuring 48.8 l x 44.7 w x 50 h cm on the
insides and equipped with a video camera suspended above the test cage. The position of the rat was continuously monitored throughout the test
session. Spontaneous motor activity was expressed as the total distance run in a 30 minute test period. In addition, habituation of activity was
evaluated. Each session was divided into 5 time blocks of 6 minutes each. Motor activity tests was recorded on DVD. Recordings were used only for
re-analysis of motor activity tests should that be necessary for technical reasons. If re-analysis of motor activity tests was not necessary, the DVDs
were removed from the study dossier after submission of the final report. Squads of up to eight animals were monitored simultaneously. Dose groupswere evenly distributed over motor activity test cages and over time as good as possible. Motor activity testing of a squad was conducted immediatelyafter functional observations for that squad have finished.
Sacrifice and (histo)pathology:
All surviving male and female Cohort 2A (between postnatal days 75 - 90) and 2B (postnatal day 22) animals were sacrificed by perfusion fixation.
Perfused animals were stored and the following organs were carefully removed.
Brains (analysed on eight levels)
Eyes with retina and optic nerve
Musculus gastrocnemius
Nose (nasal cavity)
- olfactory epithelium (nose level III)
Pituitary gland
Sciatic nerve, proximal section
Spinal cord, cervical part (C1-C6)
Spinal cord, thoracic part (Th5-Th8)
Spinal cord, lumbar part (L1-L4)
Spinal ganglia, cervical part (3x)
Spinal ganglia, lumbar part (3x)
Tibial nerve (knee) proximal section
Tibial nerve (lower leg) distal section
Trigeminal ganglia (with part of nerve)
Root fibers, dorsal (C1-C6 & L1-L4)
Root fibers, ventral (C1-C6 & L1-L4)

Activities in Cohort 2B animals
Brains (analysed on eight levels)
Pituitary gland
Trigeminal ganglia

Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin, except for the sections
of the sciatic nerve, the spinal cord, the spinal ganglia, the tibial nerve, the trigeminal ganglia and the dorsal and ventral root fibres.
These tissues were embedded in epoxy resin, sectioned and stained with toluidine blue.Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high concentration group (group 4).

Brain measurements (Cohort 2A & 2B)
Brain weight was determined after removal. The length and width of the brain was measured in all animals.
Morphometry (Cohort 2A)
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus, hippocampus, corpus callosum,
cerebellum) was performed. Measurements were carried out bilaterally in the left and right halves of the brain with the exception of the corpus callosum and the cerebellum.

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 of examinations

Details on results:
Results of Cohort 2A

CAGE SIDE OBSERVATIONS: Yes
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 the signs 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: Yes
Mean body weights of the male animals of the high-dose group of Cohort 2A were lower than the mean body weights of the control group during the entire
period, reaching the level of statistical significance on days 14, 28 and 35. Mean body weight changes of the male animals of the high-dose group was statistically significantly decreased from days 7 to 14 as compared to the control group. No statistically significant differences were observed on body weights and mean body weight changes of the females animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No statistically significant effects were observed on food consumption of male animals of Cohort 2A whereas food consumption of female animals of the high-dose group was statistically significantly decreased from days 28-35 and 42-49.
Intake of test item
The mean test item intake of male animals ranged between 80-178, 260-550 and 805-1842 mg/kg body weight/day for the low-, mid- and
high-dose groups, respectively. The mean test item intake of female animals ranged between 93-183, 269-552 and 859-1730 mg/kg body
weight/day for the low-, mid- and high-dose groups, respectively

Brain measurements
The terminal body weight of the male- and female animals of the high-dose was lower than the body weights of the control animals but the difference reached the level of statistical significance only in the female animals of the high-dose group. Mean absolute brain weight of the male animals of the high-dose group was slightly (approximately 5%), but statistically significantly, lower as compared to the control group. Since no effects were
observed on the absolute brain weight of female animals and on the relative brain weights of male and female animals the lower absolute brain weight of males of the high-dose group was considered not to be related to treatment. Brain length and brain width measurements (expressed in mm)
did not reveal any difference between the various groups.
Morphometry
Except for two slight but statistically significant effects on the thickness of the cerebellum (decreased in high-dose male animals (approximately
4%; p=0.003)) and the thickness of the left striatum (increased in high-dose females (approximately 15%; p=0.019)), no other effects were
observed in thicknesses of 10 major regions of the brain regions as measured in male and female animals of the control and highdose
group, respectively. The statistically significant effects observed on the thickness of the cerebellum and left striatum were considered as fortuitous
findings.
Macroscopic observations
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 observation
Microscopic analysis revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathologyof rats of this strain and age.

Functional observation Battery (FOB) and spontaneous motor activity
The results of the neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test item
in rats.
Auditory startle response
the results of the auditory startle response test did not indicate any neurotoxic potential of the test item in rats.

Results of Cohort 2B
Mortalities and clinical observation
No mortalities and no clinical sings were reported in the animals of Cohort 2B.
Brain measurements
The terminal body weight of the male- and female animals of Cohort 2B was comparable amongst the various groups.
Mean absolute brain weight of the male animals of the mid- and high-dose groups was slightly (approximately 5 and 6%, respectively), but statisticallysignificantly higher as compared to the control group. Since no effects were observed on the absolute brain weight of female animals and on the
relative brain weights of male and female animals the slight effects observed on the absolute weight of the male pups was considered a change findingand not related to treatment.
Brain length and brain width measurements (expressed in mm) did not reveal any difference amongst the various groups.
Macroscopic observations
Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were unremarkable and part of the background
pathology of rats of this strain and age.
Microscopic observation
Microscopic analysis revealed no treatment-related abnormalities. The findings were unremarkable and part of the background pathology of rats of this strain and age.

Applicant's summary and conclusion

Conclusions:
There were no effects of the test item on (developmental) immune toxicity parameters.