Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
2-ethylhexylal
IUPAC Name:
2-ethylhexylal
Details on test material:
Batch No.: 1204051500
Purity: 99.766 % (w/w)
Impurities: 2-Ethylhexanol (0.0826 %); Formaldehyde (not found); Water (0.0370 %)
Storage: Store in supplied bottle in dark at the temperature under 25°C. Provide local exhaust or general room ventilation. Keep container closed when not in use. Keep away from heat.
Safety precautions: Do not breathe gas, fumes, vapour and spray. Use personal protective equipment (goggles, gloves).
Stability/Expiration: 3 years / 05.04.2015

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
Selection of animal species: the preferred rodent species according to the guidelines is the rat; the test facility has long experience with this species
Species: Albino laboratory rat
Strain: Wistar Han (outbred SPF quality - guaranteed)
Supplier: SPF breeding, VELAZ s.r.o., Koleč u Kladna; Czech Republic, RČH CZ 21760152
Age at start of the study: 10 weeks
Acclimatization: 6 days
Total number of animals: 48 males and 48 females (12 females and 12 males per group)

Housing conditions:
All the study was conducted in the individually ventilated cages (IVC) – special animal room in conditions according to SOP No. 225.
Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12 hour dark cycle.
Animals were housed in individually ventilated cages, 2 rats of the same sex in one polysulfone cage (floor area – 900 cm2) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.

Identification:
Identification of animals was made by colour marks on fur (system 1 – 12), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and colour mark of group.

Diet:
Complete pelleted diet for rats and mice in SPF breeding - ST 1 BERGMAN was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.

Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.

Water:
Free access to drinking water (ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.

Additional Information
The standard pelleted laboratory animal diet are analysed for nutrients (once a year) and bacteriologicaly examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives.
Analysis of diet and water and steriliser control, did not reveal any findings that could affect study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
Olive oil (pharmaceutical quality) Manufacturer: Dr.Kulich Pharma s.r.o., Piletická 178/61, 500 03 Hradec Králové, Czech Republic; Lot No.: 5211201; Expiry date: 12/2013
Details on exposure:
Preparation of application form
The concentrations of suspension at all dose levels was adjusted to ensure the administration of 1 mL per 100 g of body weight. The test substance was administered in olive oil (pharmaceutical quality). The application form (test substance in vehicle) was prepared daily just before administration and it was mixed by magnetic stirrer (500 rpm for 10 minutes).



Details on mating procedure:
Animals were mated from the 15th day of the study. Mating 1 : 1 (one male to one female) was used in this the study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The treated groups were administered daily for the period: males and females – 2 weeks prior to the mating period and 2 weeks during the mating period. Pregnant females were administered during pregnancy and till 4th day of lactation. Males were administered till 28th day of the study, females showing no-evidence of copulation till 54thday of the study and non-pregnant females till the 25th day after confirmed mating.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Details on study schedule:
The treated groups were administered daily for the period:
- males and females – 2 weeks prior to the mating period and 2 weeks during the mating period.
- Pregnant females were administered during pregnancy and till 4th day of lactation.
- Males were administered till 28th day of the study, females showing no-evidence of copulation till 54thday of the study and non-pregnant females till the 25th day after confirmed mating. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Doses / concentrations
Remarks:
Doses / Concentrations:
160; 400 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:

To find appropriate dose range for the Reproduction/Developmental Toxicity Screening Test in the rat.

EXPERIMENTAL DESIGN

The dose-range finding experiment with 14-day application period was performed with 4 groups of treated animals.
The doses for the dose-range finding experiment were chosen with respect to the literature data as follows:

Dose levels: 125, 250, 500 and 1000 mg/kg/day

Further experimental design:

Vehicle: olive oil (pharmaceutical quality)
Administration: by gavage
Species: albino laboratory rat
Strain: Wistar Han (outbred SPF quality - guaranteed)
Housing: convention animal room
Total number of animals: 20 males and 20 females (5 males + 5 females per dose-group)
Animal arrival: 30. 5. 2012
Acclimatization period: 5 days
Application: 4. – 17. 6. 2012
Body weight: 4. 6. 2012 and then weekly
Health condition check: daily
Clinical observation: daily – repeatedly in different time intervals (for findings of maximal toxic effect of the test substance after application)
Haematology examination: after 14-day of application, basic parameters - 18. 6. 2012
Pathological examination: gross necropsy - 18. 6. 2012
Mortality observation: daily

RESULTS

Mortality observation: No animal died during the 14-day period.

Health condition and clinical observation: No serious changes of animal health status and clinical symptoms of intoxication were observed in treated animals. Since the 8th day of application the congestion of mucous membranes and skin of apical part of the body was recorded in males and females of dose levels of 125, 250, 500 and 1000 mg/kg/day.

Body weight:
Weight increments of males were similar at all dose levels for the whole time of application period.
Weight increments of females were similar at all dose levels for the whole time of application period.
Weight increments were adequate to species, sex and age of animals used in experiment.

Haematological examination: Average values of basic blood parameters of males and females of all dose levels were similar and did not markedly exceed the historical control range.

Pathological examination: No serious macroscopical changes were observed during necropsy of treated animals. Only the changes not related to the test substance administration were registered in female rats at the dose levels of 250, 500 and 1000 mg/kg.

CONCLUSION

In the dose-range finding experiment with the test substance 2 -ETHYLHEXYLAL the changes of body weight increments were not observed in treated males and females.
Health condition control and clinical observation did not detect adverse impact of the test substance on the health condition, clinical status and behaviour of animals at all dose levels.
Results of haematological examination did not show the test substance influence on blood parameters.
During pathological examination the changes were observed (uterus – dilatation), but these changes are not related to the test substance action.
No animal died during the 14-day application period.

On the basis of the results given above the following the dose levels - 160, 400 and 1000 mg/kg/day were chosen for the main Reproduction/Developmental Toxicity Screening Test.

Examinations

Parental animals: Observations and examinations:
Body Weight
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia.
Weight increment was computed as an mean per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

Food Consumption
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from mean values of each group.
The same way of calculation of mean food consumption was used for females in premating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Non pregnant and aborted females (females without parturition) were not included in calculation of mean food of pregnant females.

Mortality Control
All rats during the treatment periods were examined for vitality or mortality changes daily.

Health Condition Control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before and during application.

Clinical Observations of Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 - 14 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

Examination of Vaginal Smears
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.

Sperm parameters (parental animals):
Observation of Sperms
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperms was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 – fast progressive motility, 2 - slow progressive motility, 3 – no progressive motility, 4 – non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck ¬– were recorded.
Litter observations:
Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was
performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Postmortem examinations (parental animals):
Pathological Examination
Parental males were killed at the end of the administration period – after 28 days of administration. Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 26th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

Biometry of Reproductive Organs
The absolute weights of testes, epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Histological Technique
The following tissues and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
Organs with macroscopical changes (liver and kidneys) in males and females were histopathological examined at the middle and at the highest dose level groups.
The full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the following publications:
1. Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990);
2. Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997);
3. Russel, L.D. Ettlin, R. A., Sinha Hikim A. P., Clegg E. D., Histological and histopathological evaluation of the testis (Cache River Press, 1990);
4. Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

C) Reproduction Parameters
All females were mated (presence of sperm was found) with except one female at the highest dose level.
The numbers of females achieving pregnancy and duration of pregnancy were comparable between the control and treated groups.
The duration of mating of the dose level 160 mg/kg/day females was same as in control group. The females at the dose level of 1000 mg/kg/day mated slightly longer against control and the lowest dose level. The numbers of females bearing live pups and females with live pups at day 4 after parturition were slightly lower at all dose levels. The lowest number of females bearing live pups was observed at the dose level 1000 mg/kg/day.
The number of abortions was increased with dose. At the highest dose level 2 aborted females, at the middle dose level 1 aborted female were observed.
No statistically significant differences of the numbers of corpora lutea and implantations were detected. The numbers of corpora lutea and implantation were balanced in group 400 mg/kg/day compared to control. At the dose level 1000 mg/kg/day the number of corpora lutea and implantations was decreased against control.
The mating index (provides information on the integrated function of the neuroendocrine-gonadal axis) and gestation index (number of females delivering live young/number of females with evidence of pregnancy) were decreased at the highest dose level. The fertility index - ability of the male and female to achieve a pregnancy, was decreased at the lowest dose level compared to control.
Slight decrease of pre-implantation losses was recorded at the highest dose level but it cohered to lower number of corpora lutea at this dose levels. Slight decrease of post-implantation losses at the highest dose level is associated with lower number of implantations at this dose level. The decrease of post-natal losses at the highest dose level was connected with decreased numbers of live pups. The survival index was not changed.
Postmortem examinations (offspring):
Pathological Examination
Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight, biometry of organs and number of pups. Control group with vehicle was compared to three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.
Reproductive indices:
Implantation index: (number of implants / number of corpora lutea) x 100

Preimplantation index: ((number of corpora lutea – number of implants) / number of corpora lutea) x 100

Postimplantation index: ((number of implants – number of viable fetuses) / number of implants) x 100

Male mating index: (number of males with confirmed mating / number of males cohabited) x 100

Female mating index: ((number of sperm-positive females) / number of cohabited females) x 100

Male fertility index: (number of males impregnating a female / number of males cohabited) x 100

Female fertility index: (number of pregnant females / number of sperm-positive (cohabited) females) x 100

Gestation index: (number of females with live born pups / number of females with evidence of pregnancy) x 100

Offspring viability indices:
Survival index = (number of live pups on day 4 post partum* / number of pups born alive+) x 100

Note: * without still born pups (dead pups with anaerial lungs)
+ with dead pups with aerial lungs

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Parental Males
In treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. During the application period no clinical changes were observed in control males.
In animals of all dose levels the following changes were detected during the health condition control and clinical observation since the 2nd week of application: congestion of mucous membranes and skin of apical part of the body.

Parental females
In treated females of all dose levels no signs of disease were recorded during the check-in and acclimatisation period. During the application period no clinical changes were observed in control females.
In animals of all dose levels during the health condition control and clinical observation since the 2nd week of application were detected: congestion of mucous membranes and skin of apical part of the body. In females of the dose level of 1000 mg/kg/day salivation and piloerection were detected. The salivation was detected since the 5th week of application in two females and piloerection was observed since the 6th week of application in one female.
Mortality:
no mortality observed
Description (incidence):
Parental Males
There were no unscheduled deaths during the whole study.

Parental females
There were no unscheduled deaths during the whole study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Parental Males
Statistical analysis was performed for necropsy body weights. The statistically significant differences were not detected. Body weights of control males and males of the lowest and the middle dose level were quite balanced during the application period.
The mean body weight of treated males at the dose levels 1000 mg/kg/day was lower in comparison with the control males during the application period. The weight increments in males of the highest dose level were markedly lower compared to control in the 1st and the 3rd week of the application period.

Parental females
Before mating period
The mean body weight and the mean body weight increments of the control females and treated females were well balanced (slightly increased in the 2nd week in treated females).
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy.
The mean body weight of treated mothers at all dose levels were similar in comparison with control whereas mean body weight increments of the females at the dose levels 1000 mg/kg/day were slightly decreased against control females on the 14th and 20th day of pregnancy.

Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. The mean body weight increments of treated mothers at all dose levels were decreased against control but this difference was not dependent on dose level.

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Parental Males
The mean food consumption of treated males at all dose levels was decreased compared to the food consumption of the control males.

Parental females
Statistical analysis was performed for mean food consumption in pregnancy (7th – 20th day) and in lactation.

Pre-mating period
The mean food consumption of females at the middle dose level was lower than in control females.
Pregnancy
Females without parturition (non pregnant, aborted, non paired females) were not included in evaluation of food consumption during pregnancy.
The mean food consumption of mothers at all dose levels was decreased against control mothers (statistically significant at the 2nd week of pregnancy).



Lactation
Only mothers (without non pregnant, aborted, non paired females) were included in evaluation of food consumption during lactation period.
The mean food consumption of treated females at all dose level was statistically significantly lower than in control females (without dose dependence).

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Parental Males
Histopathological examination of the male reproductive system was performed at the control group and at the highest dose. The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels of 0 -1000 mg/kg/day further in the text.
Slight lymphocyte infiltration of the interstitium and epithelium in the epididymis was observed in 0-2 males. The germ cells in lumen of tubules in epididymis were detected in 2-2 males.
In the testes degeneration and/or atrophy of germinative epithelium in testicular tubules (less than 10 % tubules) were detected in 3-1 males. The exfoliation of germ cells to lumen of tubules were observed in 6-5 males (to 10 % tubules), in 0-1 male (more than 10 % tubules).
Lymphocyte infiltration of the interstitium was described in the prostate gland of 1-2 males. Atrophy of alveolar epithelium was detected in prostate gland of 0-1 male. The focal oedema of interstitium occurred in the prostate gland of 1-1 males. The focal desquamation of epithelium was observed in the seminal vesicles of 0-1 male, in the coagulating gland of 0-1 male. Presence of cysts in adenohypophysis was detected in pituitary gland of 2-0 males.

Histopathological examination of the liver and kidneys was performed at the middle and at the highest dose levels and is expressed in numeric form and ranged in sequence of the dose levels of 400-1000 mg/kg/day further in the text. The more often observed histopathological finding was hepatocellular hypertrophy (in 12-10 males). The focal infiltration and/or fibrosis were detected in liver of 2-3 males; vacuolation of hepatocytes in 1-0 male.
The tubular hypertrophy in cortex of kidneys was determined in 4-8 males, haemorrhage in kidneys in 0-1 male.

Parental females
The incidence of affected females is expressed in numeric form and ranged in sequence at the dose levels of 0-160-400-1000 mg/kg/day further in the text.
Histopathological examination of the organs of female genital tract (vagina, cervix, uterus, ovary) showed changes only in the uterus – in both control and treated mothers: signs of previous gravidity (focal accumulation of macrophagic elements - lipophages and siderophages in mesometrium) were recorded in 10-7-9-7 females; siderophages in mucosa in 7-1-8-5 females. Hydrometra (accumulation of ovulatory intraluminal fluid – spontaneous change during the oestrous cycle) were detected in 0-1-0-1 females. Acute endometritis (most propably of spontaneous origin) was observed in 0-0-0-1 female. Organizing hematoma was detected in 3-3-1-3 females.
Small myoepithelial adenoma (most propably of spontaneous origin) in mammary gland was found in 0-0-0-1 female.
The observation of the liver was performed at the middle and at the highest dose levels and is expressed in numeric form and ranged in sequence at the dose levels of 400-1000 mg/kg/day further in the text. In the liver parenchyma were found hypertrophy (in 6-10 females) accompanied by the presence of extramedullary hemapoiesis in 3-3 females.

Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility was similar in control and treated males (sperm motility was the same in control males and males at the dose level of 400 mg/kg/day). A fast progressive motility of sperms was observed in 11-12-11-10 males (sequence of the dose levels: 0-160-400-1000 mg/kg/day). Slow progressive motility of sperms was observed in 1-0-1-2 males. Presence of “no progressive motility” and “non-motile sperms” was not detected.
The percentual proportion of morphologically changed sperms was increased in all treated males. The most frequent changes were bent tails in sperms.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproductive performance – ability of males to successfully mate was unaffected by the test substance treatment.
Reproductive performance of females - ability to produce viable offspring was affected by the test substance treatment.

All females were mated (presence of sperm was found) with except one female at the highest dose level.
The numbers of females achieving pregnancy and duration of pregnancy were comparable between the control and treated groups.
The duration of mating of the dose level 160 mg/kg/day females was same as in control group. The females at the dose level of 1000 mg/kg/day mated slightly longer against control and the lowest dose level. The numbers of females bearing live pups and females with live pups at day 4 after parturition were slightly lower at all dose levels. The lowest number of females bearing live pups was observed at the dose level 1000 mg/kg/day.
The number of abortions was increased with dose. At the highest dose level 2 aborted females, at the middle dose level 1 aborted female were observed.
No statistically significant differences of the numbers of corpora lutea and implantations were detected. The numbers of corpora lutea and implantation were balanced in group 400 mg/kg/day compared to control. At the dose level 1000 mg/kg/day the number of corpora lutea and implantations was decreased against control.
The mating index (provides information on the integrated function of the neuroendocrine-gonadal axis) and gestation index (number of females delivering live young/number of females with evidence of pregnancy) were decreased at the highest dose level. The fertility index - ability of the male and female to achieve a pregnancy, was decreased at the lowest dose level compared to control.
Slight decrease of pre-implantation losses was recorded at the highest dose level but it cohered to lower number of corpora lutea at this dose levels. Slight decrease of post-implantation losses at the highest dose level is associated with lower number of implantations at this dose level. The decrease of post-natal losses at the highest dose level was connected with decreased numbers of live pups. The survival index was not changed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms.
Dose descriptor:
NOAEL
Remarks:
for reproduction
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Value based predominantly on decreased numbers of corpora lutea, decreased numbers of implantations and number of mothers with live born pups and increased number of the aborted females.
Dose descriptor:
NOAEL
Remarks:
for development
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: P1 (second parental generation)

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Value based predominantly on decreased numbers of corpora lutea, decreased numbers of implantations and number of mothers with live born pups and increased number of the aborted females.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The statistical evaluation of the number of live pups was performed. No statistically significant changes were recorded.
The total numbers of live pups and mean number of pups per litter (on the day of parturition/1st day after parturition and the 4th day of lactation) at all dose level was decreased in comparison with control group. At the dose level 1000 mg/kg/day decreased numbers of pups related with decreased numbers of females with live pups. At the dose levels 160 and 400 mg/kg/day the changes were not dose dependent.
Sex ratio of pups at all dose levels was balanced with the control. At the dose levels 400 mg/kg/day were detected increased portion of female pups than male pups.

Five pups from one litter at the dose level of 400 mg/kg/day and 12 pups - whole litter at the dose level of 1000 mg/kg/day were still born.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the litters at all dose levels were slightly lower compared to control but without dose dependence.
The mean body weights of the pups at all dose levels were in balance with control
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The macroscopical examination was performed in all pups. No pathological findings were recorded in pups of control group and treated groups.

Details on results (F1)

Number and Sex Ratio of Pups
The statistical evaluation of the number of live pups was performed. No statistically significant changes were recorded.
The total numbers of live pups and mean number of pups per litter (on the day of parturition/1st day after parturition and the 4th day of lactation) at all dose level was decreased in comparison with control group. At the dose level 1000 mg/kg/day decreased numbers of pups related with decreased numbers of females with live pups. At the dose levels 160 and 400 mg/kg/day the changes were not dose dependent.
Sex ratio of pups at all dose levels was balanced with the control. At the dose levels 400 mg/kg/day were detected increased portion of female pups than male pups.

Body Weight
The mean body weights of the litters at all dose levels were slightly lower compared to control but without dose dependence.
The mean body weights of the pups at all dose levels were in balance with control.

Development of Pups
Five pups from one litter at the dose level of 400 mg/kg/day and 12 pups - whole litter at the dose level of 1000 mg/kg/day were still born.

Pathological Examination
The macroscopical examination was performed in all pups. No pathological findings were recorded in pups of control group and treated groups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Based predominantly on the major manifestations of developmental toxicity death of the developing organism.

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Individual data tables of parental males and females and Data tables of pups are provided in 'attached background material' section.

Applicant's summary and conclusion

Conclusions:
The growth (body weight and food consumption), clinical status and biometry and structure of reproductive organs of parental animals were not adversely affected by the test substance treatment. The quality of sperms of parental males was not adversely influenced by the 28thlong test substance treatment.     
Reproductive performance – ability of males to successfully mate was unaffected by the test substance treatment.
Reproductive performance of females - ability to produce viable offspring was affected by the test substance treatment.
The maternal toxicity(toxic effect on a pregnant woman or nursing mother) could affect an embryo and foetus.
Sex ratio and postnatal development of pups were not changed in treated groups but is not possible to exclude influence of the test substance treatment to prenatal development of offspring. 
 
The test substance, 2 - ETHYLHEXYLAL, directly affected theliverin males and females at the dose levels of 400 and 1000 mg/kg/day but this findings has no impact on establishing of NOAELs for reproduction and development. 
 
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day.The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms.
 
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females was established as 400 mg/kg body weight/day.Value based predominantly ondecreased numbers of corpora lutea, decreased numbers of implantationsand number of mothers with live born pups and increased number ofthe aborted females. 
    
The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of pups was established as 400mg/kg body weight/day.Value based predominantly onthe major manifestations of developmental toxicity death of the developing organism. The toxic effect of the test substance on the structurnal abnormality, altered growth and functional dificiencyis not possible to exlude due to abortions and cannibalism of the developing organism.
Executive summary:

Introduction

The test substance, 2-ETHYLHEXYLAL,was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th1995.

This study provides information on the possible harmful effects of the test substance on reproduction and development after repeated exposure on parental generation and first generation pups.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in olive oil (pharmaceutical quality) using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 160, 400, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 12 males and 12 females.The dose levels for study were chosen on the basis of the results of the dose-range finding experiment with 14-day application period.

The treated groups were administered daily for the following periods:

- males and females – 2 weeks prior to the mating period and during the mating period,

- pregnant females – during pregnancy and till the 3rdday of lactation,

- males  after mating period – totally for 28 days,

- non pregnant females (mated females without parturition) – for 25 days after the confirmed mating.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

Results

The test substance, 2- ETHYLHEXYLAL, did not seriously affect growth of parental males and females at all dose levels. The results relevant to mean body weight males and females were not statistically significant.

The slightly lower food consumption was recorded in treated males. The mean body weight increments of treated mothers in lactation period at all dose levels (not dose dependent) were decreased against control, which related with decrease meanfood consumption of treated mothers in pregnancy and lactation (statistically significant at the 2ndweek of pregnancy and in lactation). The results relevant to growth could be considered not to be of toxicological importance.

 

The clinical examination revealed congestion of mucous membranes and skin of apical part of the body in all treated animals since the 2ndweek. The macroscopic changes in the liver (enlarged, light colour, marked structure) were observed in males and females atthe dose levels of 400 and 1000 mg/kg/day.The changes in the kidneys (enlarged, light colour, marked structure) were detected only in males at the dose levels of 400 and 1000mg/kg/day.

In females of the highest dose the salivation (only in 2 females) and piloerection (only in one female) were observed sporadically.

 

The biometry of organs showed only statistically insignificant differences in treated parental males and females. Decrease of absolute and relative weight of prostate gland was recorded in parental males of the highest dose level and was not considered to be toxicologically relevant because microscopic findings (focal oedema and lymphocyte infiltration in interstitium) were observed also in control males.In males at thedose level of 1000 mg/kg/daywas also observed reduction of absolute and relative weight of epididymis and pituitary gland. Changes in these organ weights were not accompanied by significant morphological changes in the same tissues.

The histopathological findings in epididymis (slight lymphocyte infiltration of the interstitium and epithelium) were observed in two males at the highest dose. The finding of germ cells in lumen of tubules (in two males at the highest dose) is very sensitive indicator of spermatogenic disturbance in the rat but the same number of affected animals was observed also in control animals.

 

The histopathological examination of the liver revealed findings in animals of the doses 400 and1000 mg/kg/day (hypertrophy, in some females accompanied by extramedullary hemopoiesis; focal infiltration and vacuolation in males) caused by the test substance. It is probably adaptation response to application of the test substanceIn male kidneys at the middle and high dose the tubular hypertrophy and in one case haemorrhage in cortex were observed.

 

The relative and absolute weights of organs in treated females were not statistically significant different versus control animals and histopathological examination of female reproductive organs and pituitary gland did not reveal any serious changes. Only changes relating to previous gravidity or proceeded oestrous cycle were recorded.

 

Absolute and relative weights of the testes were not changed in treated males and were similar to control. Histopathological examination of the testes of parental males showed some changes of testicular microscopical structure but without damage of spermiogenesis.The exfoliation of germ cells to lumen of tubules was observed in treated and also in control animals so it can not be assessed as spermatogenic disturbance factor which was caused by the test substance. 

Observation of sperm motility and sperm morphology did not reveal serious damage of sperms quality in treated males versus control. The test substance did not influence sperm motility.The percentual proportion of morphologically changed sperms was slightly increased in alltreated males. The most frequent changes were bent tails in sperms. In spite of sporadic change in sperm morphology thefertility of treated males was not affected. But the animals in the reproduction screening study are treated for the time period which is shorter than the duration of whole spermatogenic cycle so that an effect on spermatogenesis may not have had adequate time to become evident as reduced sperm counts that affect fertility.

Observation of pups revealed statistically insignificant difference. The mean body weights of pups on the day of parturition/1stday after parturition and the 4thday of lactation were comparable.Sex ratio were unaffected by the test substance treatment.

The litter size (an important indicator of overall reproduction performance) was changed – decreased number of pups at all dose levels versus control groupwas observed. Marked decreasing of mean number of pups per litter in the highest dose level was probably caused by decreased numbers of mothers delivered live pups (11-9-10-7). The parturition of stillborn pups was observed at the dose levels of 400 and 1000 mg/kg/day. Mortality of live born pups was not detected.

 

Reproduction parameters– ability of males and females to successfully mate and achieve pregnancy was not adversely influenced by the test substance treatment. The number of females achieving pregnancy and duration of pregnancy were unchanged in treated groups compared to control. The duration of mating was slightly longer at the highest dose level.

The fertility index was decreased without dose dependence and the alteration was observed also in control group. In fertility indexmay be difficult to determinate the affected sex when both sexes were dosed furthermore males were dosed for less than the duration of the spermatogenic cycle so reproductive effect may not be manifested in the fertility index.

The mating index and gestation index were decreased at the highest dose level.The numbers of aborted females were increased with doses. At the highest dose level 2 aborted females, at the middle dose level 1 aborted female were observed.

The decreased numbers ofcorpora lutea and numbers of implantations at the highest dose level manifested statistically insignificant differences. Slight decreased of pre-implantation losses, post-implantation losses and post-natal losses at the highest dose level associated with lower number of corpora lutea, implantations and number of live pups.

The survival index– an important endpoint reflects the ability of the pups to survive – was not changed.

   

Conclusion

The growth (body weight and food consumption), clinical status and biometry and structure of reproductive organs of parental animals were not adversely affected by the test substance treatment. The quality of sperms of parental males was not adversely influenced by the 28thlong test substance treatment.     

Reproductive performance – ability of males to successfully mate was unaffected by the test substance treatment.

Reproductive performance of females - ability to produce viable offspring was affected by the test substance treatment.

The maternal toxicity (toxic effect on a pregnant woman or nursing mother) could affect an embryo and foetus.

Sex ratio and postnatal development of pups were not changed in treated groups but is not possible to exclude influence of the test substance treatment to prenatal development of offspring. 

 

The test substance, 2 - ETHYLHEXYLAL, directly affected the liver in males and females at the dose levels of 400 and 1000 mg/kg/day but this findings has no impact on establishing of NOAELs for reproduction and development. 

 

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day.The conclusion is based predominantly on unaffected ability to successfully mate and unaffected quality of sperms.

 

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females was established as 400 mg/kg body weight/day.Value based predominantly on decreased numbers of corpora lutea, decreased numbers of implantations and number of mothers with live born pups and increased number ofthe aborted females. 

    

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of pups was established as 400mg/kg body weight/day.Value based predominantly onthe major manifestations of developmental toxicity death of the developing organism. The toxic effect of the test substance on the structurnal abnormality, altered growth and functional dificiencyis not possible to exlude due to abortions and cannibalism of the developing organism.

 

 

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