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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL , the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-05 until 2012-02-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
, adopted 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, EPA Health Effects Test Guidelines, July 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Hsd.Brl.Han:Wist
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male animals: 10-11 weeks old
Female animals: 10-11 weeks old
- Weight at study initiation:
Male animals: 298 – 328 g
Female animals: 195 – 223 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet (e.g. ad libitum):
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water (e.g. ad libitum):
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
6 days, satellite groups 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil, at concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations were prepared in the laboratory of Test Facility 2-3 days before the use. Analytical control of concentration of dosing solutions was performed in the laboratory of the Test Facility. The measured concentrations varied in the acceptance range in comparison to the nominal values at all analytical occasions.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is not soluble in water; therefore sunflower oil (Oleum helianthi) was used for preparing formulations appropriate for oral administration. Oleum helianthi /sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Amount of vehicle (if gavage):
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.
Details on mating procedure:
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front using a GC/MS method. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations. Aldehyde L proved to be stable in sunflower oil formulations at room temperature up to 72 hours (recovery was >90% in concentration range of 50 - 500 mg/mL). Concentration of the test item in the dosing solutions varied in the acceptance range in comparison to the nominal values at all analytical occasions.
Duration of treatment / exposure:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 49 or 63 days and satellite male animals for 45 days (14 days pre-mating, 23 days mating and 12 or 26 days post-mating; satellite animals: 14 days pre-mating, 4 days mating and 27 days post-mating), then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3, 4 or 5 (for 41 – 61 days; satellite animals for 41 – 44 days. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (for 44, 47 or 51 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.
Frequency of treatment:
Animals were treated once per day.
Details on study schedule:
Male animals were dosed for 49 or 63 days and satellite male animals for 45 days (14 days pre-mating, 23 days mating and 12 or 26 days post-mating; satellite animals: 14 days pre-mating, 4 days mating and 27 days post-mating), then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3, 4 or 5 (for 41 – 61 days; satellite animals for 41 – 44 days. The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant and not mated female animals were treated up to and including the day before necropsy (for 44, 47 or 51 days).
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 animals/sex in the control and low dose groups
12 animals/sex in the middle and high dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
The dose setting was based on findings obtained in a previous oral repeated dose toxicity study. The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
All parent animals were weighed with an accuracy of 1 g. Parent male animals were weighed on the first day of dosing (day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 20 and on postpartal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals was weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.
Oestrous cyclicity (parental animals):
Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.
Postmortem examinations (parental animals):
Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):
GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality
There was no test item related mortality during the course of study.

Clinical Observations
Daily Observations
Test item related clinical signs were not observed at any dose level (1000, 300 or 100 mg/kg bw/day) during the entire observation period. The behavior and physical condition of animals were considered to be normal.
Alopecia was noted for two male (1/15 control between days 56 and 62; and 1/15 100 mg/kg bw/day between days 27 and 48) and one non-pregnant female (1/15 100 mg/kg bw/day between days 38 and 46) animal on the forelimb, on thigh, scrotum, abdomen and on the back, respectively. Alopecia is a common findings in this strain of experimental rats and occurred in the control and low dose treated animals, therefore had no toxicological meaning in this study.

Detailed Weekly Observations
There were no test item related clinical signs during the weekly detailed observations in male animals at any dose level during the entire observation period (pre-mating, mating and post-mating).
Alopecia as described above was also observed and recorded for two male and one female animal at the weekly clinical observations.

Body Weight
The body weight development was undisturbed in the test item treated animals with respect to controls.
In the male animals, the mean body weight was similar to that of the control group during the observation period. The slight but statistically significant differences indicated a lower mean body weight gain at 100 mg/kg bw/day between days 13 and 20 and a slightly higher mean body weight gain in all test item treated groups between days 20 and 27 with respect to controls.
There were no significant differences in the mean total body weight gain between the control and test item treated group at any dose level.
The higher mean total body weight gain in the female animals dosed with 1000 and 300 mg/kg bw/day resulted in a higher mean body weight on day 13. Statistical significances were also noted for the higher body weight values at 300 mg/kg bw/day on gestation days 0 and 14 and for a higher mean total body weight gain at 100 mg/kg bw/day during the lactation period.
All these statistically significant differences with respect to control were not considered to be of toxicologically significance because the effects were only present in the low dose group or body weight values exceeded the control. Therefore these effects were considered to be of no toxicological relevance.

Food Consumption
The mean daily food consumption was not affected by the treatment with the test item in male and female animals at any dose level. The slightly statistically significantly higher mean food consumption of male animals dosed with 1000 mg/kg bw/day between days 20 and 27 was not judged to be toxicologically significant.

Delivery Data of Dams
There were no test item related differences between the control and dosed groups in the delivery data of dams. The duration of pregnancy, the mean of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss and total intrauterine mortality was similar in the control and all test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of total births, live-borns, stillborns and viable pups.

Reproductive Performance
There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance. The percentage of fertile male animals was slightly less and the percentage of infertile male animals was slightly higher with respect to controls at 1000 and 100 mg/kg bw/day (no statistical significance), however it was not considered to be related to test item influence as there were no similar findings at 300 mg/kg bw/day and the difference from the control was higher at the lower dose than for the higher dose.
There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval were similar to the appropriate control values in all test item treated groups.

Necropsy
No test item related alterations were found at the macroscopic observations of organs and tissues at the necropsy.
Smaller than normal seminal vesicles (unilateral) were observed in two high dose treated male rat (2/12) at 1000 mg/kg bw/day.
In one male animal dosed with 300 mg/kg bw/day (1/12), one side hydronephrosis and accompanying enlargement and pyelectasia of the other side kidney were observed.
Alopecia was noted for one male animal in 100 mg/kg bw/day (1/12) and in the control (1/12) groups.
Macroscopic findings were not detected in dams and in single pregnant female rat not delivered.
In the non-pregnant females alopecia on the back (1/4) and moderate hydrometra (1/4) were observed at 100 mg/kg bw/day dose level.
In the only not mated female rat (1/12) dosed with 100 mg/kg bw/day, one side ovarian cyst and moderate hydrometra were seen.

In summary, no macroscopic findings related to the test item effect were detected. The smaller than normal seminal vesicles were present in the high dose treated animals however histopathological examination did not reveal any lesion thus these were judged to be toxicologically not relevant. Hydronephrosis, enlarged and pale kidneys, alopecia ovarian cyst and hydrometra are species-specific alterations commonly occurring in experimental rats, so these were considered to be individual changes irrespective of the treatment.

Organ Weight
The absolute and relative organ weights did not demonstrate any test item related alterations. There were no toxicologically significant differences between the control and test item treated groups in the examined organ weights.
Statistical significances was noted for the testes weights (absolute and relative to the brain weight) at 1000 mg/kg bw/day. However, the mean and individual testes weight remained within the historical control ranges.
(Historical control values of absolute testes weight:
mean: 3.48 g; SD: 0.31 g; n = 18; min = 3.04 g; max = 4.20 g;
testes weight relative to brain weight:
mean: 170.95 g; SD: 13.56 g; n = 18; min = 155.1 g; max = 201.92 g;)

Also, statistical significances was noted for ovaries weight relative to the body weight at 300 mg/kg bw/day. Regarding the ovaries weight, similar findings were not found at the high dose (1000 mg/kg bw/day).
The degree of weight change for testis and ovaries was only slight and no abnormal histopathological findings were observed. Therefore these effects were not considered to be toxicologically relevant.

Histopathology
Histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles with coagulating gland) and pituitary did not reveal any toxic or other test item related alterations at 1000 mg/kg/bw/day dose.
In the male animals the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated animals. Histologically, epididymides, and pituitary gland were normal in all cases as well.
In the female animals including not mated and non-pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.





Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Mortality
There was no test item effect on pup’s mortality.
Extra uterine mortality did not occur in the 1000 mg/kg bw/day group. Single pups were found dead or were missing at 100, 300 and 1000 mg/kg bw/day group in ratios of 1/160, 1/100 and 1/110, respectively, between days 0 and 4. The survival indices were similar between all groups and it was 100 % in the 1000 mg/kg bw/day group.

Sex Distribution
There were no significant differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4

Clinical Observations
Test item related clinical signs did not appear in pups. The percent of pups without findings exceeded the control value at 1000, 300 and 100 mg/kg bw/day. Cold pups were only found in the control group.

Body Weight
The mean litter weight and mean pup weight were similar in the control and test item treated groups, no test item related changes were found.

Necropsy
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Key result
Reproductive effects observed:
not specified
Conclusions:
The reprotoxic properties of Aldehyde L were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL , the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.
Executive summary:

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Aldehyde L on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. The study was conducted in accordance with OECD guideline No. 421.

Four groups of Hsd.Brl.Han:Wist rats (n=15/sex in the control and low dose group and n= 12 in the middle and high dose groups) were administered orally (by gavage) once a day at 0 (vehicle only),100, 300 and 100 and 1000 mg/kg bw/day at concentrations of 50, 150 and 500 mg/mL corresponding to 2 mL/kg bw dose volume. Group of satellite animals (3 animals/sex/group; control and 100 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters at the low dose. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation day 3, 4 or 5, i.e. up to the day before the necropsy.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. The dams were allowed to litter, and rear their young up to termination on days 4, 5 or 6 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups.

Results  

Mortality

There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day).  

Clinical observation

No toxic signs related to the test item were found at general daily and detailed weekly clinical observations. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).

Body weight and body weight gain

The body weight development of parental male and female animals was undisturbed in the course of the entire study. No test item-related body weight, or body weight gain changes were observed with respect to controls at any dose level during the pre-mating, mating, post-mating, gestation and lactation periods.  

Food consumption

The mean daily food consumption was not influenced by the test item.  

Reproduction

There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.  

Necropsy

Specific macroscopic alterations related to the test item were not found during the necropsy.  

Organ weight

There were no test item related changes in testes and epididymides weights.  

Histopathology Histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level.  

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs body weight, necropsy findings) were not detected between postnatal days 0 and 4(occasionally to days 5, 6).

Conclusion

Under the conditions of the present study Aldehyde L did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 (occasionally to days 5, 6) post-partum after repeated dose oral administration at 100, 300 or 1000 mg/kg bw/day.    

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:  

NOAEL for male and female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the male and female rats:1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproduction/Developmental toxicity screening test


The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Aldehyde L on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. The study was conducted in accordance with OECD guideline No. 421.


Four groups of Hsd.Brl.Han:Wist rats (n=15/sex in the control and low dose group and n= 12 in the middle and high dose groups) were administered orally (by gavage) once a day at 0 (vehicle only),100, 300 and 100 and 1000 mg/kg bw/day at concentrations of 50, 150 and 500 mg/mL corresponding to 2 mL/kg bw dose volume. Group of satellite animals (3 animals/sex/group; control and 100 mg/kg bw/day group) were involved in the study to ensure the appropriate number of litters at the low dose. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation day 3, 4 or 5, i.e. up to the day before the necropsy.


Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. The dams were allowed to litter, and rear their young up to termination on days 4, 5 or 6 postpartum. Pups were weighed and observed for possible abnormalities. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups.


 


Results  


Mortality


There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day).  


 


Clinical observation


No toxic signs related to the test item were found at general daily and detailed weekly clinical observations. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods).


 


Body weight and body weight gain


The body weight development of parental male and female animals was undisturbed in the course of the entire study. No test item-related body weight, or body weight gain changes were observed with respect to controls at any dose level during the pre-mating, mating, post-mating, gestation and lactation periods.  


 


Food consumption


The mean daily food consumption was not influenced by the test item.  


 


Reproduction


There were no differences between the control and test item treated groups in the reproductive performance of male and female animals and in delivery data of dams.  


 


Necropsy


Specific macroscopic alterations related to the test item were not found during the necropsy.  


Organ weight


There were no test item related changes in testes and epididymides weights.  


Histopathology examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level.  


 


Offspring


Negative effects of the test item on offspring development (mortality, clinical signs body weight, necropsy findings) were not detected between postnatal days 0 and 4(occasionally to days 5, 6).


 


Conclusion


Under the conditions of the present study Aldehyde L did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 (occasionally to days 5, 6) post-partum after repeated dose oral administration at 100, 300 or 1000 mg/kg bw/day.    


Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:  


NOAEL for male and female rats: 1000 mg/kg bw/day


NOAEL for reproductive performance of the male and female rats:1000 mg/kg bw/day


NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

Based on the results of the OECD 414 study the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 May 2015 and 23 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.,1103 Budapest Cserkesz u. 90., Hungary
- Age at study initiation: females (10 weeks of age at start of the mating period), males (25-26 weeks of age at start of the mating period)
- Weight at study initiation: 167-224 g
- Housing: before mating: 1-3 females per cage, 1-2 males per cage; mating: 1 male and 1-3 females/cage; during gestation: 2-3 sperm positive females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days for females, 119 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations will be prepared in the laboratory daily to every three days.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore Sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 103 % of nominal concentrations at both analytical occasions confirming proper dosing.
Formulation samples are diluted with hexane and then analysed by a GC-FID method in the presence of heptadecane internal standard. Five samples were taken from the formulations. Two replicate dilutions were prepared from each sample. One dilution was carried out from the control samples. The samples were diluted with hexane in two consecutive steps. A volume of 0.5 mL of the internal standard solution was added in the second step before the samples were filled up to 10 mL final volume.
Gas chromatograph: Clarus 580 GC, No: 580S11081708
Balance: AB54-S, Mettler Toledo, No.: 1122092721

GC conditions:
Column: Elite-5MS, 30 m x 0.32 mm x 0.25 μm, No: 926895
Temperature program: 1 min hold, 165°C to 200°C at 10°C/min, 1 min hold
Injector: 250°C
Detector: FID, 250°C
Carrier gas: nitrogen, 2 mL/min, constant flow
Split ratio: 20
Injection volume: 1 μL
Retention times: Heptadecane 2.5 min, test item: 4.3 min
Evaluation: Ratio of the peak areas is calculated and used for the computations
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:3
- Length of cohabitation: The females were paired to males in the mornings for two to four hours until the number of sperm positive females per group achieves at least twenty four.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
7 days per week every day at similar time
Duration of test:
Treatment period: from gestational day 5 to 19
No. of animals per sex per dose:
24 sperm positive females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on findings obtained in a previous GLP Reproduction/Developmental Toxicity Screening Test with the test item in the rat (OECD 421, Toxi-Coop Zrt. 66.421.2959)
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (observations for signs of morbidity and mortality were made twice daily, at the beginning and end of the working period)
- Examinations: signs of morbidity, mortality, toxicity as well as behaviour and general conditions

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule: between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and ovary

OTHER:
- Examination of placental signs: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Historical control data:
The results were compared to the laboratory's historical control data.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. No treatment related reduction on food consumption or body weight was observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were two fetuses with different types of malformations during external examination.
Cleft palate observed in one fetus at 1000 mg/kg bw/day and umbilical hernia in another single fetus at 100 mg/kg bw/day were judged to be without a relationship to the treatment with the test item considering the historical control database and the experience with this strain at the lab as well as in the international scientific literature. During visceral examination malformations were found only in the control group. The malformations found sporadically in single fetuses during skeletal examination were judged to be unrelated from the treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Conclusions:
Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room for 7 days at the concentrations of 50 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 103 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 22 evaluated litters each in the control, 100 and 1000 mg/kg bw/day and 20 in the 300 mg/kg bw/day group.

None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed.

There was no treatment related reduction on food consumption and body weight indicated.

The test item did not influence the number of implantations, intrauterine mortality and sex distribution of the fetuses. With regard to fetal examinations, there were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations.

There were two fetuses with different types of malformations during external examination.

Cleft palate observed in one fetus at 1000 mg/kg bw/day and umbilical hernia in another single fetus at 100 mg/kg bw/day were judged to be without a relationship to the treatment with the test item considering the historical control database and the experience with this strain at the lab as well as in the international scientific literature.

During visceral examination malformations were found only in the control group.

The malformations found sporadically in single fetuses during skeletal examination were judged to be unrelated from the treatment.

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP conform study according to guideline
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity study


The test item was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.


A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room for 7 days at the concentrations of 50 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 103 % of nominal concentrations at both analytical occasions confirming proper dosing.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.


After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.


In total, on gestation day 20 there were 22 evaluated litters each in the control, 100 and 1000 mg/kg bw/day and 20 in the 300 mg/kg bw/day group.


None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed.


There was no treatment related reduction on food consumption and body weight indicated.


The test item did not influence the number of implantations, intrauterine mortality and sex distribution of the fetuses. With regard to fetal examinations, there were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations.


There were two fetuses with different types of malformations during external examination.


Cleft palate observed in one fetus at 1000 mg/kg bw/day and umbilical hernia in another single fetus at 100 mg/kg bw/day were judged to be without a relationship to the treatment with the test item considering the historical control database and the experience with this strain at the lab as well as in the international scientific literature.


During visceral examination malformations were found only in the control group.


The malformations found sporadically in single fetuses during skeletal examination were judged to be unrelated from the treatment.


 


Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:


NOAEL (maternal toxicity): 1000 mg/kg bw/day


NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Justification for classification or non-classification

Based on the test results, no classification and labelling is required according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.

Additional information