Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to Reproduction (screening): oral: gavage, rat (Wistar) m/f, 12/sex/dose, 0, 100, 300, 1000 mg/kg bw/d in sunflower oil, (OECD TG 422, GLP): no adverse effects in adults or offspring noted, NOAEL = 1000 mg/kg (systemic toxicity), NOAEL = 1000 mg/kg (reproductive toxicity)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental phase: 2018-08-01 - 2018-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP
Reason / purpose:
reference to same study
Remarks:
OECD 422
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No. 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test adopted 29 July 2016.
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Justification for study design:
As indicated in the guideline
Specific details on test material used for the study:
The test item of a suitable chemical purity, analytical certificate, test item data sheet and specification of the product was supplied by the Sponsor. All precautions required in the handling and disposal of the test item was outlined by the Sponsor.
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males & females: 83 – 85 days
- Weight at study initiation:
Males: 343 – 405 g; Females: 206 – 234 g; The weight variation did not exceed ± 20 per cent of the mean weight.
- Fasting period before study:
no
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals / cage
Cage type: Type III polypropylene/polycarbonate; Size: 22 x 32 x 19 cm (width x length x height)
Bedding: Certified laboratory wood bedding (Lignocel 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany).
The bedding is suitable as nesting material. Details of quality of bedding material were reported.
The cages and bedding were changed once or twice a week.
- Diet (e.g. ad libitum):
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. Food was changed at weekly intervals.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
- Water (e.g. ad libitum):
Animals received tap water, as for human consumption, ad libitum. Fresh drinking water was given daily. Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).
- Acclimation period:
20 days; only healthy animals were used for the study. Healthy status was certified by the breeder.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30 - 70 %
- Air changes (per hr):
Above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod (hrs dark / hrs light):
Artificial light, from 6 a.m. to 6 p.m.
Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.

IN-LIFE DATES: From: 2018-07-12 To: 2018-09-27
Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:

Hexanal was formulated in the vehicle (sunflower oil) in concentrations of 20, 60 and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at room temperature until use.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified. The recovery of the test item from the vehicle was within the acceptance criteria.
Hexanal proved to be stable in sunflower oil at the intended concentrations at room temperature and at 5 ± 3 °C for three days.
- Concentration in vehicle:
0, 20, 60, 200 mg/ml
- Amount of vehicle (if gavage):
5 ml/kg bw
- Lot/batch no. (if required):
1804-4239
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1-7 days
- Proof of pregnancy: Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at room temperature until analysis.
Date of sampling: August 08 and September 12, 2018
Date of analysis: August 09 and September 13, 2018
Concentration of the test item in the dosing formulations varied between the range of 91 % and 101 % in comparison to the nominal values.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified. The recovery of the test item from the vehicle was within the acceptance criteria.
Hexanal proved to be stable in sunflower oil at the intended concentrations at room temperature and at 5 ± 3 °C for three days.
Duration of treatment / exposure:
Dosing of both sexes begun after 20 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy. Rats of this strain reach full sexual maturity at the age of 10 weeks. The mating phase started after 14-days treatment (pre-mating) period.
Male animals were dosed for 42 days (14 days pre-mating and 1-7 days mating in animals plus 21-27 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-7 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Frequency of treatment:
The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours).
Details on study schedule:
- Age at mating of the mated animals in the study: Rats of this strain reach full sexual maturity at the age of 10 weeks.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Concentration of the test item in the dosing formulations varied between the range of 91 % and 101 % in comparison to the nominal values.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Concentration of the test item in the dosing formulations varied between the range of 91 % and 101 % in comparison to the nominal values.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Concentration of the test item in the dosing formulations varied between the range of 91 % and 101 % in comparison to the nominal values.
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were determined on the basis of the findings obtained from a previously performed oral preliminary toxicity screening test with Hexanal in rats. Doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
- Rationale for animal assignment (if not random): random
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). General clinical observations were made on parental animals once a day, after the administration at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
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 behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of all parental animals was determined with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated. Body weight was measured on the day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated according to measurement days and for the study overall (latter named also as summarized body weight gain).

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the given and non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13 and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

OTHER: see IUCLID chapter 7.5.1
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were prepared and evaluated in each female animal on the day of necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, detailed histological examination of testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender 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 litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on day and 13 post-partum. Litter weight were determined with an accuracy of 0.1 g. The litter weight was calculated by the individual weight of pups on postnatal day 4. Any abnormal behavior of the offspring was recorded.
All litters were checked and recorded daily for the number of viable and dead pups.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS:
Dead pup was subjected to necropsy by macroscopic examination on postnatal day 1.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Blood samples for determination of serum levels of thyroid hormones (FT4 and TSH) were drawn in the morning hours from 2-7 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter) and from 3-7 pups per litter on post-partal/post-natal day 13.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal terminally (one day after the last treatment).
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”) and were subjected to gross necropsy as follows:
Parental male animals: after the optionally extended post-mating period on Day 42.
Dams on post-partum days 14, 15 or 16 (on Days 51, 54 or 57).
Non-pregnant female animals on Days 42 and 44.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected from each group:

Adrenal glands
Aorta
Bone with bone marrow and joint (femur)
Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
Eyes (lachrymal gland with Harderian glands)
Mammary gland
Heart
Kidneys
Large intestines (caecum, colon, rectum),
Liver
Lungs (with main stem bronchi; inflation with fixative and then immersion;)
Lymph nodes (submandibular, mesenteric)
Muscle (quadriceps)
Esophagus
Pituitary
Pancreas
Salivary glands (submandibular)
Sciatic nerve
Sex organs (testes, epididymides, prostate seminal vesicle with coagulating gland, ovaries, uterus with cervix and fallopian tube, vagina)
Skin
Small intestines (duodenum, ileum, jejunum, including Peyer’s patches)
Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
Spleen
Sternum
Stomach
Thymus
Thyroid + parathyroid
Trachea
Urinary bladder
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.
Organ weight: At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight was not determined as no related changes were detected.
Paired organs were weighed together.

HISTOPATHOLOGY: Yes
Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
These organs were processed and examined histologically in non-pregnant female and male these females were cohabited with in the mid dose group.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day) groups.
In male animals, the esophagus (1/12 at 100 mg/kg bw/day), liver (1/12 at 300 mg/kg bw/day) were processed and evaluated histologically on the basis of necropsy observations).
In the female animals, kidneys (1/12 at 100 mg/kg bw/day), liver (1/12 at 100 mg/kg bw/day) and uterus (1/12 at 100 mg/kg bw/day and 3/11 dam and 1/12 non-pregnant female at 300 mg/kg bw/day) were processed and evaluated histologically.
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Postmortem examinations (offspring):
SACRIFICE
Gross necropsy was performed on each animal terminally (one day after the last treatment). In offspring, on postnatal day 13 or shortly thereafter.

GROSS NECROPSY
Pups euthanized on day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
Statistics:
The statistical evaluation of appropriate data was 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) is 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.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Copulatory index, Fertility index, Gestation index
Offspring viability indices:
Post-implantation mortality (intrauterine mortality), Post-natal mortality, Survival Index, Sex ratio
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs in any group, i.e. parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 1000 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Alopecia was noted for two control female animals (2/12). In one of them, alopecia was detected on the thorax, approximately 1 cm in diameter, from gestational day 18 up to lactation day 15. In the other one, alopecia was observed on the fore limbs and on the left side of the abdomen – approximately 2 cm in diameter – from gestational day 12 up to and including lactation day 14.
Alopecia was detected at the weekly clinical observations on gestation days 14 (1/12) and 21 (2/12) as well as on lactation days 0, 4 and 13 (2/12, on lactation days)
Alopecia is a common dermal change in experimental rats of this strain with similar age and it was only noted for control animals in this study. Therefore, this finding was not considered toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was undisturbed in male and female animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods.
Statistical significance with respect to the control was only detected at the lower mean body weight gain in the male animals at 100 mg/kg bw/day between Days 34 and 41 and at 1000 mg/kg bw/day between Days 7 and 13, as well as bw/day between Days 34 and 41.
These sporadic changes in the body mean weight gain were with minor degree and did not resulted in significant changes in the mean body weight of male. Therefore, this finding was considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no test item related adverse effect in the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day.
The mean daily food consumption was comparable in the control and test item treated groups at 100, 300 or 1000 mg/kg bw/day during pre-mating and post mating periods in male animals and during the pre-mating, gestation and lactation periods in female animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.
Statistically significant difference with respect their control was detected at the higher mean percentage of eosinophil granulocytes (EOS) in male animals at 100 mg/kg bw/day.
At 300 mg/kg bw/day, higher mean percentage of neutrophil granulocytes (NEU), lower mean percentage of lymphocytes (LYM) and higher mean platelet count (PLT) were observed when compared to their control in male animals.
The mean corpuscular volume (MCV) and mean hemoglobin content (MCH) were slightly lower in male animals at 1000 mg/kg bw/day compared to their control.
The examined hematological and blood coagulation parameters were comparable in female animals in the control and test item treated groups except for the slightly lower mean white blood cell count (WBC) at 300 mg/kg bw/day.
These differences were considered to be toxicologically not relevant due to the minor degree and in the lack of dose dependency (WBC, EOS, NEU, LYM, PLT). Values of MCV and MCH in male animals at 1000 mg/kg bw/day were within or marginal to the historical control ranges and there were no related changes in the red blood cell parameters. Therefore, these findings were judged to be not relevant.,
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
The examined clinical chemistry parameters were similar to their control in male animals at 100, 300 and 1000 mg/kg bw/day.
In the female animals, statistical significance with respect to the control was detected at lower mean concentration of cholesterol at 100 and 1000 mg/kg bw/day and at the lower mean sodium concentration (Na+)
The statistically significant differences in CHOL and Na+ in female animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 100, 300 or 1000 mg/kg bw/day groups at the end of the treatment period (on Day 42 for male animals and on Day 50 for female animal).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia on the thorax of one control animal (1/5) selected for the examination was detected at the functional observations, too.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examinations did not induce any toxic or other test item related lesions in the investigated reproductive and other organs of experimental male and female animals at 1000 mg/kg bw/day.
In the male animals of control and 1000 mg/kg bw/day groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each). 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 control and treated animals.
The histology picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control and high dose groups (12/12, both).
In the female animals belonging to the high dose (1000 mg/kg bw/day) treated and control groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (12/12, in control and high dose groups). The cortical region of ovaries 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.
In some dam showing hydrometra at the necropsy, – 1/12 control, 1/1 at 100 mg/kg bw/day, 4/4 at 300 mg/kg bw/day and 2/12 at 1000 mg/kg bw/day – dilatation of uterine horns was observed. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5 in both groups) animals.
In animals, selected for full histological examinations, no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.
Minimal or mild alveolar emphysema in the lungs (1/5 male control, 1/5 male and 1/5 female at 1000 mg/kg bw/day) were detected sporadically. The pulmonary emphysema was considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs in minimal or mild degree in some animal (2/5 control male; 1/5 male and 1/5 female at 1000 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Focal fibrosis in the Glisson’s capsule of the liver – in one male animal at 300 mg/kg bw/day (1/1) and in one female at 100 mg/kg bw/day (1/1) – was in connection with the mechanical irritation due to the diaphragmatic hernia.
One side pyelectasia was observed in single female animal at 100 mg/kg bw/day without other histopathological lesions (degeneration, inflammation, fibrosis): Therefore, this renal alteration was considered to be an individual lesion, without toxicological significance. Pyelectasia is a common finding in experimental rats of this strain with similar age.
The abscess formation close to the esophagus was present in one male animal (1/1 at 100 mg/kg bw/day) in the low dose group. Therefore, this phenomenon was considered as individual disorder, without toxicological significance.
No morphological evidence of acute or subacute test item related injury (degeneration, inflammation, necrosis etc.) of the gastrointestinal tract, the liver, the pancreas, the respiratory system, the cardiovascular system, the urinary system, the lymphoid system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system, the eyes, the lachrymal glands, and the integumentary system, was observed.
The cytomorphology of endocrine glands was the same in the animals belonging to the control and treated.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (100, 300 or 1000 mg/kg bw/day).
Statistical significance was observed at the slightly lower mean number of the days in pre-estrous in female animals at 300 mg/kg bw/day when compared to the control.
There were no statistically or biologically significant differences between the control and test item treated groups (100, 300 and 1000 mg/kg bw/day) in the other examined parameters of estrous cycle: all animal shoved regular cycles, the mean number of cycles, the mean length of cycles, mean number of days estrous or diestrus were similar in all groups and there were no animals with prolonged estrous or diestrous during the pre-mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the male animals of control and 1000 mg/kg bw/day groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each). 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 control and treated animals.
The histology picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control and high dose groups (12/12, both).
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the test item at 100, 300 or 1000 mg/kg bw/day in male or female animals.
There was no difference between the control and test item treated groups in the gestation or copulatory indices, in the mean pre-coital interval or mean conceiving days.
The statistical significances were considered to be toxicologically not relevant at the percentage of fertile/infertile male animals, pregnant/non-pregnant female animals and at the lower fertility indices in male animal and female animals at 300 and 1000 mg/kg bw/day. This finding met the historical control as mating of one pair at 300 and 1000 mg/kg bw/day (1/12, both) did not result in pregnancy.
There were no toxicologically relevant differences in the evaluated parameters of delivery between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day).
The mean number of implantation sites per dams and the mean of post-implantation loss were comparable 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 duration of pregnancy, in the mean number of total births, liveborns, stillborns or viable pups on post-partal day 0 and in the percentage of dams with viable offspring on post-partal day 0. The live birth index was 100 % in each group (100, 300 or 1000 mg/kg bw/day).
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:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Dose descriptor:
LOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
at the limit dose of 1000 mg/kg
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
Clinical signs (no milk in the stomach and cold, alopecia, hemorrhage on the nose, found dead, missing) were observed with variable incidence in the control, 100 or 1000 mg/kg bw/day group and with the highest incidence at 100 mg/kg bw/day. Hemorrhage on the nose, alopecia, death or missing pups were observed in single litters in control, 100 or 1000 mg/kg bw/day groups.
These signs are commonly observed in offspring of not treated dams of this strain therefore these observations were considered to have no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
There were no extrauterine mortality in offspring in 300 and 1000 mg/kg bw/day groups. One male pup in the control group was found dead on post-natal day 1 and one male and one female pup in two litters were missing at 100 mg/kg bw/day on post-natal day 1.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development of the offspring was undisturbed during the observation period.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.
Statistical significance was observed at the slightly lower mean body weight of male and female pups at 1000 mg/kg bw/day comparing to their control on postnatal day 4. These differences with respect to the control were minor and these were considered to be toxicologically not relevant.
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:
no effects observed
Description (incidence and severity):
There were no test item related differences between the control and all test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not adversely affected by the test item treatment at 100, 300 and 1000 mg/kg bw/day.
The mean absolute and normalized anogenital distances were comparable to their control at 100, 300 and 1000 mg/kg bw/day (male and female).
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on postnatal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Description (incidence and severity):
One control male pup was found dead and subjected to necropsy on post-natal day 1. There were no macroscopic changes in the organs or tissues of this pup and milk in the stomach was detected.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
The thyroid hormone (free T4 and TSH) levels were not affected by the test item in offspring sampled on postnatal day 13.
Statistical significance was only detected at the lower mean concentration of TSH level of PN13 offspring at 100 mg/kg bw/day compared to their control. Similar finding was not detected at the high dose group, therefore was considered to be toxicologically not relevant.
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:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
developmental immunotoxicity
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
developmental immunotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
at the limit dose of 1000 mg/kg
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential reproductive toxicity (and repeated dose toxicity) of the test item.
Under the conditions of the present study, Hexanal administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats.
There were no signs of systemic toxicity in male or female animals at 100, 300 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) by active ingredient were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day
In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the Wistar rat. The test item showed no evidence of being an endocrine disruptor. No necessity for classification as reproductive toxicant is given.
Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day compared to control animals according to OECD 422 under GLP.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects 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 13 post-partum associated with administration of repeated maternal doses.

Hexanal was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 20, 60 and 200 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (sunflower oil) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Hexanal was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature or in a refrigerator (at 5 ± 3 °C) for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Hexanal concentrations in the dosing formulations varied within the range of 91 % and 101 % in comparison to the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-57 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 3-7 pups per litter at termination on post‑partum/post-natal day 13 and from all parent male animals at termination.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. In addition, organs showing macroscopic findings at the necropsy were processed and examined histologically in animals of the low and mid dose groups.

 

The results of this study were summarized as follows:

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

Clinical and functional observation: Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain: The body weight development was not adversely affected by the test item in male or in female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Food consumption: The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study.

Estrous cycle: A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day).

Reproductive performance: There were no significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day).

Hematology and blood coagulation: Hematological and blood coagulation investigation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.

Clinical chemistry: There were no test item related effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).

Serum thyroid hormones: There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

Necropsy: Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day.

Organ weight: There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level.

The weights of organs of selected animals were comparable in the control and test item treated groups (male and female).

Histopathology: There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) of male or female animals administered with 1000 mg/kg bw/day.

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

Offspring: The offspring’s development was undisturbed at 100, 300 and 1000 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Conclusion

Under the conditions of the present study, Hexanal administered at 100, 300 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine (thyroid hormone) effects in parental male and female Han:WIST rats.

There were no signs of systemic toxicity in male or female animals at 100, 300 or 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 nor indication for an endocrine effect (thyroid hormone) was note after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.Based on these observations the No Observed Adverse Effect Levels (NOAEL) by active ingredient were determined as follows:

NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day

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

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

The test item showed no evidence of being an endocrine disruptor. No necessity for classification as reproductive toxicant is given.

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:
Study was conducted on the registered substance itself acc. OECD TG 422 under GLP. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

See above, Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422), no adverse effects in adults or offspring noted.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on lack of any adverse effects on reproductive toxicity (and general systemic toxicity), it is impossible to hypothesize a concrete mode of action.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the Repeated dose / reproduction toxicity screening test on rats, the no observed adverse effect level (NOAEL) of the test item was determined. The oral (gavage) administration of the test item to Wistar rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any test item related effects in adults or offspring. So, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day as well as the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

No effects on fertility were noted up to the limit dose of 1000 mg/kg, and no systemic toxicity was noted. Consequently the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.