Registration Dossier

Administrative data

Description of key information

Short-term toxicity studies (4 weeks) on hexanal in Sprague-Dawley rats (10 animals per sex per dose) were carried out similar to OECD TG 407. Hexanal was administered at the doses 1, 10, 100, and 1000 mg/L in the drinking water. The actual doses received were 0.1 - 95.7 mg/kg bw/day for female and 0.1 - 124.7 mg/kg bw/day for male rats. It was shown that hexanal produced no overt toxic effects. Although treatment-related morphological changes were observed in the highest dose groups; these were considered to be mild and adaptative in nature, and could not be related to any functional changes. The only biochemical parameter affected by treatment was the reduced LDH activity in hexanal treated female rats. However, the biological significance of this change is uncertain. The growth rate and hematological parameter were not affected (Komsta et al. 1988).

Repeated Dose toxicity: Subacute study in Wistar rats, m/f, 42 resp. 51-57 days, oral: gavage (OECD TG 422, GLP): NOAEL = 1000 mg/kg

Hexanal was repeatedly administered orally (by gavage) to rats at doses of 0, 100, 300 and 1000 mg/kg bw/day to Wistar rats (12/sex/group) over 42 resp. 51-57 days. There was no test item related mortality or clinical signs at any dose level (100, 300 and 1000 mg/kg bw/day). No test-item related effects were noted on Body weight and body weight gain, Food consumption, Hematology and blood coagulation, Clinical chemistry or Serum thyroid hormones. Necropsy revealed no macroscopic findings related to the effect of the test item at any dose level, no effects on organ weights were noted. 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. The NOAEL for systemic toxicity of male/ female rats was determined to 1000 mg/kg bw/day (Szakonyiné 2019).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
The purity and chemical identification of the test compound was confirmed by thinlayer chromatographic, gas chromatographic (Hewlett Packard, model 5820A), and gas chromatography-mass spectrometric techniques (Finnigan mass spectrometer, model 4000, data system 6000).
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: "young"
- Weight at study initiation: 200 g (female) and 300 g (male)
- Housing: individually in stainless steel mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2°C
- Humidity (%): 40 - 60 %
Route of administration:
oral: drinking water
Details on route of administration:
Animals were given the test chemicals in their drinking water at concentrations of 1.0, 10.0, 100.0, or 1000.0 mg/L for a period of 4 weeks.
Vehicle:
other: 0.5% w/v Emulphor
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Hexanal, being slightly soluble in water, was initially solubilized with 0.5% w/v Emulphor followed by dilution with tap water to appropriate concentrations.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no analytical verification
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
1 mg/L drinking water
Dose / conc.:
10 mg/L drinking water
Dose / conc.:
100 mg/L drinking water
Dose / conc.:
1 000 mg/L drinking water
Dose / conc.:
0.1 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
0.9 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
8.6 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
95.7 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
0.1 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
1.2 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
12.6 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
124.7 mg/kg bw/day (actual dose received)
Remarks:
males
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control:
no positive control
Observations and examinations performed and frequency:
Clinical observations were made daily; body weight gain and food and water consumption were measured weekly.
Sacrifice and pathology:
At the termination of chemical exposure all animals were lightly anesthetized with ether and exsanguinated via the abdominal aorta.

GROSS PATHOLOGY: Yes. All animals were examined grossly at the time of necropsy. The brain, heart, liver, spleen and kidneys were excised and weighed.

HISTOPATHOLOGY: Yes. Blood samples were analyzed for the following hematological parameters: hemoglobin, packed cell volume, erythrocyte count (Baker 7000), total and differential leukocyte counts and platelet count. Serum biochemical profiles were determined in a Technicon microanalyzer (Model 12/60 micro) and included sodium, potassium, inorganic phosphate, total bilirubin, alkaline phosphatase (AP), aspartate aminotransferase (AST), total protein, calcium, cholesterol, glucose, uric acid and lactic dehydrogenase (LDH). Hepatic microsomal aniline hydroxylase (AH), aminopyrine demethylase (APDM), and ethoxyresorufin deethylase (ER) activities were determined based on published methods and adapted to automated instruments. A selection of tissues from the control and the highest dose groups was taken and fixed in 10% buffered formalin (pH 7.4) for routine histological examination. The tissues examined histologically included brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, pancreas, colon, kidney, spleen, bone marrow, mesenteric and mediastinal lymph nodes, testes and epididymis in the male, ovaries and uterus in the female, skeletal muscle and heart. Potential fatty change in the liver was determined in frozen sections
Statistics:
The data were treated statistically with one-way analysis of variance. When a significant difference (p<0.05) was noted among the groups, the data were further analyzed by Duncan's multiple range test in order to determine which groups were different.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed.
Mortality:
no mortality observed
Description (incidence):
All animals survived the entire chemical exposure period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight gain of all treated groups was not significantly different from those of control rats and vehicle controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all treated groups was not significantly different from those of control rats and vehicle controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption of all treated groups was not significantly different from those of control rats and vehicle controls. Based on the water intake data the amount of chemicals ingested by the rats ranged from 0.1 to 124.65 mg/kg body wt./day for the males and 0.10 to 95.66 mg/kg body wt./day for the females (Table 1).
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters determined in this study were within normal ranges established for the Sprague-Dawley rats in our laboratories.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
LDH activity was reduced in female rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male animal treated with 10 mg/L and one female in the 100 mg/L group had dilated kidney pelves while one female in the 1000 mg/L group had unilateral hydronephrosis. These gross changes were of a sporadic nature and could not be related to any specific chemical treatment.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Only the tissues from the control and the highest dose groups were examined histopathologically and even at this dose level the changes were generally mild in nature. The thyroid, liver and kidney were the target organs affected by treatment. Mild thyroid changes, which consisted of increased epithelial height, reduced colloid density and angular collapse of follicles was observed. Hepatic changes were characterized by mild increases in perivenous cytoplasmic homogeneity and periportal cytoplasmic density. Animals also appeared to have more incidences of anisokaryosis. Morphological alterations in the kidney were confined to the proximal tubules and glomeruli. Tubular changes consisted of eosinophilic inclusions, pyknosis and central displacement of nuclei. In some cases the inclusions protruded into the lumen. Glomerular changes were characterized by adhesions of the visceral and parietal layers of Bowman's capsules, and were observed in both control and treated animals but the changes appeared to be more prevalent in the treated groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
conc. level: histopathological changes
Effect level:
95.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
conc. level: histopathological changes
Effect level:
124.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

TABLE 1 Body weights and water consumption of rats fed hexanal chemicals via drinking water for 28 days.a'

MALE

FEMALE

Treatment mg/L in drinking

Initial body weight (g)

Weight gain(g)

Water Consumption (g/rat/day)

Approximate amount of chemical Ingested (mg/kg/day)

Initial body weight (g)

Weight gain (g)

Water Consumption (g/rat/day)

Approximate amount of chemical ingested (mg/kg/day)

Control-Water

133±15

214±14

26±3

0

116±10

88 ±12

18.8 ± 3

0

Vehicle Control (5% Emulphor)

134±17

210±14

25±2

0

116±9

102 ±18

20.9 ± 4

0

Hexanal

1.0

136±15

200 ± 21

24±2

0.1

116±11

95+13

21±5

0.1

10.0

134±18

197 ± 21

24±2

1.2

116±11

98 ±15

19±3

0.9

100.0

137±16

203 ±23

25±3

12.6

117±12

102±18

19±1

8.6

1000.0

136±19

210 ±18

25±2

124.7

119±8

104 ± 23

22±4

95.7

a) Values represent mean ± S.D. obtained from 10 animals

Table 2 Serum Lactate Dehydrogenase activity (mU/mL) of rats fed hexanal via drinking water for 28 days.a

 

Treatment mg/L in drinking water

MALE

FEMALE

 

Control - Water

892±151

1276±248

 

Vehicle Control -0.5% Emulphor

1089±275

1262±401

  

Hexanal

 

1.0

820±202

1038±205

 

10.0

882±208

893± 208b

 

100.0

840±296

836± 324b

 

1000.0

702±301

915± 197b

 

a)  Values represent mean ± SD obtained from 10 animals

b)  Statistically different from control at p < 0.05

 

TABLE 3 Prevalence of histological changes In rats fed drinking water containing hexanal at 1000 mg/La

 

MALE

FEMALE

Tissues

Control Water

Vehicle Control - 0.5 % Emulphor

Treatment

Control Water

Vehicle Control - 0.5 % Emulphor

Treatment

THYROID

-Reduced follicular size

- Increased epithelial height

- Reduced colloid density

5/0

1/0

0/0

1/0b

4/0

0/0

6/0

5/1

3/0

2/0

0/0

0/0

3/0

0/0

0/0

4/0

1/0

0/0

LIVER

-Anisokaryosls

- Increased cytoplasmic homogeneity

0/0

3/0

0/0

5/1

3/0

10/0

0/0

1/0

2/0

3/2

2/0

0/0

KIDNEY

-Glomerular adhesions

-Tubular cytoplasmic Inclusions

6/0

2/0

3/1

1/1

6/0

3/2

8/0

0/0

9/0

0/0

5/1

4/1

a)Number examined was10

b)Values denote

(number of animals with minimal-mild changes)/(number of animals with moderate to severe changes)

Conclusions:
Data presented above indicated that administration of hexanal via drinking water up to 1000 mg/L produced no overt toxic effects. Although treatment-related morphological changes were observed in the highest dose groups; these were considered to be mild and adaptative in nature, and could not be related to any functional changes. The only biochemical parameter affected by treatment was the reduced LDH activity in hexanal treated female rats. However, the biological significance of this change is uncertain. The growth rate and hematological parameter were not affected.
Executive summary:

Short-term toxicity studies (4 weeks) on hexanal in Sprague-Dawley rats (10 animals per sex per dose) were carried out similar to OECD TG 407. Hexanal was administered at the doses 1, 10, 100, and 1000 mg/L in the drinking water. The actual doses received were 0.1 - 95.7 mg/kg bw/day for female and 0.1 - 124.7 mg/kg bw/day for male rats. It was shown that hexanal produced no overt toxic effects. Although treatment-related morphological changes were observed in the highest dose groups; these were considered to be mild and adaptative in nature, and could not be related to any functional changes. The only biochemical parameter affected by treatment was the reduced LDH activity in hexanal treated female rats. However, the biological significance of this change is uncertain. The growth rate and hematological parameter were not affected.

Endpoint:
short-term repeated dose toxicity: oral
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
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 oral 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
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).
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
Observations and examinations performed and frequency:
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).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment (i.e. on the day of necropsy).
- Anaesthetic used for blood collection: Yes, blood samples were harvested from the retro-orbital venous plexus under Isofluran Cp® anesthesia.
- Animals fasted: Yes, animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
- Parameters checked in table "Hematology parameters examined" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment (i.e. on the day of necropsy).
- Animals fasted: Yes, animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy).
- Parameters checked in table "Clinical chemistry parameters examined" were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week (on Day 41 for male animals and Day 50 for female animals). General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: Blood samples for determination of serum levels of thyroid hormones (FT4 and TSH) were drawn in the morning hours.
- How many animals / Dose groups that were examined: Blood samples were collected from all dams on post-partal day 13 and from all parent male animals at termination on Day 42.
- Parameters checked in table "Thyroid hormone parameters examined" were examined.

OTHER: Reproductive parameters. See IUCLID chapter 7.8.
Sacrifice and pathology:
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.
Other examinations:
Reproductive parameters (see IUCLID chapter 7.8)
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.
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.
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:
effects observed, non-treatment-related
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
Description (incidence and severity):
The thyroid hormone (free T4 and TSH) levels were not affected by the test item in parental male animals (100, 300 and 1000 mg/kg bw/day) sampled on postnatal day 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no adverse effects on the weights of examined organs in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day).
The weights of examined organs (absolute and relative to body and brain weights) were comparable to their control at 100, 300 mg/kg bw/day (male and female) and at 1000 mg/kg bw/day (male).
Minor change in the thymus weight relative to body weight in female animals at 1000 mg/kg bw/day compared to their control were considered to be of little or no biological relevance in the lack of related histological alterations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Specific macroscopic alterations were not detected in the organs or tissues at any dose levels (100, 300 or 1000 mg/kg bw/day) at the necropsy.
There were no macroscopic findings in the organs or tissues in male animals in the control and 1000 mg/kg bw/day groups (12/12).
Necropsy observation revealed 1x0.5 cm formation with yellow fluid content close to the esophagus in one male animal at 100 mg/kg bw/day (1/12) and Hernia diaphragmatica in one animal at 300 mg/kg bw/day (1/12).
In the control group, alopecia was observed on the skin of thorax (1/12) in one female animal and on the limbs (fore limbs and left hind limb) and abdomen (1/12) in other one female animal.
In female animals at 100 mg/kg bw/day, renal pyelectasia (1/12), Hernia diaphragmatica (1/12) and moderate hydrometra (1/12) were seen at the necropsy.
At 300mg/kg bw/day, moderate hydrometra was noted for three dams (3/11) and marked hydrometra was detected in one non-pregnant female animal (1/1)
Marked hydrometra was observed in one dam (1/11) and in non-pregnant female animal (1/1) at 1000 mg/kg bw/day.
Moderate hydrometra was also note for one control female animal (1/12).
Alopecia, pyelectasia and Hernia diaphragmatica are common observations in untreated experimental rats of this strain with similar age. These occurred independently from doses and with low incidence, therefore these findings were considered to be toxicologically not relevant.
Hydrometra, related to the female sexual cycle and alopecia are frequent observations in untreated experimental rats of this strain and the frequency noted here fit the ranges seen for control animals. In the lack of related histopathological alterations (inflammatory or other pathological lesion) the above-mentioned findings were judged to be toxicologically not relevant.
Neuropathological findings:
not examined
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
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
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
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
behaviour (functional findings)
immunology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
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

14-Day Oral Gavage Dose Range Finding Study with Hexanal in Rats (Study no: 919-400-3737, 2018-07-23)

A 14-Day Oral Gavage Dose Range Finding Study was conducted in rats. The purpose of this study was to obtain first information on the toxic potential of Hexanal in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (main study according to TG OECD 422).

The dose setting with 0, 100, 300 and 1000 mg/kg bw/day has been chosen in agreement with the Sponsor and based on information available from studies with similar substances.

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

The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation.

Measured concentrations of formulations applied in the study varied in the acceptable range (between 94 % and 103 % of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Detailed clinical observations were performed daily after the treatment. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed.

The results of this study were summarized as follows:

Mortality:There was no mortality in control, 100, 300 or 1000 mg/kg bw/day groups.

Clinical observations:Hexanal did not induce clinical signs at 100, 300 or 1000 mg/kg bw/day in male or female animals during the two weeks treatment period. The behavior and physical condition of all animals were considered to be normal.

Body weight and body weight gain: The body weight development was undisturbed in male or female animals at 100, 300 or 1000 mg/kg bw/day during the observation period.

Food consumption:The mean daily food consumption was comparable in the control and test item treated groups – 100, 300 or 1000 mg/kg bw/day, male and female.

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

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

Organ pathology:Specific macroscopic alterations of the organs or tissues or changes in the investigated organ weights related to the test item were not detected.

Conclusion:

Under the conditions of the present study, Hexanal did not cause adverse effects in male or female Han:WIST rats after the consecutive 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day.

Based on the observations made in this toxicity study, the dose levels for a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) were determined as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

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 repeated dose toxicity (and reproductive toxicity) of the test item, especially taking into account the prolonged exposure duration (minimum 5 weeks) compared to a regular subacute 28-day study and the fact that pregnant animals are in general more susceptible for toxic effects.

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 rats: 1000 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 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. Further, no evidence for classification as STOT RE was given.
Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item 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.

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.

Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH) from all dams 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.

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).

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.

 

Conclusion

Under the conditionsof the present study, Hexanal administered at 100, 300 or 1000 mg/kg bw/dayby 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

The test item showed no evidence of being an endocrine disruptor. Further, no evidence for classification as STOT RE was given.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is a Klimisch 1 OECD TG 422 study available, showing no test item related effects up to 1000 mg/kg, and an additional 4 week study consistently supporting the finding that hexanal is relatively non-toxic when administered repeatedly. Hence, the database is of high quality.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Both available studies provide no indication for specific target organ toxicity.

In the key study, the NOAEL was found to be 1000 mg/kg due to the lack of any test-item related effects.

According to Regulation 1272/2008, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s). Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance values for classification into category 2 are 10 < C100 mg/kw bw/d in a subchronic study, corresponding to300 mg/kg in subacute studies. The oral administration of the test item to rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated and did not result in any adverse toxicological findings, no LOAEL up to the limit dose could be determined. Hence, no classification according to Regulation 1272/2008 is triggered.