Reaction mass of undec-8-enal and undec-9-enal and undec-10-enal

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-10-2009 to 30-11-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Intreleven aldehyde.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-10-2009 to 30-11-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Intreleven aldehyde.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment the animals were 12 weeks old instead of 10 weeks. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Weight at study initiation: no data
- Fasting period before study: no
- Housing:
Pre-mating:Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating:Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating:Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During the motor activity test, males were caged individually and females were caged with their pups.No cage-enrichment was provided during activity monitoring.
- Health check F0: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.6°C
- Humidity (%): 31 - 69%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.

- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 13 October 2009 To: 30 November 2009

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (0.832 g/ml; i.e. the mean of the range indicated by the sponsor), and for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX project 492005). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Results:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for at 42-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Two females of Group 3 were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study (NOTOX Project 492083). See Attachment.
- 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology:
Males: the first 5 males per group
Females: with live offspring only

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily detailed clinical observations were made in all animals, at least between approximately 1 and 2 hours after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- For one female of Group 1 no body weight was determined during the post-coitum period as mating of this female was overlooked. Body weights were determined during the mating period.

FOOD CONSUMPTION : Yes
- Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- For one female of Group 1 no food consumption was determined during the post-coitum period as mating of this female was overlooked. Sufficient food consumption data is available to make a good assessment.

FOOD EFFICIENCY: Yes
- (food consumption per animal per day/ average body weightper cage)*1000

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group (females: with live offspring only)
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group (females: with live offspring only)
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females (with live offspring) were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea was recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group (females: with live offspring only):

Identification marks (not processed), Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Coagulation gland, Colon, Duodenum, Epididymides *, Eyes with optic nerve (if detectable) and Harderian gland *, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular,mesenteric), (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes *, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

*Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes

The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group (females: with live offspring only): Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*
* weighed when fixed for at least 24 hours.

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- All gross lesions of all animals (all dose groups).

Based on (possible) treatment-related changes in the stomach histological examination was extended to that particular organ of all selected animals of groups 2 and 3 (males and females).

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values. No statistical analysis was performed on histopathology findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant lower food consumption of females at 1000 mg/kg between Days 0-4 of the post-coitum phase was of a temporary and slight nature, and therefore considered to be of no toxicological relevance.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant higher potassium value was measured for males at 1000 mg/kg bw/day, but the mean remained within the range considered normal for rats of this age and strain. In females at 1000 mg/kg bw/day, a higher mean cholesterol level was recorded (being slightly outside the range considered normal for rats of this age and strain) along with notably higher bile acid levels in some females (means not statistically significant).

Other statistically significant variations in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.
These variations consisted of higher or lower alkaline phosphatase activity (ALP) in males and females at 300 mg/kg bw/day, lower total bilirubin level in males at 300 mg/kg bw/day, higher urea level in males at 300 mg/kg bw/day, higher cholesterol level in males at 300 and 1000 mg/kg bw/day, and lower urea level in females at 1000 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A higher liver weight and liver to body weight ratio, and lower prostate and prostate to body weight ratio was noted for males at 1000 mg/kg bw/day (statistically significant for liver to body weight ratio only). These changes were only slightly outside the normal range for rats of this age and strain.

The higher prostate to body weight ratio of males at 100 mg/kg/day occurred in the absence of a dose-related trend, and the mean remained within the range considered normal for rats of this age and strain. No toxicological relevance was ascribed to this change.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following necropsy findings were considered to be related to treatment with the test substance:
- An irregular surface of the forestomach in all males at 300 and 1000 mg/kg bw/day, and in 6 females at 300 mg/kg bw/day and nine females at 1000 mg/kg bw/day.
- Foci (black or red) in the forestomach in two males at 300 mg/kg bw/day.
- Yellowish discolouration of the forestomach in one male at 300 mg/kg/day and one female at 1000 mg/kg bw/day.
- Reddish discolouration/reddish foci in the glandular mucosa in two males at 1000 mg/kg bw/day.
- Thickened glandular mucosa in three females at 1000 mg/kg bw/day.
Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested (as a bolus) by humans.

Incidental findings among control and treated animals included reddish foci on the lungs, a nodule on the liver, epididymides or clitoral glands, agenesis of the testes and epididymides, reduced size of the seminal vesicles or preputial glands, tan foci on the preputial glands, a greenish/red-brown foci on the clitoral glands, a yellowish nodule on the uterine adipose tissue, scab formation on the skin, alopecia and exophthalmus.
The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and the incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related microscopic findings in the stomach at 100 mg/kg bw/day and higher:
- Lymphogranulocytic inflammation of the forestomach at 100 mg/kg bw/day in 4/5 males (minimal), at 300 mg/kg bw/day in 10/10 males (1: minimal, 9: slight) and 5/6 females (4: minimal, 1: slight), and at 1000 mg/kg bw/day in 10/10 males (3: minimal, 6: slight, 1: moderate) and 8/9 females (6: minimal, 2: slight).
- Hyperplasia of the squamous epithelium of the forestomach at 100 mg/kg bw/day in 3/5 males and 4/5 females (minimal), at 300 mg/kg bw/day in 10/10 males (8: slight, 2: moderate) and 6/6 females (3: minimal, 3: slight) and at 1000 mg/kg bw/day in 10/10 males (4: slight, 6: moderate) and 9/9 females (2: minimal, 6: slight, 1: moderate).
- Ulcer of the forestomach in 3/10 males at 300 mg/kg bw/day (2: minimal, 1: slight) and in 1/10 males of Group 4 (minimal). This was the microscopic correlate to the red/black foci in the forestomach recorded at necropsy.
- Congestion of the glandular stomach in 2/10 males at 1000 mg/kg/day.
Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested (as a bolus) by humans.

All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

local effects observed in the stomach

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

other: forestomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested as a bolus by humans.

Conclusions:

Based on the results of the OECD TG 422 study with doses: 100, 300 and 1000 mg/kg bw/day, the NOAEL for Undecanal for systemic toxicity was determined to be 1000 mg/kg/day and for local toxicity a LOAEL of 100 mg/kg bw/day was derived, based on effects in forestomach.

Executive summary:

The repeated dose toxicity of Undecanal was examined in an OECD TG 422 study according to GLP. Undecanal was administered by daily oral gavage to male and female Wistar Han rats (10 animals/sex and dose) at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-48 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Clinical signs:

No toxicologically relevant changes were noted during clinical or functional observations or in body weight and food intake during treatment up to 1000 mg/kg bw/day.

Haematological parameters:

Haematology parameters were normal in all groups.

Clinical chemistry:

Blood analysis at 1000 mg/kg bw/day revealed slightly higher potassium values in males (within normal ranges), higher mean cholesterol levels in females (only slightly outside normal ranges) and higher bile acid levels in some females. Given that these changes were generally slight in nature, were not present as a group response (bile acids) and occurred in the absence of supportive morphological changes, these were considered to be of no toxicological relevance.

Organ weight:

The higher liver weight and liver to body weight ratio, and lower prostate and prostate to body weight ratio for males at 1000 mg/kg bw/day was not supported by any histopathological changes. Moreover, since these changes were slight in nature (only slightly outside the normal range), these were considered to be of no toxicological relevance.

Histopathology:

Histopathological changes were confined to the stomach, and consisted of lymphogranulocytic inflammation and hyperplasia of the squamous epithelium of the forestomach in both sexes at 100, 300 and 1000 mg/kg bw/day, and ulcer formation in the forestomach in three males at 300 mg/kg bw/day (correlating to red/black foci in the forestomach of two males) and in one male at 1000 mg/kg bw/day. Hyperplasia of the squamous epithelium was the microscopic correlate to the irregular surface and yellowish discolouration of the forestomach recorded at necropsy at 300 and 1000 mg/kg bw/day. Congestion of the glandular stomach (correlating to red discolouration of the glandular mucosa) was observed in two males at 1000 mg/kg bw/day. There was no microscopic correlate to thickening of the glandular mucosa recorded in three females at 1000 mg/kg bw/day.

To summarize, based on these results, the NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day and for local toxicity a LOAEL of 100 mg/kg bw/day was found.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-10-2009 to 30-11-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Intreleven aldehyde.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment the animals were 12 weeks old instead of 10 weeks A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Weight at study initiation: no data
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During the motor activity test, males were caged individually and females were caged with their pups.No cage-enrichment was provided during activity monitoring.
- Health check F0: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.6°C
- Humidity (%): 31 - 69%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.

- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 13 October 2009 To: 30 November 2009

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (0.832 g/ml; i.e. the mean of the range indicated by the sponsor), and for specific gravity of the vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 days was allowed for mating. All females had mated within the mating period. After 12 days of mating, one female of group 1 had not shown evidence of mating and was subsequently separated from the male. This female delivered live offspring.
- Proof of pregnancy: confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Between Days 7 and 8 of the mating period, one female of group 1 was not cohabitated with a male. No vaginal smear was therefore made for this female on Day 11. The female did deliver live offspring
-Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX project 492005). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for at 42-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Two females of Group 3 were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study (NOTOX Project 492083).
- Results of the dose range finding study are described in End point study record: Repeated dose toxicity: oral. notox 492004
- 5 animals/sex/group were randomely selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs):
Males: the first 5 males per group
Females: with live offspring only

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily detailed clinical observations were made in all animals, at least between approximately 1 and 2 hours after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- For one female of Group 1 no body weight was determined during the post-coitum period as mating of this female was overlooked. Body weights were determined during the mating period.

FOOD CONSUMPTION : Yes
- Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- For one female of Group 1no food consumption was determined during the post-coitum period as mating of this female was overlooked. Sufficient food consumption data is available to make a good assessment.

FOOD EFFICIENCY: Yes
- (food consumption per animal per day/ average body weightper cage)*1000

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group (females: with live offspring only)
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group (females: with live offspring only)
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females (with live offspring only)
were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in all male parental animals:
testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance.

GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea was recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group (females: with live offspring only):
Identification marks (not processed), Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Coagulation gland, Colon, Duodenum, Epididymides *, Eyes with optic nerve (if detectable) and Harderian gland *, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular,mesenteric), (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes *, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

All remaining animals:
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes *, Epididymides *, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions, Identification marks (not processed)
*Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate*,
Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*
* weighed when fixed for at least 24 hours.

All remaining males: Epididymides and Testes

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).

Based on (possible) treatment-related changes in the stomach histological examination was extended to that particular organ of all selected animals of groups 2 and 3 (males and females).

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-6.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
no

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.
The number of implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.

Reproductive indices:

For each group, the following calculations were performed:

Percentage mated females: Number of females mated/Number of females paired x 100

Fertility index: Number of pregnant females/Number of females paired x 100

Conception index: Number of pregnant females/Number of females mated x 100

Gestation index: Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check:
Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check:
Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Checkx 100

Viability index (%):
(Number of live pups on Day 4 of lactation/Number of pups born alive) x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant lower food consumption of females at 1000 mg/kg between Days 0-4 of the post-coitum phase was of a temporary and slight nature, and therefore considered to be of no toxicological relevance.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant higher potassium value was measured for males at 1000 mg/kg/day, but the mean remained within the range considered normal for rats of this age and strain. In females at 1000 mg/kg/day, a higher mean cholesterol level was recorded (being slightly outside the range considered normal for rats of this age and strain) along with notably higher bile acid levels in some females (means not statistically significant).

Other statistically significant variations in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.
These variations consisted of higher or lower alkaline phosphatase activity (ALP) in males and females at 300 mg/kg/day, lower total bilirubin level in males at 300 mg/kg/day, higher urea level in males at 300 mg/kg/day, higher cholesterol level in males at 300 and 1000 mg/kg/day, and lower urea level in females at 1000 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related microscopic findings in the stomach at 100 mg/kg/day and higher:
- Lymphogranulocytic inflammation of the forestomach at 100 mg/kg/day in 4/5 males (minimal), at 300 mg/kg/day in 10/10 males (1: minimal, 9: slight) and 5/6 females (4: minimal, 1: slight), and at 1000 mg/kg/day in 10/10 males (3: minimal, 6: slight, 1: moderate) and 8/9 females (6: minimal, 2: slight).
- Hyperplasia of the squamous epithelium of the forestomach at 100 mg/kg/day in 3/5 males and 4/5 females (minimal), at 300 mg/kg/day in 10/10 males (8: slight, 2: moderate) and 6/6 females (3: minimal, 3: slight) and at 1000 mg/kg/day in 10/10 males (4: slight, 6: moderate) and 9/9 females (2: minimal, 6: slight, 1: moderate).
- Ulcer of the forestomach in 3/10 males at 300 mg/kg/day (2: minimal, 1: slight) and in 1/10 males of Group 4 (minimal). This was the microscopic correlate to the red/black foci in the forestomach recorded at necropsy.
- Congestion of the glandular stomach in 2/10 males at 1000 mg/kg/day.
Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested (as a bolus) by humans.

All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

No toxicologically significant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

local effects observed in the stomach. Local effects in (fore)stomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested as a bolus by humans.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

other: forestomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of small size, red spots on the head/nose and a pale appearance. No relationship with treatment was established for these observations and they were considered to be of no toxicological relevance.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

A total of three pups of the control group, and one pup each at 100, 300 and 1000 mg/kg/day were found dead or missing during lactation. No toxicological relevance was ascribed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Incidental macroscopic findings among pups found dead included autolysis, cannibalism and absence of milk in the stomach. Some surviving pups were small in size. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No toxicologically significant effects on developmental parameters were observed.

Gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Key result

Critical effects observed:

no

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of the OECD TG 422 study, Undecanal exerted no reproductive/developmental toxicity at any dose tested and therefore the reproductive and developmental NOAEL was established to be 1000 mg/kg bw/day.

Executive summary:

The reproductive toxicity of Undecanal was examined in an OECD TG 422 study according to GLP. Undecanal was administered by daily oral gavage to male and female Wistar Han rats (10 animals/sex and dose) at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-48 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Clinical signs:

Parental results: No toxicologically relevant changes were noted during clinical or functional observations or in body weight and food intake during treatment up to 1000 mg/kg bw/day.

Haematological parameters:

Haematology parameters were normal in all groups.

Clinical chemistry:

Blood analysis at 1000 mg/kg bw/day revealed slightly higher potassium values in males (within normal ranges), higher mean cholesterol levels in females (only slightly outside normal ranges) and higher bile acid levels in some females. Given that these changes were generally slight in nature, were not present as a group response (bile acids) and occurred in the absence of supportive morphological changes, these were considered to be of no toxicological relevance.

Organ weight:

The higher liver weight and liver to body weight ratio, and lower prostate and prostate to body weight ratio for males at 1000 mg/kg bw/day was not supported by any histopathological changes. Moreover, since these changes were slight in nature (only slightly outside the normal range), these were considered to be of no toxicological relevance.

Histopathology:

Histopathological changes were confined to the stomach, and consisted of lymphogranulocytic inflammation and hyperplasia of the squamous epithelium of the forestomach in both sexes at 100, 300 and 1000 mg/kg bw/day, and ulcer formation in the forestomach in three males at 300 mg/kg bw/day (correlating to red/black foci in the forestomach of two males) and in one male at 1000 mg/kg bw/day. Hyperplasia of the squamous epithelium was the microscopic correlate to the irregular surface and yellowish discolouration of the forestomach recorded at necropsy at 300 and 1000 mg/kg bw/day. Congestion of the glandular stomach (correlating to red discolouration of the glandular mucosa) was observed in two males at 1000 mg/kg bw/day. There was no microscopic correlate to thickening of the glandular mucosa recorded in three females at 1000 mg/kg bw/day.

Reproductive/Developmental results:

No reproductive/developmental toxicity was observed at any dose level. There were no morphological findings in the reproductive organs of either sex, which could be attributed to the test substance. Furthermore, gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.

To summarize, based on these results, the parental NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day and for local toxicity a LOAEL of 100 mg/kg bw/day was found. The reproductive and developmental NOAEL were established to be 1000 mg/kg bw/day. 

Cross-reference 3

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

The read across justification is presented in the endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Key result

Dose descriptor:

LOAEL

Remarks:

Local effects

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: local effects observed in the stomach

Remarks:

Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested as a bolus by humans.

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The developmental toxicity of Intreleven aldehyde was evaluated by read across from the source study (OECD TG 422) for Undecanal. Given at doses 100, 300 and 1000 mg/kg bw/day, it did not exert any reproductive/developmental toxicity at any dose level in male and female rats and caused no morphological findings in the reproductive organs of either sex. The reproductive and developmental NOAEL was established to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPP TS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test,

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

Undecanal

EC Number:

203-972-6

EC Name:

Undecanal

Cas Number:

112-44-7

Molecular formula:

C11-H22-O

IUPAC Name:

Hendecanaldehyde

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment the animals were 12 weeks old instead of 10 weeks A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During the motor activity test, males were caged individually and females were caged with their pups. No cageenrichment was provided during activity monitoring.
- Health check F0: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.6°C
- Humidity (%): 31 - 69%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 13 October 2009 To: 30 November 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (0.832 g/ml; i.e. the mean of the range indicated by the sponsor), and for specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX project 492005). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 days was allowed for mating. All females had mated within the mating period. After 12 days of mating, one female of group 1 had not shown evidence of mating and was subsequently separated from the male. This female delivered live offspring.
- Proof of pregnancy: confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum. Between Days 7 and 8 of the mating period, one female of group 1 was not cohabitated with a male. No vaginal smear was therefore made for this female on Day 11. The female did deliver live offspring
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
Females were exposed for at 42-48 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation. Two females of Group 3 were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study (NOTOX Project 492083).
- Results of the dose range finding study are described in end point study record: Repeated dose toxicity: oral. notox 492004
- 5 animals/sex/group were randomely selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs): Males: the first 5 males per group, Females: with live offspring only.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early morning/late afternoon).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily detailed clinical observations were made in all animals, at leastbetween approximately 1 and 2 hours after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- For one female of Group 1 no body weight was determined during the post-coitum period as mating of this female was overlooked. Body weights were determined during the mating period.
FOOD CONSUMPTION : Yes
- Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- For one female of Group 1no food consumption was determined during the post-coitum period as matin g of this female was overlooked. Sufficient food consumption data is available to make a good assessment.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Lactation Day 5-6.
- Organs examined: All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea was recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Selected 5 females/group (females: with live offspring only):

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

Each litter was examined to determine the following, if practically possible:

Mortality I Viability
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death
were evaluated.

Clinical signs
Live pups were weighed on Days 1 and 4 of lactation.

At least once daily, detailed clinical observations were made in all animals.

Body weights
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance.


Live pups were weighed on Days 1 and 4 of lactation. Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many -to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values. No statistical analysis was performed on histopathology findings. The number of implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test (Krusk al, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.

Indices:

Reproductive indices:
For each group, the following calculations were performed:
Percentage mated females: Number of females mated/Number of females paired x 100
Fertility index: Number of pregnant females/Number of females paired x 100
Conception index: Number of pregnant females/Number of females mated x 100
Gestation index: Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices
Percentage live males at First Litter Check:
Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check:
Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage of postnatal loss Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Checkx 100
Viability index (%):
(Number of live pups on Day 4 of lactation/Number of pups born alive) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant lower food consumption of females at 1000 mg/kg between Days 0-4 of the post-coitum phase was of a temporary and slight nature, and therefore considered to be of no toxicological relevance.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, a higher mean cholesterol level was recorded (being slightly outside the range considered normal for rats of this age and strain) along with notably higher bile acid levels in some females (means not statistically significant). Other statistically significant variations in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following necropsy findings were considered to be related to treatment with the test substance:
- An irregular surface of the forestomach in 6 females at 300 mg/kg/day and nine females at 1000 mg/kg bw/day.
- Yellowish discolouration of the forestomach in one female at 1000 mg/kg bw/day.
- Thickened glandular mucosa in three females at 1000 mg/kg bw/day.
Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested (as a bolus) by humans.

Incidental findings among control and treated animals included foci on the clitoral glands, a greenish/red-brown foci on the clitoral glands and alopecia.
The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and the incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related microscopic findings in the stomach at 100 mg/kg bw/day and higher:
- Lymphogranulocytic inflammation of the forestomach at 300 mg/kg bw/day in 5/6 females (4:minimal, 1: slight), and at 1000 mg/ kg/day in 8/9 females (6: minimal, 2: slight).
- Hyperplasia of the squamous epithelium of the forestomach at 100 mg/kg bw/day in 4/5 females (minimal), at 300 mg/kg/day in 6/6 females (3: minimal, 3: slight) and at 1000 mg/kg bw/day in 19/9 females (2: minimal, 6: slight, 1: moderate).
Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested (as a bolus) by humans.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

No toxicologically significant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time and number of corpora lutea and implantation sites were unaffected by treatment.
No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

2

Key result

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: local effects observed in the stomach

Remarks:

Local effects in forestomach are not regarded as relevant to humans, as anatomical situation in humans is different from rats and because the substance is not ingested as a bolus by humans.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

A total of three pups of the control group, and one pup each at 100, 300 and 1000 mg/kg bw/day were found dead or missing during lactation. No toxicological relevance was ascribed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

DEVELOPMENTAL DATA:
No toxicologically significant effects on developmental parameters were observed.

Gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by treatment.
Incidental clinical symptoms of pups consisted of small size, red spots on the head/nose and a pale appearance. No relationship with treatment was established for these observations and they were considered to be of no toxicological relevance.
Incidental macroscopic findings among pups found dead included autolysis, cannibalism and absence of milk in the stomach. Some surviving pups were small in size. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of toxicity noted and this was the highest dose tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of the OECD TG 422 study, the parental NOAEL for Undecanal for systemic toxicity was determined to be 1000 mg/kg bw/day and for local toxicity a LOAEL of 100 mg/kg bw/day was found. The reproductive and developmental NOAEL were established to be 1000 mg/kg bw/day.

Executive summary:

The developmental toxicity of Undecanal was examined in an OECD TG 422 study performed under GLP. Undecanal was administered by daily oral gavage to male and female Wistar Han rats (10 animals/ sex and dose) at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-48 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results: No toxicologically relevant changes were noted during clinical or functional observations or in body weight and food intake during treatment up to 1000 mg/kg bw/day. Blood analysis at 1000 mg/kg bw/day revealed slightly higher potassium values in males (within normal ranges), higher mean cholesterol levels in females (only slightly outside normal ranges) and higher bile acid levels in some females. Given that these changes were generally slight in nature, were not present as a group response (bile acids) and occurred in the absence of supportive morphological changes, these were considered to be of no toxicological relevance. Haematology parameters were normal in all groups. The higher liver weight and liver to body weight ratio, and lower prostate and prostate to body weight ratio for males at 1000 mg/kg bw/day were not supported by any histopathological changes. Moreover, since these changes were slight in nature (only slightly outside the normal range), these were considered to be of no toxicological relevance. Histopathological changes were confined to the stomach and consisted of lymphogranulocytic inflammation and hyperplasia of the squamous epithelium of the forestomach in both sexes at 100, 300 and 1000 mg/kg bw/day, and ulcer formation in the forestomach in three males at 300 mg/kg bw/day (correlating to red/black foci in the forestomach of two males) and in one male at 1000 mg/kg bw/day. Hyperplasia of the squamous epithelium was the microscopic correlation to the irregular surface and yellowish discolouration of the forestomach recorded at necropsy at 300 and 1000 mg/kg bw/day. Congestion of the glandular stomach (correlating to red discolouration of of the glandular mucosa) was observed in two males at 1000 mg/kg bw/day. There was no microscopic correlation to thickening of the glandular mucosa recorded in three females at 1000 mg/kg bw/day.

Reproductive/Developmental results: No reproductive/developmental toxicity was observed at any dose level. There were no morphological findings in the reproductive organs of either sex, which could be attributed to the test substance. Furthermore, gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were unaffected by the treatment.

Based on these results, the parental NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day and for local toxicity a LOAEL of 100 mg/kg bw/day was found. The reproductive and developmental NOAEL were established to be 1000 mg/kg bw/day.