2-methylundecanal

Endpoint summary

Description of key information

Dietary administration of Aldehyde C12 MNA Pure for at least 90 days to male and female rats resulted in a No Observed Adverse Effect Level (NOAEL) of 15000 ppm, corresponding to dose levels of 1046 mg/kg (males) and 1211 mg/kg (females).
  
  
  
    
    
    
    
    

Aldehyde C12 MNA was assessed for repeated dose oral toxicity according to OECD 408. The No Observed Adverse Effect Level (NOAEL) for the rat was considered to be 15000 ppm,corresponding to dose levels of 1046 mg/kg (males) and 1211 mg/kg (females).


























 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Identification: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
Batch: VE00487208
Test substance storage: At room temperature protected from light container flushed with nitrogen
Stable under storage conditions until: 09 May 2018 (expiry date)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. The animals were 6 weeks old at initiation of dosing and weighed between 120 and 184 g.
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Identification
At study assignment, each animal was identified using an earmark and tattoo.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Housing
On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne
GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. The room(s) in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating test facility study no., group, animal number(s), and sex. Cages were arranged on the racks according to a Latinsquare model.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 50 to 71%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Prepared diet was provided ad libitum in stainless steel containers, covered by a stainless steel grid to prevent spillage, except during designated procedures. Diets remained in the food hopper for a maximum of one day, each day the remaining food in the food hopper was replaced with new room temperature-acclimated diet retained from the freezer. Food hoppers were shaken on a daily basis to divide any sawdust equally over the diet in order to facilitate food consumption. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there are no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Route of administration:

oral: feed

Details on route of administration:

The test and control item was administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 91 days. The first day diets were available to the animals was designated as Day 1.
The amount of test item incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test article intake was estimated based on the body weight and food consumption values.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The oral route of exposure via dietary inclusion was selected because this is the intended route of human exposure.
The dose levels were selected based on results of a (14-day repeated dose toxicity study with oral exposure of Aldehyde-C12 MNA Pure in rats, Test Facility Study No. 517136), and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

Duration of treatment / exposure:

The test and control item was administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 91 days

Frequency of treatment:

The test and control item was administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 91 days

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for all study animals.

Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Arena Observations
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.

Body Weights
Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

Food Consumption
Food consumption was quantitatively measured daily, starting on Day 1, throughout the dosing period.

Water Consumption
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Ophthalmic Examinations
The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during Pretreatment in all study animals, and at the end of the Dosing Period in Week 13 in all Group 1 and 4 study animals.

Functional Tests
Functional tests were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed shortly before or after completion of clinical observations.
The following tests were performed:
- Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
- Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Sacrifice and pathology:

Laboratory Evaluations
Clinical Pathology

Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) of fasted animals. After collection all samples were transferred the appropriate laboratory for analysis - Hematology, Coagulation and Clinical chemistry.

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.

Scheduled Euthanasia
Main study and recovery animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Organ Weights
The organs identified in the following table were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair may be taken and entered as a tissue comment. Organ to body weight ratio (using the terminal body weight) were calculated.

Tissue Collection and Preservation
Representative samples of the tissues identified in the following table were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Histology
Tissues identified in the table above (except animal identification, nasopharynx (body cavity), femur, clitoral gland, lacrimal gland, preputial gland, skeletal muscle, and tongue) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology
All tissues as defined under Histology were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.

Statistics:

All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Incidental scabs and chromodacryorrhoea is considered a background finding for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed this was considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality occurred during the study period.
One female (no. 80, receiving 15000 ppm) died during animal handling, which was considered to be an accidental death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment.
Statistically significant changes in body weight for males at 1500 ppm (from Week 4 onwards) and body weight gain for females at 5000 ppm (Weeks 3, 4, 7 and 8 only) were considered to be unrelated to treatment since no trend was apparent regarding dose.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were seen in food consumption before or after correction for body weight.
Any statistically significant changes in food consumption before or after correction for body weight were considered to not toxicologically significant since no clear trend was apparent regarding dose and duration of treatment or have arisen as a result of slightly high control values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in haematological parameters.
Statistical significance is noted in monocytes count for males treated at 15000 ppm, which was very slight and since no corroborative findings were present in the opposite sex, this is considered to represent no change of biological significance.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight changes were noted in clinical laboratory investigations. Alkaline phosphatase levels were higher in males treated at 5000 ppm and in males and females treated at 15000 ppm compared to their controls. This increase was seen in absence of any other elevation in liver enzymes and any other macroscopic or microscopic findings in the liver, therefore this elevation was considered to be a non-adverse finding.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Any differences, including those that reached statistical significance were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination revealed increased macrophage aggregates in the mesenteric lymph node in males (1/10) and females (4/9) treated at 15000 ppm. Histologically this was characterized by increased number of small, multifocal aggregates of epithelioid macrophages in the cortical parenchyma of the lymph node. This increase was considered to be non-adverse as the change was minimal to slight in severity, represented a slight exacerbation of a background change that can be present in control rats, and without evidence of structuralalteration or degenerative change to the affected organ. There were no macroscopic orclinical pathology correlates to this microscopic change.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Description (incidence and severity):

Functional Observations
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and treated animals.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related adverse effect observed up to the highest dose tested

Critical effects observed:

no

Dietary analyses confirmed that diets were prepared accurately and homogenously.

No toxicologically significant changes were noted in the following parameters investigated in this study; clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, haematology parameters, macroscopic examination and organ weights.

Histopathological examination revealed increased macrophage aggregates in the mesenteric lymph node in one male and four females treated at 15000 ppm. This increase was considered to be non-adverse as the change was minimal to slight in severity, represented a slight exacerbation of a background change that can be present in control rats, and without evidence of structural alteration or degenerative change to the affected organ. There were no macroscopic or clinical pathology correlates to this microscopic change.

Slight changes were noted in clinical laboratory investigations. Alkaline phosphatase levels were higher in males treated at 5000 ppm and 15000 ppm and in females treated at 15000 ppm compared to their controls. This increase was seen in absence of any other elevation in liver enzymes and any other macroscopic or microscopic findings in the liver, therefore this elevation was considered to be a non-adverse finding.

Other alteration in coagulation and biochemical parameters (i.e. prothrombin time in males treated at 5000 and 15000 ppm, bilirubin levels and urea levels in males treated at 15000 ppm and total protein levels, glucose levels and inorganic phosphate levels in females treated at 15000 ppm) were considered as non-adverse, since the alterations were slight in nature and occurred without any other macroscopic or microscopic findings that could be correlated to these findings.

Conclusions:

Dietary administration of Aldehyde-C12 MNA Pure for at least 90 days to male and female Wistar Han rats resulted in a No Observed Adverse Effect Level (NOAEL) of 15000 ppm, corresponding to dose levels of 1046 mg/kg (males) and 1211 mg/kg (females).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 046 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Very good quality: GLP, OECD 408 on the substance itslef.

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 selection of repeated dose toxicity via oral route - systemic effects endpoint:
The test substance was assessed for repeated oral dose toxicity according to OECD 408.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
According to Regulation (EC) No. 1907/2006, Annex IX, subchronic testing should be proposed "having regard to the likely route of human exposure".
The vapour pressure is low; 2.4 Pa at 20 °C so exposure via the inhalation route is unlikely.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
According to Regulation (EC) No. 1907/2006, Annex IX, subchronic testing should be proposed "having regard to the likely route of human exposure".
The vapour pressure is very low; 2.4 Pa at 20 °C so exposure via the inhalation route is unlikely.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
According to Regulation (EC) No. 1907/2006, Annex IX, subchronic testing should be proposed "having regard to the likely route of human exposure". However, the substance is a skin irritant, and is of low toxicity. Further dermal testing is therefore not proposed. This is also in line with requirements to minimise further in vivo testing on animal welfare grounds.
The classification as a skin irritant should apply to all mixtures containing the substance at ≥ 10 % per table 3.2.3 of Regulation (EC) No. 1272/2008. Additionally, the substance is classified as a skin sensitiser. This classification should apply to all mixtures containing the substance at > 1.0 % w/w, per Table 3.4.3 of Regulation (EC) No. 1272/2008. Appropriate risk management measures should be implemented to avoid dermal exposure to the substance at or above these levels. Skin contact in production and/or use is therefore unlikely. Any one-off accidents would represent acute exposure to the substance, not subchronic or chronic repeat doses. Repeat exposure via the dermal route should therefore be ruled out at these concentrations, although may still occur at lower concentrations. We do not consider dermal exposure to be the most likely route of human exposure. Human exposure via the dermal route is therefore unlikely.
Furthermore, the available acute oral and acute dermal toxicity studies indicate high LD₅₀ values of > 5000 mg/kg bw and >8280 mg/kg bw respectively. There were no systemic effects in the acute oral toxicity. In the skin irritation study, although positive, there were no signs of systemic toxicity. The eye irritation study was negative and included no signs of systemic toxicity.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
According to Regulation (EC) No. 1907/2006, Annex IX, subchronic testing should be proposed "having regard to the likely route of human exposure". However, the substance is a skin irritant, and is of low toxicity. Further dermal testing is therefore not proposed. This is also in line with requirements to minimise further in vivo testing on animal welfare grounds.
The classification as a skin irritant should apply to all mixtures containing the substance at ≥ 10 % per table 3.2.3 of Regulation (EC) No. 1272/2008. Additionally, the substance is classified as a skin sensitiser. This classification should apply to all mixtures containing the substance at > 1.0 % w/w, per Table 3.4.3 of Regulation (EC) No. 1272/2008. Appropriate risk management measures should be implemented to avoid dermal exposure to the substance at or above these levels. Skin contact in production and/or use is therefore unlikely. Any one-off accidents would represent acute exposure to the substance, not subchronic or chronic repeat doses. Repeat exposure via the dermal route should therefore be ruled out at these concentrations, although may still occur at lower concentrations. We do not consider dermal exposure to be the most likely route of human exposure. Human exposure via the dermal route is therefore unlikely.
Furthermore, the available acute oral and acute dermal toxicity studies indicate high LD₅₀ values of > 5000 mg/kg bw and >8280 mg/kg bw respectively. There were no systemic effects in the acute oral toxicity. In the skin irritation study, although positive, there were no signs of systemic toxicity. The eye irritation study was negative and included no signs of systemic toxicity.

Justification for classification or non-classification

Aldehyde C12 MNA was assessed for repeated dose oral toxicity according to OECD 408. The No Observed Adverse Effect Level (NOAEL) for the rat was considered to be 15000 ppm, corresponding to dose levels of 1046 mg/kg (males) and 1211 mg/kg (females).

Based on these results, no classification is required for repeated dose toxicity.