Amines, di-C16-18-alkyl

Endpoint summary

Description of key information

A 90 day oral dosing study according to OECD Guideline 408 with Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is available, with reliability rating 1. The results seen in the study, leads to a NOAEL of 5 mg/kg bw based on granulomatous inflammation in the mesenteric lymph nodes observed at an increased severity and incidence at the higher dose levels. These findings are also present in the available 28 day study on the same substance, where similar effects were seen at higher doses.

Key value for chemical safety assessment

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

Study period:

24-Sep-2013 to 03-Jul-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Guideline:

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

Deviations:

no

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 10 males and 10 females in each of groups 1 to 3 and 15 males and 15 females in group 4
- Age (at Delivery): Ca. 7 weeks
- Body Weight Range (at Acclimatization): Males between 177 and 221 g (mean 202 g) and females between 127 to 155 g (mean 145 g)
- Identification: Acclimatization: Cage card and tail mark (later ear tattoo)
- Treatment: Cage card and individual ear tattoo
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred (including on 01 and 02-Feb-2014, when the osmosis equipment malfunctioned) and are considered not to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of two or three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 29/13, 35/13 and 53/13) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants. Results of respective analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of respective samples were included in the study report.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

DOSE FORMULATIONS

The test item was used as supplied by the Sponsor. The purity was not corrected.

The dose formulations were prepared daily. Based upon the results of dose formulation analyses performed during a previous GLP study (Harlan Laboratories study C55117 confirming 4-hour stability), the stability of the test item formulations was considered to require daily preparation.

Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was weighed into a glass beaker on a tared Mettler balance. A small amount of vehicle was added and the remaining vehicle was added. The mixtures were stirred using an Ultra-Turrax homogenizer and used at room temperature (20 ± 5 °C).

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

The analyses were repeated under GLP for this study.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels: 0 mg/kg/day (Group 1), 5 mg/kg/day (Group 2), 15 mg/kg/day (Group 3) and 50 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous toxicity study in Wistar rats, Harlan Laboratories study C55117.
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1), 1 mg/mL/day (Group 2), 3 mg/mL/day (Group 3) and 10 mg/mL/day (Group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

The analysis was performed by Harlan Laboratories Ltd. using a LC/MS method provided by the Sponsor. Analytical Standard: The test item served as analytical standard/reference item.

Samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. The samples were delivered on cool pads to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) and stored at -20 ± 5 °C until analysis. The backup samples were delivered to the analytical department on cool pads.

Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director.

Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start. Additionally, during week 6 of treatment, concentration and homogeneity of the dose formulations were determined.

RESULTS

The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by power regression analysis. The coefficients of determination (R2) calculated were found to be better than 0.99.

The Amines, di-C16-18 (even-numbered) alkyl peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no significant peak appeared at the retention time of Amines, di-C16-18 (even-numbered) alkyl and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.

The Amines, di-C16-18 (even-numbered) alkyl concentrations in the dose formulations ranged from 81.1% to 120.4% with reference to the nominal and were within the accepted range of ±20%, except for sample of group 2 prepared on 06-November-2013 (top 77.6%, middle 73.1% and bottom 70.6%) and for sample of group 4 prepared on 02-October-2013 (top 131.3%).

The homogeneous distribution of Amines, di-C16-18 (even-numbered) alkyl in the preparations was approved because single results found did not deviate more than 10.9% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value, except for sample of group 4 prepared on 02-October-2013 (14.4%).

Duration of treatment / exposure:

92/93 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 5, 15 and 50 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1 (0 mg/kg/day): 10 males and 10 females
Group 2 (5 mg/kg/day): 10 males and 10 females
Group 3 (15 mg/kg/day): 10 males and 10 females
Group 4 (50 mg/kg/day): 15 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this oral toxicity study was to assess the cumulative toxicity of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) when administered daily to rats by gavage for a period of 92/93 days. The reversibility of test item-related changes was assessed after a treatment-free 42-day recovery period.

This study should provide a rational basis for toxicological risk assessment in man.

In this subchronic toxicity study, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 5, 15 and 50 mg/kg body weight/day for a period of 92/93 days. A control group was treated similarly with the vehicle, corn oil, only. The groups comprised 10 animals per sex which were sacrificed after 92/93 days of treatment. Additional 5 rats per sex were used at 50 mg/kg. These animals were treated for 92/93 days and then allowed a 42-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during weeks 13 and 19. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

From the animals of the low and middle dose groups, the mesenteric lymph nodes were examined to establish a no-effect level.

Positive control:

Not requirded

Observations and examinations performed and frequency:

VIABILITY/MORTALITY

Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS

The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 92/93 (twice daily during days 1 - 3) during the treatment period and once daily during days 1 to 42 of the recovery period.

WEEKLY BEHAVIORAL OBSERVATIONS

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter. More details on the investigations are detailed in the section “Any other information on materials and methods incl. results”.

FUNCTIONAL OBSERVATIONAL BATTERY (SCREEN)

During weeks 13 and 19, relevant parameters from a modified Irwin screen test were evaluated in all animals. More details on the investigations are detailed in the section “Any other information on materials and methods incl. results”.

- Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Three measurements were recorded for fore- and hind legs; the means were calculated and recorded.

- Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.

Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION

The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

BODY WEIGHTS

Body weights were recorded once at the beginning acclimatization for random allocation by body weight, once during acclimatization following allocation, and weekly during the treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

OPHTHALMOSCOPY

The eyes of all rats were examined with a direct ophthalmoscope once during the acclimatization period, and of control and high dose rats during week 13 of treatment.

VAGINAL SMEAR FOR ESTRUS STAGE

Vaginal smears were taken over fourteen days from all females, and the stage of estrus was evaluated, if possible during weeks 12 - 13.

CLINICAL LABORATORY INVESTIGATIONS

Blood and urine sampling were performed after week 13 in allocation A and B animals and after week 19 in allocation B animals.

Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

In the summary and individual tables the names of some parameters have been abbreviated.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

The following hematology parameters were determined:

1. Complete Blood Cell Count

- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count

2. Hemoglobin Derivatives

- Methemoglobin
- Heinz bodies

3. Coagulation

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

The following urine parameters were determined:

1. Physical Examination

- Urine volume (18 hour)
- Specific gravity (relative density)
- Color
- Appearance

2. Chemical Examination

- pH value
- Nitrite
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Erythrocytes
- Leukocytes

Sacrifice and pathology:

NECROPSY

After Week 13: 02/03-Jan 2014 (allocation A)
After Week 19: 13-Feb-2014 (allocation B)

All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in modified Davidson's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pharynx
- Pituitary gland
- Prostate gland and seminal vesicles incl. coagulating glands
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in modified Davidson's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea
- Ureters
- Urinary bladder, filled w/formalin at necropsy
- Uterus (incl. oviducts, cervix and vagina)
- All gross lesions

ORGAN WEIGHTS

The organs from allocation A and B animals listed below were weighed before fixation and recorded on the scheduled dates of necropsy.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Epididymides (fixed in modified Davidson's solution)
- Heart including auricles
- Kidneys
- Liver
- Ovaries
- Prostate gland and seminal vesicles incl. coagulating glands
- Spleen
- Testes (fixed in modified Davidson's solution)
- Thymus
- Uterus (incl. oviducts, cervix and vagina)

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

HISTOTECHNIQUE

All organ and tissue samples listed under “Necropsy” above were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin. Additionally, slides of the testes of control and high-dose males were stained with Periodic Acid-Schiff (PAS).

HISTOPATHOLOGY

Slides of all organs and tissues listed under “Necropsy” above collected at scheduled sacrifices from allocation A animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. The mesenteric lymph nodes of allocation A males and females treated with 5 or 15 mg/kg/day were examined as well as PAS-stained testes of allocation B males.

Detailed qualitative and quantitative examinations of the testes were made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted if present.

Attempts were made to correlate gross observations with microscopic findings.

A peer review was performed by W. Henderson. The findings of the study pathologist and the peer-reviewing pathologist compared favorably. A peer review report was prepared.

Statistics:

The following statistical methods were used to analyze body weight, food consumption, ophthalmoscopic findings, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test .

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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Findings in both genders at 50 mg/kg/day and late effects still present after recovery.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose related increase in incidence and severity of granulomatous foci in the mesenteric lymph nodes remaining in evidence after recovery, and minor degrees of lymphoid hyperplasia in test item treated groups after treatment.

Histopathological findings: neoplastic:

not examined

Details on results:

1. IN-LIFE DATA

MORTALITY

All animals survived the scheduled treatment or recovery periods.

DAILY OBSERVATIONS

During the treatment and recovery periods, there were no test item-related clinical signs in any male or female at any dose level.

One female treated with 5 mg/kg/day showed transient hair loss; all other females were unaffected.

In two males (nos. 29 and 30) treated with 15 mg/kg/day, transient hair loss and dermal scabbing were noted.

Transient local hair loss was noted in one male treated with 50 mg/kg/day.

WEEKLY BEHAVIORAL OBSERVATIONS

No abnormal findings were noted during weekly observations (weeks 1 - 12) in males and females at any dose level.

FUNTIONAL OBSERVATIONAL BATTERY (SCREEN)

No abnormal findings were noted during the functional observational battery (week 13) in males and females at any dose level.

- Grip Strength: There were no test item-related differences in the mean fore- and hind limb grip strength values. The males treated with 15 mg/kg/day and 50 mg/kg/day showed significantly elevated values for the forelimbs (p<0.01 and p<0.05, respectively). Females treated with 5 mg/kg/day showed significantly elevated mean fore- and hind limb grip strength values (both p<0.05) when compared with the controls, whereas females treated with 15 mg/kg/day showed only significantly greater fore limb grip strength (p<0.05). Such differences are generally not associated with toxicological relevance and there was no dose response relationship. The values recorded during the recovery period did not show any trend for late effects.

- Locomotor Activity: There were no test item-related effects on locomotor activity. During week 13, males treated with 50 mg/kg/day showed a transient, statistically significant reduction (p<0.01) of locomotor activity during 40 - 50 minutes. Females were unaffected. There was no evidence of any late effects at the end of the recovery period.

FOOD CONSUMPTION

After 13 weeks of treatment, there were no differences of toxicological relevance between the mean food consumption values of test item-treated and control animals of either sex. The mean relative food consumption values were unaffected.

During the 6-week recovery period, the mean food consumption of the males and females that were previously treated with 50 g/kg/day was flat.

BODY WEIGHTS

After 13 weeks of treatment, there were no differences of toxicological relevance between the mean body weights or mean body weight gain of test item-treated and control animals of either sex.

During the 6-week recovery period, the males which had been treated with 50 mg/kg/day gained 51 grams (+12% from day 1 of recovery) and the females gained 17 grams (+6.7% from day 1 of recovery).

OPTHALMOSCOPIC EXAMINATIONS

During week 12, neither males nor females showed any test item-related ophthalmoscopic findings. A small number of typical dose-unrelated background findings (uni- or bilateral corneal opacities, lenticular vacuoles) were noted, as well as other changes (persistent hyaloid vessel and/or persistent pupillary membrane) generally associated with young rats that were unrelated to the test item treatment.

ESTRUS CYCLE DETERMINATION

The mean duration of estrus was similar in test item-treated and control animals.

At 5 mg/kg/day, the number of acyclic females was higher than that of the control females but this difference was not dose-related and therefore considered to be without toxicological relevance.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

After the treatment period, there was a small number of test item-related differences in hematology parameters. Males and females treated with 50 mg/kg/day showed elevated mean relative neutrophils (p<0.05 and p<0.01, respectively), and females showed reduced mean absolute lymphocytes (p<0.01). Additionally, females treated with 50 mg/kg/day showed elevated mean absolute neutrophils (p<0.01) when compared with the control group.

All other differences were either unrelated to dose, within the range of historical control values or were not corroborated by commensurate changes in related parameters.

After the 6-week recovery period, males and females treated with 50 mg/kg/day showed a clear shift in the reticulocyte maturity indices toward low-fluorescent cells. With the exception of the reduced high-fluorescence reticulocyte counts in recovery females, these differences exceeded the ranges of the historical control data. There were also reductions in the mean absolute and relative reticulocyte counts of males and females. The mean absolute numbers of neutrophils and monocytes were elevated in males, whereas the mean absolute number of lymphocytes was marginally reduced in males and females. These differences were considered to be late effects.

CLINICAL BIOCHEMISTRY

A small number of statistically significant differences to the control values were noted after 13 weeks of treatment. These were considered to be without toxicological relevance. All other differences were either unrelated to dose, within the range of historical control values or were not corroborated by commensurate changes in related parameters.

After the 6-week recovery period, the clinical biochemistry parameters showed no toxicologically relevant differences to the historical control data.

URINALYSIS

The urinalysis parameters were unaffected during the treatment and recovery periods.

3. PATHOLOGY

ORGAN WEIGHTS

There were no test item-related changes in the mean absolute or relative organ weights after 13 weeks of treatment. Males treated with 5 mg/kg/day had significantly reduced mean absolute epididymis weights (p<0.05) but these were unrelated to dose and considered to be incidental.

In females treated with 5 mg/kg/day and 15 mg/kg/day, significantly increased mean absolute liver weights (both p<0.05) and significantly increased mean liver-to-brain weight ratios (both p<0.01) were noted. The mean liver-to-body weight ratio was significantly increased in females treated at 5 mg/kg/day (p<0.01) only.

Females treated with 15 mg/kg/day had increased mean absolute spleen weights (p<0.05) and increased spleen-to-brain weight ratios (p<0.01) when compared with the controls.

The mean absolute and relative organ weights of the males and females treated with 50 mg/kg/day were similar to those of the respective controls.

In the absence of control values, no comparison was possible after recovery.

MACROSCOPIC FINDINGS

Macroscopic findings at the end of the main study and following the recovery period were unremarkable.

MICROSCOPIC FINDINGS

Treatment-related microscopic findings were recorded in the mesenteric lymph nodes. At the end of the main study these findings consisted of:

- Granulomatous foci were noted at minimal degree in one group 1 (0 mg/kg/day), at minimal or slight severity in seven group 2 (5 mg/kg/day), minimal to moderate in all ten group 3 (15 mg/kg/day) and at slight to severe severity in all ten group 4 (50 mg/kg/day) males. In females this finding occurred in two group 1 animals at minimal severity, in six group 2 at minimal or slight, in all ten group 3 at minimal to severe and in all ten group 4 animals at moderate or severe severity.
- Lymphoid hyperplasia at minimal degree was recorded in two group 3 and at minimal or slight severity in three group 4 males. In females a minimal degree of this finding was noted in one animal each in groups 1, 2 and 3 and at minimal or slight degree in eight group 4 animals.

Findings in group 4 animals following the forty-two day recovery period were:

- Granulomatous foci were noted at slight to severe degree in all five males and in all five females.
- Lymphoid hyperplasia at slight or moderate degree was recorded in two males and at minimal degree in one female.

The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. Staging of spermatogenesis revealed no treatment related effects.

Dose descriptor:

NOEL

Remarks:

could not be defined

Sex:

male/female

Basis for effect level:

other: Changes in some hematology parameters and a dose related increase in incidence and severity of granulomatous foci along with minor degrees (minimal or slight) of lymphoid hyperplasia

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

From the available 90-day study on Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) a NOAEL of 5 mg/kg bw can be derived. At higher dose levels the findings of granulomatous foci in the mesenteric lymph nodes increase both in severity and incidence. ‘Granuloma’ in the mesenteric lymph nodes are generally considered to be non-adverse, but as the severity and incidence increases with dose and the fact that no reversibility can be shown in the 42 days recovery group, the lowest dose is used as NOAEL.

Executive summary:

Oral administration of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) to Wistar rats at doses of 5, 15 and 50 mg/kg/day, for 92/93 days resulted in no deaths, no test item-related clinical signs, no differences in the functional observational battery (including no effects on grip strength or locomotor activity), no effects on food consumption or body weights, no ophthalmoscopic findings, no changes in the estrus cycles, no effects on clinical biochemistry or urinalysis parameters, no differences in organ weights or ratios and no macroscopical findings of toxicological relevance. Staging of spermatogenesis revealed no treatment related effects.

 

Test item-related findings were generally restricted to changes in some hematology parameters: At 50 mg/kg/day, males and females showed elevated neutrophils, whereas only females showed reduced mean absolute lymphocytes after 13 weeks of treatment. Late effects were noted after the end of the 6 week recovery period: males and females treated with 50 mg/kg/day showed a clear shift in the reticulocyte maturity indices towards low-fluorescent cells, and both genders showed reductions in the mean absolute and relative reticulocyte counts. Neutrophils and monocytes were elevated in males only, whereas lymphocytes were marginally reduced in males and females.

 

At the end of the main study microscopical findings consisted of a dose related increase in incidence and severity of granulomatous foci from one occurrence at minimal degree in control males to all ten males at 50 mg/g/day at slight to severe severity and in females from two occurrences at minimal severity in controls to all ten animals at moderate or severe degree. Associated with this finding were minor degrees (minimal or slight) of lymphoid hyperplasia in two males at 15 mg/kg/day and three males at 50 mg/kg/day, and in one animal each in the control group, 5 mg/kg/day and 15 mg/kg/day, and in eight animals at 50 mg/kg/day. These findings remained in evidence in the latter group following the forty-two day recovery period - granulomatous foci at slight to severe degree in all ten animals and lymphoid hyperplasia at slight or moderate degree in two males and at minimal degree in one female.

 

Based on the results of this study, a No Observed Adverse Effect Level (NOAEL) of 5 mg/kg bw/day was estabilished, although a NOEL for Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) could not be defined.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study is GLP compliant and has validity rating 1.

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

Quality of whole database:

Due to the low hazardous profile of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), local effects after possible exposure to the substance is considered to be limited. The substance has a low vapour pressure of 0.002 Pa at 20°C and a boiling point of > 300°C and the use of the substance is not expected to generate respirable aerosols.

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

Oral

A 90 day oral toxicity study according to OECD 408 has been performed with Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4). The dose levels were 5 mg/kg/day, 15 mg/kd/day and 50 mg/kg/day. The most significant dose response related finding was the findings of granulomatous foci in the mesenteric lymph nodes, which increased both in severity and incidence.‘Granuloma’ in the mesenteric lymph nodes are generally considered to be non-adverse, but as the severity and incidence increases with dose and the fact that no reversibility can be shown in the 42 days recovery group, the lowest dose is used as NOAEL. There were no indications of any systemic specific toxic effects such as serious organ damage.

 

This study was performed in accordance to the current OECD 408 and EU method B26 protocols, with full GLP compliance and with a well defined test substance, so the NOAEL of 5 mg/kg/day is a good basis for the calculation of DNEL values for use in Exposure assessments and the chemical safety report. No further testing is therefore proposed on the Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4).

 

Dermal

There is a 90 day oral toxicity study according to OECD 408 available for Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4). This study allows the determination of DNEL values for oral, inhalation and dermal exposure following the ECHA guidance document for deriving DNEL values. As appropriate DNEL values can be calculated using the oral dosing study data, which will allow the exposure scenarios to be performed and the Chemical safety report to be prepared, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

 

Inhalation

There is 90 day oral toxicity study according to OECD 408 available forAmines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4). This study allows the determination of DNEL values for oral, inhalation and dermal exposure following the ECHA guidance document for deriving DNEL values. As appropriate DNEL values can be calculated using the oral dosing study data, which will allow the exposure scenarios to be performed and the Chemical safety report to be prepared, it is not justified on animal welfare grounds to perform a repeat dose inhalation toxicity study.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Only available study of longest duration (90-days) of high validity.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Likelihood of exposures via inhalation is low considering use and physical properties: The substance has a low vapour pressure of 0.002 Pa at 20°C and a boiling point of > 300°C and the use of the substance is limited to industrial settings under controlled conditions, not expected to generate respirable aerosols.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Likelihood of exposures via skin is low considering use and physical properties: The use is limited to industrial settings under controlled conditions and with a molecular mass of greater than 500 and a calculated log Kow of 16.5, absorption is expected to be low.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Due to the low hazardous profile of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), local effects after possible exposure to the substance is considered to be limited. Likelihood of exposures via skin is low considering the use is limited to industrial settings under controlled conditions.

Justification for classification or non-classification

The toxic effects seen in the 90 day oral toxicity study is mainly the granulomatous inflammation in the mesenteric lymphnodes, which is generally considered to be non-adverse and generally a non-systemic effect. But as the severity and incidence increases with dose and the fact that no reversibility can be shown at the high dose level in the 42 days recovery group, only the lowest dose is used as NOAEL. At higher dose levels the findings of granulomatous foci in the mesenteric lymph nodes is more profound and together with the effects seen at higher doses in the 28 day oral toxicity study on the same substance, where granulomatous inflammation was also seen in the liver at the top dose, a STOT RE Cat 2 classification is considered relevant.