Registration Dossier

Administrative data

Description of key information

One repeated dose-range finding study over 14 days and two full 28 day oral toxicity studies are available on C16-22-(even numbered)alkylamines (CAS no 68037-92-3). They are all performed under GLP and have reliability rating 1.  The one available repeated dose oral toxicity study performed at the lowest doses on C16-22-(even numbered)alkylamines (CAS no 68037-92-3) was chosen as key study, identifying the lowest dose of 3.5 mg/kg bw as a LOAEL. 

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:
2012-10-25 to 2013-05-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008
Commission Directive 2001/59/EC of 6 August 2001
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of
the treatment period: males: 6-7 weeks old, females: 6-7 weeks old.
Body weight at the
allocation of the animals
to the experimental groups: males: 153 - 188 g (mean: 172.23 g, ± 20% = 137.79 – 206.68 g)
females: 121 - 146 g (mean: 133.53 g, ± 20% = 106.83 – 160.24 g).
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.


Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1039)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at
regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 011012 and 101112)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions
Route of administration:
oral: gavage
Vehicle:
other: Sesame oil
Details on oral exposure:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (sesame oil) was added to give the appropriate final concentration of the test item. The formulation vials were placed on Vortex machine for short period to ensure proper homogenistation of the formulation.
The vehicle was selected as suggested by the sponsor and on the basis of the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.

The test item formulation or vehicle was administered at a single dose to the animals by oral gavage at an application volume of 4 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

According to the results of the dose range finding study (BSL study no. 113873) and 28 day repeated dose toxicity study (BSL study no. 120952)
and in consultation with the sponsor the following doses (were selected for the 3 dose groups (LD, MD, HD) and 1 control group (C):

Dosage:
Control (C) and recovery control (CR): 0 mg /kg bw /day
Low Dose (LD): 3.5 mg /kg bw/day
Medium Dose (MD): 10 mg /kg bw/day
High Dose (HD) and recovery HD (HDR): 30 mg /kg bw/day

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received sesame oil using the same volume as used for the high dose group.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The animals of the main groups were treated with the test item or vehicle on 7 days per week basis.
The animals of the main and recovery groups (control and high dose) were dosed for 28 days.
After 28 days of administration the animals of the recovery groups were observed for a period of 28 days (recovery period).
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 3.5, 10, 30 mg/kg body weight/day
Basis:
other: Nominal in Sesame oil
No. of animals per sex per dose:
40 animals (20 males and 20 females) were included in the main study (5 male and 5 female animals per group). The main study included one control (C) and three dose groups (Low Dose = LD, Medium Dose = MD, High Dose = HD).
In addition, 20 animals (5 male and 5 female animals per group) were included in the control and high dose groups to be observed for a period of 28 days following the last administration.
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Clinical Observation
All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.


Functional Observation
Once before the first exposure and once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests . These tests were conducted in all animals.

Haematology
One day after the last administration, blood was sampled from all surviving animals for a haematological evaluation. Blood from the recovery animals was sampled at the end of the recovery period. After overnight fasting of the animals, blood from the abdominal aorta was collected in EDTA-coated tubes prior to or as part of the sacrifice of the animals.
The following haematological parameters were examined (haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), prothrombin time (PT), activated partial thromboplastin time (aPTT)

Clinical Biochemistry
One day after the last administration, blood was sampled from all surviving animals for an evaluation of the clinical biochemistry. Blood from the recovery animals was sampled at the end of the recovery period. After overnight fasting of the animals, blood from the abdominal aorta was collected in serum separator tubes just prior to or as part of the sacrifice of the animals. The following parameters of clinical biochemistry were examined (alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. The following parameters
(specific gravity, nitrite, ph-value (ph), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leucocytes) were measured
using qualitative indicators (Heiland Urine Stripes URI 10SL). Additionally, urine colour/ appearance were recorded.





Sacrifice and pathology:
Pathology
One day after the last administration (study day 29) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 57) were sacrificed using anesthesia (ketamine, medistar Arzneimittel, lot no: 00312, expiry date: 03/2014 and xylazin, Serumwerk, lot no. 00312, expiry date: 05/2014 (animals of treatment period) and Serumwerk, lot no. 00512, expiry date: 07/2014 (animals of recovery period) and subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Necropsy was also performed on animals found dead or animals euthanised for ethical reasons in a condition of morbidity.


The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain,
prostate with seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females), liver, vagina
(females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with coagulating glands as a whole
(males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes (mesentric and axillary), thyroid, peripheral
nerve (e.g. sciatic nerve) with skeletal muscle, spleen, bone with bone marrow (sternum), lung and trachea, pituitary gland, mammary glands, oesophagus, skin) from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for 24 hours before transferring to 10% neutral buffered formalin.

Histopathology
The afore-listed organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. A histopathological evaluation were carried out on all animals of the control and high dose groups (of the main study) which were sacrificed at the end of the treatment period. In addition, liver, spleen, lung, mesenteric lymph node, adrenal gland, stomach, duodenum, jejunum, ileum, Peyer’s patch and uterus from low, mid and recovery animals were evaluated histopathologically.

Any gross lesion macroscopically identified was examined microscopically. Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd. (test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.

Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
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
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
histocytic infiltration into liver, spleen and mesenteric lymph node
Histopathological findings: neoplastic:
no effects observed
Details on results:
Animal Survival
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Observations
No test item related clinical signs were observed in any male and female animal during the entire study period. Few spontaneous clinical signs were observed occasionally in male and female animals
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment and recovery period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, no statistically or biologically significant effect was observed on body weight and body weight gain in treatment groups during the entire study period. All group mean values for body weight and body weight gain were comparable with respective controls and were within the normal range of variation for this strain. The mean body weight increased with the progress of the study in all groups.

Food Consumption
In correlation to the body weight and body weight gain, the food consumption (g/day) in both males and was comparable to controls in all groups. However, from week 2 in the treatment groups, the food consumption was marginally decreased during treatment period when compared with controls.

Haematology and Blood Coagulation

In males, at the end of the treatment period, significant increase in neutrophils, monocytes in HD group and significant decrease in lymphocytes in HD group was observed when compared with controls although statistical significance was only achieved for increase in monocytes in HD group. At the end of recovery period, statistically significant increase in neutrophils, monocytes and significant decrease in lymphocytes was observed in HDR group when compared with controls.
In females, at the end of the treatment period, significant decrease in lymphocytes and increase in neutrophils and monocytes in MD and HD was observed when compared with control although statistical significance was not achieved for neutrophils. At the end of recovery period, statistically significant decrease in MCV, MCH, decrease in lymphocytes (statistical significance was not achieved) and significant increase in monocytes and RBC was observed in HDR group when compared with controls.

Clinical Biochemistry
In males, statistical analysis of clinical biochemistry data at the end of treatment period revealed significant increase in TBIL in LD and MD group when compared with controls. At the end of recovery period, statistically significant increase in urea was observed in HDR group when compared with controls.
In females, at the end of the treatment period, statistically significant decrease in Alb in LD and HD group and decrease in cholesterol in HD group was observed when compared with controls. At the end of recovery period, statistically significant decrease in Alb was observed in HDR group when compared with control group.


Urinalysis
High erythrocytes levels were found in the urine of two males (27 and 30 in HDR) and one female (41 in HD). All other urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed in females and recovery animals.

Pathology
Few specific gross pathological changes were recorded in female animals at terminal or recovery sacrifice and were not considered to be treatment-related. Findings were assumed to be common background findings in this strain.
Predominant macroscopic changes in female animals were fluid filled uterus (1/5 in LD, MD and HD), liver lobe protrusion in diaphragm (1/5 in HD) and fluid distension in uterus (1/5 in CR and 1/5 in MD).

Organ Weight
In males, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) spleen weight was observed in HD group. At the end of recovery period, statistically significant increase in absolute and relative (to brain and body weight) spleen and testes weights, increase in relative (to body weight) epididymides weight was observed in HDR group when compared to the controls.
In females, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) spleen weight was observed in HD group. Statistical analysis of organ weight data from females sacrificed at the end of recovery period revealed statistically significant increase in absolute and relative (to brain and body weight) spleen and decrease in absolute and relative (to brain and body weight) thyroid/parathyroid weight was observed in HDR group when compared to the controls


Histopathology
At terminal sacrifice, test item-related changes were seen in the small intestine, Peyer's patch, mesenteric lymph node, spleen and liver.
In view of the fact that mesenteric lymph node changes were seen at a minimal or mild degree in all animals of the lowest dose group and did not show any reversibility after 28 days they were considered adverse in all three dose groups. As a conclusion, under the conditions of this study, no NOAEL (No Observed Adverse Effect Level) could be established for pathology.


Dose Formulation Analytics
Concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD group were 108.9%, 96.7% and 97.1% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study week 1 for all dose groups. The mean recoveries observed for LD group were 106.8%, for MD group 88.8%, and for HD group 89.1% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) in LD group were 2.6%, in MD group 4.2%, and in HD group 2.0%.
Dose descriptor:
NOAEL
Remarks:
general
Effect level:
>= 30 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Remarks:
pathology
Effect level:
3.5 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Under the conditions of the present study, the repeated oral administration of C16-22-(even numbered)alkylaminesto male and female Wistar rats at doses of 3.5, 10 and 30 mg/kg body weight for 28 days was not associated with major signs of toxicity and mortality. In addition, the following conclusions can be made:

No test item related effect on clinical signs, body weight, food consumption, urine parameters, clinical biochemistry parameters and gross pathological findings was observed up to 30 mg/kg body weight/day.
Significant increase in male and female hematology parameters like neutrophils, lymphocytes, monocytes in HD group was indicative of inflammatory changes and a regenerative process /recovery from inflammation.

The statistical significant difference in absolute and relative spleen weight in both males and female HD group at the end of treatment period and recovery period could be attributed to lysosomal storage disorders. Minor histological changes in spleen at terminal sacrifice were completely regressed after the recovery period.

Mesenteric lymph node changes like histiocytic/foam cell infiltrates were seen at a minimal or mild degree in all animals of the lowest dose group and did not show any reversibility after 28 days and therefore no NOAEL (No Observed Adverse Effect Level) could be established for pathology. Although the effects seen have no structural or functional organ changes in animals of any group, the non reversible histocytic infiltration into liver, spleen and mesenteric lymph node is considered to be adverse.

Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of C16-22-(even numbered)alkylamines is considered to be 30 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats. However, this NOAEL is for the toxicological changes except pathology and based on pathology no overall NOAEL could be established.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of C16-22-(even numbered)alkylamines via oral administration to rats over a period of 28 days. In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 28 days following the last administration.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received sesame oil, the vehicle used in this study. The 4 groups comprised of 5 male and 5 female Wistar rats Crl: WI(Han).

The following doses were evaluated:

Control:                        0        mg/kg body weight

Low Dose:                    3.5      mg/kg body weight

Medium Dose:              10      mg/kg body weight

High Dose:                    30      mg/kg body weight

 

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in sesame oil and administered daily during a 28-day treatment period to male and female animals by oral gavage. Dose volumes were adjusted individually based on a twice-weekly body weight measurement.The application volume for all groups was 4 mL/kg body weight.

During the period of dose administration, the animals wereobservedprecisely each day for signs of toxicity. Body weight was measured twice a week and food consumption was measured weekly. At the end of the treatment period, all main study animals and at the end of recovery period, all recovery animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was determined and a set of organs/tissues was preserved in 10% neutral buffered formalin.

A histopathological evaluation was carried out on all animals of the control and high dose groups (of the main study) which were sacrificed at the end of the treatment period. In addition,liver, spleen, lung, mesenteric lymph node, adrenal gland, stomach, duodenum, jejunum, ileum, Peyer’s patch and uterus from low, mid and recovery animals were evaluated histopathologically. Organs showing gross alterations were also examined histopathologically.

Haematological and clinical biochemistry examinations were made on blood samples obtained from the overnight fasted main and recovery animals at the terminal sacrifice.

 

Summary Results:

Animal Survival

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

 

Clinical Signs

No test item related clinical signs were observed in anymale and femaleanimal during the entire study period.Few spontaneous clinical signs were observed occasionally in male and female animals During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment and recoveryperiod. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development

In both males and females, no statistically or biologically significant effect was observed on body weight and body weight gain in treatment groups during the entire study period. All group mean values for body weight and body weight gain were comparable with respective controls and were within the normal range of variation for this strain. The mean body weight increased with the progress of the study in all groups.

Food Consumption

In correlation to the body weight and body weight gain, the food consumption (g/day) in both males and was comparable to controls in all groups. However, from week 2 in the treatment groups, the food consumption was marginally decreased during treatment period when compared with controls.

Haematology and Blood Coagulation

In males, at the end of the treatment period, significant increase in neutrophils, monocytes in HD group and significant decrease in lymphocytes in HD group was observed when compared with controls although statistical significance was only achieved for increase in monocytes in HD group. At the end of recovery period, statistically significant increase in neutrophils, monocytes and significant decrease in lymphocytes was observed in HDR group when compared with controls.

In females, at the end of the treatment period, significant decrease in lymphocytes and increase in neutrophils and monocytes in MD and HD was observed when compared with control although statistical significance was not achieved for neutrophils. At the end of recovery period, statistically significant decrease in MCV, MCH, decrease in lymphocytes (statistical significance was not achieved) and significant increase in monocytes and RBC was observed in HDR group when compared with controls.

Clinical Biochemistry

In males, statistical analysis of clinical biochemistry data at the end of treatment period revealed significant increase in TBIL in LD and MD group when compared with controls. At the end of recovery period, statistically significant increase in urea was observed in HDR group when compared with controls.

In females,at the end of the treatment period, statistically significant decrease in Alb in LD and HD group and decrease in cholesterol in HD group was observed when compared with controls. At the end of recovery period, statistically significant decrease in Alb was observed in HDR group when compared with control group.

Urinalysis

High erythrocytes levels were found in the urine of two males (27 and 30 in HDR) and one female (41 in HD). All other urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed in females and recovery animals.

Pathology

Few specific gross pathological changes were recorded in female animalsat terminal or recovery sacrificeand were not considered to be treatment-related. Findings were assumed to be common background findings in this strain.

Predominant macroscopic changes in female animals were fluid filled uterus (1/5 in LD, MD and HD), liver lobe protrusion in diaphragm (1/5 in HD) and fluid distension in uterus (1/5 in CR and 1/5 in MD).

Organ Weight

In males, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) spleen weight was observed in HD group. At the end of recovery period, statistically significant increase in absolute and relative (to brain and body weight) spleen and testes weights, increase in relative (to body weight) epididymides weight was observed in HDR group when compared to the controls.

In females, at the end of treatment period, statistically significant increase in absolute and relative (to brain and body weight) spleen weight was observed in HD group. Statistical analysis of organ weight data from females sacrificed at the end of recovery period revealed statistically significant increase in absolute and relative (to brain and body weight) spleen and decrease in absolute and relative (to brain and body weight) thyroid/parathyroid weight was observed in HDR group when compared to the controls

Histopathology

At terminal sacrifice, test item-related changes were seen in the small intestine, Peyer's patch, mesenteric lymph node, spleen and liver.

In view of the fact that mesenteric lymph node changes were seen at a minimal or mild degree in all animals of the lowest dose group and did not show any reversibility after 28 days they were considered adverse in all three dose groups. As a conclusion, under the conditions of this study, no NOAEL (No Observed Adverse Effect Level) could be established for pathology.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD group were 108.9%, 96.7% and 97.1% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 for all dose groups. The mean recoveries observed for LD group were 106.8%, for MD group 88.8%, and for HD group 89.1% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) in LD group were 2.6%, in MD group 4.2%, and in HD group 2.0%.

   

Conclusion:

Under the conditions of the present study, the repeated oral administration ofC16-22-(even numbered)alkylaminesto male and female Wistar rats at doses of 3.5, 10 and 30 mg/kg body weight for 28 days was not associated with major signs of toxicity and mortality.In addition,the following conclusions can be made:

No test item related effect on clinical signs, body weight, food consumption, urine parameters, clinical biochemistry parameters and gross pathological findings was observed up to 30 mg/kgbody weight/day.

Significant increase in male and female hematology parameters like neutrophils, lymphocytes, monocytes in HD group was indicative of inflammatory changes and a regenerative process /recovery from inflammation.

The statistical significant difference in absolute and relative spleen weight in both males and female HD group at the end of treatment period and recovery period could beattributed to lysosomal storage disorders. Minor histological changes in spleen at terminal sacrificewere completely regressed after the recovery period.

Mesenteric lymph node changes like histiocytic/foam cell infiltrates were seen at a minimal or mild degree in all animals of the lowest dose group and did not show any reversibility after 28 days and therefore no NOAEL (No Observed Adverse Effect Level) could be established for pathology. Although the effects seen have no structural or functional organ changes in animals of any group, the non reversible histocytic infiltration into liver, spleen and mesenteric lymph node is considered to be adverse.

Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of C16-22-(even numbered)alkylaminesis considered to be 30 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male andfemale rats.However, this NOAEL is for the toxicological changes except pathology and based on pathology no overall NOAEL could be established.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
3.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is performed according to OECD 407 guideline and under GLP and has reliability rating 1. It is sufficient to cover the information requirements in Annex VIII.

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

Two 28 day oral toxicity studies performed according to OECD 407 and under GLP are available. The doses tested were in the two tests were the following doses tested: 3.5 mg/kg bw, 10 mg/kg bw, 15 mg/kg bw, 30 mg/kg bw, 60 mg/kg bw and 240 mg/kg bw, with recovery groups in the control, 30 mg/kg bw and 240 mg/kg bw groups respectively.Observations included mortality / viability, clinical signs (daily), functional observations and locomotor activity, body weight and food consumption, clinical pathology and macroscopy at termination, organ weights and histopathology on a selection of tissues. Formulations were analyzed for accuracy and homogeneity.

 

The highest dose tested caused two deaths, one which was considered to be related to dosing accident and the second one was considered to be treatment related. Based on all the available data from these two studies, a NOAEL value cannot be established due to the presence ofhistiocytes / foam cells in the mesenteric lymph nodes in all dose groups tested. This is considered as an adverse effect although the findings in the mesenteric lymph nodes are considered to be of local nature. The reasoning behind this is because at higher dose groups the histiocytes / foam cells are seen in duodenum, jejunum and ileum as well and no clear reversibility was observed in recovery periods up to 28 days. Histiocytes accumulating in the lymph node sinuses may impair the lymph drainage from the intestine and is seen as a local inflammatory response on the treatment withC16-22-(even numbered)alkylamines (CAS no 68037-92-3). However at 3.5 mg/kg bw the results were minimal and mild, and only located to the mesenteric lyphnodes, compared to moderate and marked effects at the highest dose in the same study. Therefore 3.5 mg/kg bw is considered to be approaching the bottom of the dose effect curve. In the first 28 day study performed, using higher doses, the effects seen at 15 mg/kg bw are only one mild effect in the males and two minimal effects in the females. No obvious explanation to the difference in response between the two studies has been found. One plausible explanation can be biological variability between the animals tested, or some concentration related difference in bioavailability. However taking the results together supports 3.5 mg/kg bw as a conservative LOAEL.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The one available repeated dose oral toxicity study performed at the lowest doses on C16-22-(even numbered)alkylamines (CAS no 68037-92-3). It is performed according to OECD 407 guideline under GLP conditions and has reliability rating 1.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
There is no repeat dose inhalation study on on C16-22-(even numbered)alkylamines (CAS no 68037-92-3) however the substance has low vapour pressure 0.022 Pa at 20 °C. Significant exposure would be unlikely since the handling is not expected to result in aerosols, particles or droplets of an inhalable size. Therefore it is considered to not be scientifically valid to conduct a repeat dose inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons. The guidance of REACH allows the inhalation long term DNEL to be calculated based on the oral repeat dose NOAEL.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
There is no repeat dose inhalation study on C16-22-(even numbered)alkylamines (CAS no 68037-92-3) however the substance has low vapour pressure 0.022 Pa at 20 °C. Significant exposure would be unlikely since the handling is not expected to result in aerosols, particles or droplets of an inhalable size. Therefore it is considered to not be scientifically valid to conduct a repeat dose inhalation study. Also the corrosive nature of the substance would make it difficult to perform such a test for animal welfare reasons. The low possibility of inhalation makes such a test scientifically unjustified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
There is no repeat dose dermal study on C16-22-(even numbered)alkylamines (CAS no 68037-92-3), and due to the corrosive properties of the substance it is not possible to conduct repeat dose dermal toxicity studies due to animal welfare considerations. It is also considered very unlikely that dermal absorption would exceed oral absorption, so it would be expected than the oral NOAEL would be lower than a corresponding value from a dermal study. Results from the available repeat dose oral study can be used in the setting of DNELs in accordance with the REACH guidelines. As appropriate DNEL values can be calculated using the oral dosing study data, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
There is no repeat dose dermal study on C16-22-(even numbered)alkylamines (CAS no 68037-92-3), and due to the corrosive properties of the substance risk management measures such as wearing appropriate gloves and protective clothing will prevent any significant dermal contact. Local effects on the skin would be expected to be limited to local irritation /corrosion, which would be dependent on the local concentration rather than the dose or duration of exposure. This is a medium hazard and does not justify the requirement for an additional dermal animal study to establish a local NOAEL for dermal exposure.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: other

Justification for classification or non-classification

The EU CLP (GHS) criteria for classification of Specific Target Organ Toxicity (STOT) are based on data from a 90 day study, for Category 1 the range for such effects are ≤ 10 mg/kg bw and for Category 2 the range for such effects is 10-<100 mg/kg/day. Where the data used are from a subacute study these levels are multiplied by three. 

 

Based on the available data on C16-22-(even numbered)alkylamines (CAS no 68037-92-3) the substance should be classified as STOT-RE Cat 1. The reasoning behind this is because at doses of 30 mg/kg bw and higher the histiocytes / foam cells are seen in duodenum, jejunum and ileum as well and no clear reversibility was observed in recovery periods up to 28 days. Histiocytes accumulating in the lymph node sinuses may impair the lymph drainage from the intestine and is therefore considered as an adverse effect within the classification range generating a STOT-RE Cat 1 classification.