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Description of key information

For C36-alkylenediamine an OECD 422 study is available, involving the dosing of males up to 29 days and female animals up to 43-57 days. A NOAEL of 50 mg/kg was derived from this study, based on effects observed of foamy macrophages in the mesenteric lymphnodes appearing as the first effect at low dose and in lamina propria of intestines at increasing dose levels. This effect is also referred to as phospholipidosis, and is considered at its lowest severity to be a local effect, as consequence to route of exposure. Only from 500 mg/kg an increased WBC was the only additional toxic effect observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March - 20 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 293 gr (males) or 201 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.
Temporary deviations from the minimum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.


IN-LIFE DATES: From: 25 March - 20 May 2012
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and density of the test substance and vehicle. No correction was made for the purity of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe B.V. and on information provided by the Sponsor.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion after the treatment phase, since a validated method (Project 498062) was not yet available during the in-life phase. Samples of formulations prepared in an identical manner as during the in-life phase were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for Group 4 or 85-115% of the target concentration for Groups 2 and 3. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Accuracy of preparation results:
The concentrations analysed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
For the formulation of Group 2, the mean accuracy was above the target concentration (i.e. 117% of target). Since the deviation above 115% was relatively small and since it was known from the method validation (see NOTOX project 498062) that recoveries can vary more than normal due to the properties of the substance, this was accepted.
A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared after in-life phase. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.

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

Stability results:
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-57 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Four females (all of different Groups) were not dosed during littering.
Frequency of treatment:
Once daily, 7 d/w
On Day 36, one female of Group 3 was dosed twice (within 30 minutes; 88% of the intended dose at the first dosing, and 12% of the intended dose at the second dosing). A clinical observation was conducted only after the first dosing. The animal eventually received the correct volume and dose. Dosing an animal twice within 30 minutes is considered to have no adverse effect on the outcome of the study given its incidental occurrence, and since oral gavage is not an invasive procedure. The observation was conducted after the animal received the larger part of the dose (i.e. after the first dose), and since no peak effect of occurrence of clinical signs was noted in the dose range finding study (NOTOX Project 498061), this was considered sufficient for the purpose of this study.
One female of Group 1 was dosed on the day of necropsy (lactation Day 7). This did not adversely affect any subsequent examinations conducted for this animal. This animal was not selected for clinical pathology determinations and was hence not fasted overnight prior to necropsy/dosing.
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10; an extra 5 males in Groups 1 and 4 were allowed 2 weeks of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In consultation with the sponsor and based on the results of the dose range finding study (NOTOX Project 498060: Based on the results of this range finding study, dose levels for the main study were 50, 150 and 500 mg/kg body weight. No clinical signs were observed at the highest selected dose for the main study (500 mg/kg). Therefore, clinical observations in the main study were conducted immediately after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.)
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS:
Daily detailed clinical observations were made in all animals, within 0-30 minutes after dosing between Days 1-18, and within 0-15 minutes after dosing from day 19 onwards. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
On the following days, clinical observations were conducted later than protocolled: Day 15, females (start of observations approximately 6 minutes later); Day 17, males (start of observations approximately 3 minutes later); Daily from Day 18 onwards: maximum 23 minutes later. The slightly later time point of observations did not adversely affect the interpretation of the study results, given the absence of a peak effect of occurrence of clinical signs (based on the dose range finding study (NOTOX Project 498061).
No arena observations were performed in weeks 8 and 9 of the study. The daily observations for clinical signs consist of a series of extensive examinations including the type of observations that are also assessed in the arena observations, and as such, the likelihood of having missed any relevant observations is minimal.

BODY WEIGHT:
- Males and females were weighed on the first day of exposure and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected Main males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling). Since (potential) treatment-related findings were noted in motor activity of males at 500 mg/kg bw, motor activity measurements were also conducted for all Recovery males at the end of the recovery phase.
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes
Terminal body weight and organ weights were recorded from a non-selected female of Group 2, and from one selected female of Group 4 no terminal body weight and organ weights were recorded. The animal of Group 2 showed a higher liver weight since this animal was not fasted as it was not selected. Other organ weights of this animal were comparable to control weights. Overall, more organs were recorded for this group than required according to protocol, which provides additional information. For Group 4, sufficient organ weight data were available for adequate interpretation of the study results.


HISTOPATHOLOGY:
- According to test guidelines
A few organs were not available for histopathology. Missing tissues are listed in raw data and pathology report. Sufficient data was available for histopathological evaluation.
A limited instead of full list of tissues/organs was collected from one female of Group 1 and one female of Group 4. A full list of tissues/organs was processed for histopathological examination from another animal of the same Group, who had also delivered live offspring. Hence, a full list of tissues/organs was examined histopathologically from 5 animals/sex for this group as per protocol. For Group 4, sufficient data were available for adequate histopathological evaluation.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Ref. 5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Urinalysis findings:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 500 mg/kg was found dead on Day 14 of the post-coitum period. Macro- and microscopic findings observed for this animal indicated that its death was gavage-related. In addition, in the absence of similar findings or signs of moribundity among other animals of this dose group, this death was considered to be unrelated to toxicity of the test substance.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 500 mg/kg and at lower incidence at 150 mg/kg during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Incidental findings that were noted included alopecia, a broken tail apex and rales. One female at 50 mg/kg showed piloerection, hunched posture and a lean appearance during two weeks prior to scheduled necropsy. Piloerection was also noted for one female each at 50 and 500 mg/kg. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
No toxicologically significant effects on motor activity were noted. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Motor activity of males at 500 mg/kg at the end of the treatment period was slightly higher than control animals, achieving a level of statistical significance for ambulation counts. Motor activity of females at 500 mg/kg during the lactation period showed the opposite effect, i.e. being lower than control animals, achieving a level of statistical significance for total movements and ambulation counts. At the end of the recovery period (assessed for males only), motor activity of males at 500 mg/kg was similar to control levels. Since the change in motor activity was reversible in males, the motor activity habituation profile appeared similar between the groups, clinical observations revealed no supportive findings in both sexes and functional observation tests were normal, these changes in motor activity were considered not to be of toxicological relevance.

BODY WEIGHTS
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period (both genders) and recovery period (males).

FOOD CONSUMPTION
Food consumption before or after correction for body weight was similar between treated and control animals during the treatment period (both genders) and recovery period (males). One female at 50 mg/kg showed a notable reduction in food intake over Days 11-20 of the post-coitum period, coinciding with occurrence of clinical signs (see respective paragraph). Given the incidental nature of this change, this was considered to be of no toxicological relevance.

HAEMATOLOGY
At the end of the treatment period, a higher mean white blood cell count occurred in males and females at 500 mg/kg (not statistically significant for females), which was essentially due to a higher mean neutrophil count (relative and absolute counts increased with statistical significance). At the end of the recovery period (assessed for males only), the mean white blood cell count and mean relative and absolute neutrophil count remained increased over the control mean.

Other (statistically significant) changes in haematological parameters were considered not to be of toxicological relevance:
-The lower mean relative lymphocyte count in males and females at 500 mg/kg was due to the higher white blood cell counts, and mean lymphocyte counts were similar to control levels when expressed in absolute terms.
- The statistically significant higher mean relative and absolute monocyte count in males at 500 mg/kg at the end of the treatment and recovery period was minor in nature (well within the range considered normal for rats of this age and strain). The relative mean monocyte count at 150 mg/kg was similar to the mean at 500 mg/kg, but no difference to the control mean was seen when expressed in absolute terms.
- Any statistically significant changes in red blood cell parameters of males at the end of the treatment and recovery period occurred in the absence of a dose-related trend, were minor in nature (i.e. essentially within the range considered normal for rats of this age and strain), and occurred in the absence of any morphological correlates. At the end of the treatment period, these changes consisted of higher red blood cell counts, haemoglobin and haematocrit levels, and lower mean corpuscular haemoglobin (MCH) level and mean corpuscular haemoglobin concentration (MCHC) in males at 50, 150 and/or 500 mg/kg. At the end of the recovery period (assessed for males only), haematological changes consisted of higher reticulocyte counts, lower haemoglobin and lower mean corpuscular haemoglobin (concentration) level.
- The shortened activated partial thromboplastin time (APTT) in males at 500 mg/kg and in females at 50 and 150 mg/kg at the end of the treatment period was considered to be of no toxicological relevance since the opposite effect (i.e. a prolonged activated partial thromboplastin time) would be expected in case of target organ toxicity, these changes occurred in the absence of a treatment-related distribution (females) and remained within the range considered normal for rats of this age and strain.

CLINICAL BIOCHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters at the end of the treatment period in males were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes consisted of higher alanine and aspartate aminotransferase activity levels (ALAT/ASAT) at 150 and/or 500 mg/kg, higher alkaline phosphatase activity levels (ALP) at 150 mg/kg, higher albumin levels at 50 and 150 mg/kg, lower glucose levels at 150 and 500 mg/kg, and higher sodium and chloride levels at 150 mg/kg.

MACROSCOPIC EXAMINATION
Necropsy did not reveal any toxicologically relevant alterations. Macroscopic findings in the female at 500 mg/kg found dead on Day 14 of the post-coitum period consisted of gray-white discolouration of the left side of the lungs and reddish fluid in the thoracic cavity. The nature of these findings, and absence of similar findings among other animals of the same group indicate that these were gavage-related. The incidence of other necropsy findings among surviving control and treated animals at the end of the treatment and recovery period was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in absolute and relative organ weights. Any statistically significant changes in organ weights of treated and control animals were considered not to be a sign of toxicity since these changes did not show a dose-related trend, changes remained within the range considered normal for rats of this age and strain, and/or were absent at the end of the treatment period (for changes noted at the end of the recovery period for males). These changes consisted of a higher absolute and relative spleen, testes and epididymides weight and higher relative kidney weight of males at 500 mg/kg at the end of the treatment and recovery period, higher adrenal (absolute) and spleen (absolute and relative) weight in females at 150 mg/kg, and higher absolute and relative uterus weight in females at 150 and 500 mg/kg.

MICROSCOPIC EXAMINATION
The following microscopic findings were considered to be related to treatment with the test substance:
- Duodenum: foamy macrophage foci in the lamina propria were present in 3/5 500 mg/kg (3 minimal) treated Main group male and 1/5 500 mg/kg (minimal) treated Main Group female rats.
- Jejunum: foamy macrophage foci in the lamina propria were present in 2/5 500 mg/kg (2 minimal) treated male rats and in 1/5 500 mg/kg (minimal) treated female rats. In Recovery Group 4 males this finding was present at minimal degree in 1/5 rats.
- Ileum: foamy macrophage foci in the lamina propria were present in 2/5 150 mg/kg (2 minimal) and in 5/5 500 mg/kg (5 minimal) treated male rats and in 1/5 150 mg/kg (1 minimal) and in 4/5 500 mg/kg (4 minimal) treated female rats. In Recovery group 4 males this finding was present in 4/5 rats (3 minimal, 1 slight).
- Mesenteric lymph node:
- Macrophage foci were present at increased incidence and severity in 4/5 50 mg/kg (1 minimal, 2 slight, 1 moderate), in 5/5 150 mg/kg (1 minimal, 3 slight, 1 moderate) and in 4/4 500 mg/kg (3 slight, 1 moderate) treated male rats compared to 1/5 control (slight) treated male rats and in 5/5 50 mg/kg (4 slight, 1 moderate), in 5/5 150 mg/kg (1 slight, 3 moderate, 1 marked) and in 5/5 500 mg/kg (1 minimal, 1 slight, 3 moderate) treated female rats (no foci in control females). In Recovery Group rats macrophage foci were present at increased incidence and severity in 5/5 Recovery Group 4 rats (2 slight, 3 moderate), compared to 2/5 Recovery Group 1 rats (1 minimal, 1 slight).
- Necrosis was present in 1/5 150 mg/kg treated Main Group males (slight) and in 1/5 Recovery Group 4 males (slight).
- Fibrosis was present in 1/5 500 mg/kg treated Main Group male (minimal) and 2/5 500 mg/kg treated female rats (1 minimal, 1 slight) and in 3/5 Recovery Group 4 males (2 slight, 1 moderate).
- Sinus histiocytosis was present in 1/5 50 mg/kg treated male (minimal) and in 3/5 50 mg/kg treated female (3 minimal) rats, in 2/5 male rats at 150 mg/kg (1 minimal, 1 slight), in 5/5 female rats at 150 mg/kg (2 minimal, 3 slight), in 4/4 male rats at 500 mg/kg (1 slight, 3 moderate) and in 5/5 female rats at 500 mg/kg (2 slight, 3 moderate). In Recovery group 4 males this finding was present in 5/5 rats (4 slight, 1 moderate).
After the 14-day treatment free recovery period, the lesions in the ileum and mesenteric lymph node did not show any clear degree of recovery, whilst findings in the macrophages of the lamina propria of the duodenum and jejunum showed full or partial recovery, respectively.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: macrophage foci and sinus histiocytosis in mesenteric lymph nodes accompanied by foamy macrophages in the lamina propria of the duodenum and/or ileum, and necrosis and fibrosis in the mesenteric lymph nodes at 150 mg/kg bw/d
Critical effects observed:
not specified
Conclusions:
In conclusion, treatment with Amines, C36-alkylenedi-, by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg revealed parental toxicity at 50 mg/kg and higher. At 50 mg/kg, findings were confined to macrophage foci and sinus histiocytosis in the mesenteric lymph nodes of both genders (minimal to moderate degree). At 150 and/or 500 mg/kg, these findings were noted at higher incidence and/or severity, along with foamy macrophages in the lamina propria of the duodenum and/or ileum, and necrosis (one male at 150 mg/kg) and fibrosis (one male and two females at 500 mg/kg) in the mesenteric lymph nodes, and higher white blood cell (neutrophil) counts at 500 mg/kg. The nature of changes at 150 and 500 mg/kg (in particular necrosis and fibrosis in the mesenteric lymph nodes, albeit occurring at low incidence), indicate that these effects are to be considered adverse in toxicological terms at those dose levels.

Based on these results, the parental NOAEL was established to be 50 mg/kg.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Longest repated dose study available on C36-alkylenediamine. Study is GLP compliant with Klimisch score 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

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Available information suggests that dermal route is not the most sensitive route of exposure. Following the severe irritating / corrosive properties of the substance, dermal testing should not be performed also based on animal welfare grounds.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
C36-alkylenediamine has proven to be severely irritating to skin showing to be irreversible within the 14-day observation period, and corrosive to the eyes.

Additional information

Oral

Evaluation for the oral route of C36-alkylenediamine (Dimerdiamine) involved a 14-day range finding followed by a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 and under GLP.

In the range finding study groups of 3 females were dosed 500 or 1000 mg/kg bw/day for 14 days. At 500 mg/kg no effects were observed. At 1000 mg 1/3 animals died, and a second group of 3 animals were dosed in order to evaluate whether this was accidental or related to treatment, also leading to 1/3 animals sacrificed. The surviving animals showed hunched posture, piloerection and/or salivation, with normal weight gain with slightly reduced food intake over days 1-4. No abnormalities were noted in the surviving animals upon macroscopic examination.

 

Following the results from a 14-day range finding study, the dose levels for the OECD 422 were selected. Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 150 and 500 mg/kg/day. An extra 5 males in Groups 1 and 4 were allowed 2 weeks of recovery. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-57 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Evaluated were: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/ developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Results:

Males and females at 500 mg/kg showed a higher white blood cell count at the end of the treatment period and recovery period (males), which was essentially due to a higher neutrophil count. The only other effects observed involve the occurrence of foamy macrophages mesenteric lymph nodes and in lamina propria of intestines at higher dose levels:

At 50 mg/kg, findings were confined to macrophage foci and sinus histiocytosis in the mesenteric lymph nodes of both genders (minimal to moderate degree). At 150 and/or 500 mg/kg, these findings were noted at higher incidence and/or severity, along with foamy macrophages in the lamina propria of the duodenum and/or ileum, and necrosis (one male at 150 mg/kg) and fibrosis (one male and two females at 500 mg/kg) in the mesenteric lymph nodes, and higher white blood cell (neutrophil) counts at 500 mg/kg. The nature of changes at 150 and 500 mg/kg (in particular necrosis and fibrosis in the mesenteric lymph nodes, albeit occurring at low incidence), indicate that these effects are to be considered adverse in toxicological terms at those dose levels.

Based on these results, the parental NOAEL was established to be 50 mg/kg.

 

The finding of “foamy macrophages” refers to phospholipidosis, a common effect observed following administration of cationic amphiphilic materials, including marketed pharmaceuticals. It has been observed in most of the repeated dose studies involving alkyl amine derived substances, including for primary fatty amines. Further studies involving 90-day and even a chronic 2-year study with the more potent of these substances shows that these effects of foamy macrophages remains the critical endpoint and that the levels do not change with increasing of the duration of the study.

 

The data obtained on C36-alkylenediamine can be compared to available data from repeated dose studies available for the primary fatty amines (PFA). Cross-reading to Primary alkyl amines can be considered on the basis of similarities of structure with same functional groups, leading to common biological activity, and common metabolic degradation.For full justification see document “Justification in support of cross-reading from primary fatty amines (PFA) to Dimerdiamine”.

For the oral route of exposure information from GLP and guideline-compliant sub-acute 28 -day studies are available for Oleyl-alkylamines and Tallow-alkylamines which demonstrated the existence of a closely related toxicity profile.

Leading health effects after oral exposure were mortalities at higher dose-levels associated with precedent bad general health status, erosions of the mucosa of the gastrointestinal tract, accumulation of histiocytes in the submucosa of the distal parts of the small intestine and in the mesenteric lymph nodes, and mild toxic effects in liver and kidneys.

With respect to local effects, the available data clearly demonstrate that primary alkylamines cause damage along the exposure route, i. e. the mucosa of the gastrointestinal tract. The data also indicate a cytotoxic potential at any site of contact along the exposure route which can be explained by the strong dermal irritative / corrosive nature of the primary alkylamines.

With Tallow-alkylamine, at the low dose (12.5 mg/kg), histopathological changes were limited to slight histiocytic vacuolation in mesenteric lymph nodes and small intestine, without abnormal accumulation of histiocytes.

 

The most sensitive NOAEL for the endpoint `repeated dose toxicity` is used for the assessment which was derived from the valid oral 28-day study on Oleyl-alkylamines. The NOAEL from this study was 3.25 mg/kg body weight per day, based on the significantly reduced body weight gain observed at 12.5 mg/kg bw/d.

This approach is supported by findings from a 2-year chronic toxicity study in rats and a 1-year chronic feeding study in dogs which revealed oral NOAELs of about 10 mg/kg body weight per day. Based hereupon, additional testing was not considered necessary in the existing EU risk assessment on primary alkylamines.

 

 

Based on the information above, it is apparent that the toxicity of the dimerdiamine is much lower than that of the primary alkyl amines. The observation of foamy macrophages is the first critical effect that appears at low dose, and experience shows that the dose level at which this becomes apparent does not vary with increasing study duration. This effect is also referred to as phospholipidosis, and is considered at its lowest severity a local effect, as consequence to route of exposure.

The available OECD 422 study involved dosing of female animals up to 43-57 days. A study of longer duration (90-day), is not likely to bring additional information on unexpected increased toxicity, nor is it expected to result to lower NOAEL.

 

C36-alkylenediamine is mainly used as intermediate in industrial settings where adequate PPE are applied, limiting direct dermal exposures as consequence to its irritative / corrosive properties. Consequently, exposures are very low, leading already to very low RCR.Based on thelow exposures it is therefore proposed to waive the 90-day study in accordance with the Annex XI(3) of the REACH regulation setting the guidelines for substance-tailored exposure-driven testing.

 

Dermal:

C36-alkylenediaminehas proven to be severely irritating to skin showing to beirreversible within the 14-day observation period, and corrosive to the eyes. Following these dermal irritative / corrosive properties, dermal exposure has to be limited / minimized.C36-alkylenediamineis only used as intermediate in industrial settings where adequate PPE are applied, limiting direct dermal exposures. Consequently, skin contact in production and/or use is not likely. Testing by the dermal route is therefore not appropriate as stipulated in Annex IX, point 8.6.2, column 2 of REACH regulation (EC) 1907/2006.

Also available information suggests that dermal route is not the most sensitive route of exposure. Furthermore, following the severe irritating / corrosive properties of the substance, dermal testing should not be performedalso based on animal welfare grounds.

 

Inhalation:

Additional testing of C36-alkylenediamine (Dimerdiamine) for inhalation toxicity is not appropriate, as exposure of humans via inhalation is not likely. Due to the very low vapour pressure (2.2 x10-5 hPa @ 20°C) inhalation of vapours leading to irritation of airways will not occur. Also aerosol exposures are not likely to occur in view of its only use as industrial intermediate under conditions that do not lead to aerosol formation. Testing for toxicity by inhalation route is therefore generally not needed according to REACH regulation (EC) 1907/2006 (Annex IX, point 8.6.2, column 2).

Furthermore, testing would also not be justified because of animal welfare reasons. C36-alkylenediamine is severely irritating / corrosive to skin and is classified as corrosive to the eyes, whereas its systemic toxicity is low. Due the very low vp (2.2 x10-7 Pa @ 20°C) and viscosity of substance, inhalation exposures would only be possible in the form of aerosol, consisting of larger droplets depositing in upper airways. It can be expected that aerosol levels which would lead to severe local damage in the airways will not lead to systemic toxicity following absorption from larger and smaller bronchi, as most of the inhaled amounts will deposit in larger airways and will be ingested after mucociliary transport to the pharynx.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only repeated dos estudy avaibale for C36-alkylenediamine

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
testing would also not be justified because of animal welfare reasons. C36-alkylenediamine is severely irritating / corrosive to skin and is classified as corrosive to the eyes, whereas its systemic toxicity is low. Due the very low vp (2.2 x10-7 Pa @ 20°C) and viscosity of substance, inhalation exposures would only be possible in the form of aerosol, consisting of larger droplets depositing in upper airways. It can be expected that aerosol levels which would lead to severe local damage in the airways will not lead to systemic toxicity following absorption from larger and smaller bronchi, as most of the inhaled amounts will deposit in larger airways and will be ingested after mucociliary transport to the pharynx.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Waiving: Additional testing of C36-alkylenediamine (Dimerdiamine) for inhalation toxicity is not appropriate, as exposure of humans via inhalation is not likely. Due to the very low vapour pressure (2.2 x10-5 hPa @ 20°C) inhalation of vapours leading to irritation of airways will not occur. Also aerosol exposures are not likely to occur in view of its main use as industrial intermediate under conditions that do not lead to aerosol formation. Testing for toxicity by inhalation route is therefore generally not needed according to REACH regulation (EC) 1907/2006 (Annex IX, point 8.6.2, column 2).

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

Justification for classification or non-classification

The available OECD 422 study involving dosing by oral route for 28 days or more (females even over 45 days) did even at the highest dose of 500 mg/kgbw/day not show significant toxic effects.

C36-alkylenediamine therefore does not need to be classified STOT-RE.

 

There are no appropriate repeated dose studies by dermal route available for C36-alkylenediamine. Available information however suggests that dermal route is not the most sensitive route of exposure, and classification STOT-RE is thus not required.

 

Also no information is available from testing of C36-alkylenediamine via inhalation route. However, exposures via inhalation are not expected in view of very low vapour pressure, and following its use as industrial intermediate under conditions that do not lead to aerosol formation.