Registration Dossier

Administrative data

Description of key information

28 day oral (OECD 407, GLP): NOAEL 30 mg/kg bw/day based on increased ALAT, reduced BW gain, changes in thymus weight, and microscopic findings in mesenteric lymph node, stomach, thymus and lung seen at 100 mg/kg bw/day.
90 day oral (OECD 408, GLP): NOAEL 10 mg/kg bw/day based on histopathological findings at 30 mg/kg consisting of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles, increased severity of macrophage foci in the lungs (in one male only), lower relative reticulocyte counts and higher alanine aminotransferase activity in males.

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:
26 March - 26 June 2013
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.
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 153 grams; females: 122 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- 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 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. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 26 March - 26 June 2013
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
From Day 1 to 30, formulations (w/w) were prepared daily within 5 hours prior to dosing. From Day 31 onwards, formulations (w/w) were prepared for a maximum of 8 days prior to dosing. Formulations were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

DOSE VOLUME:
10 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:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations in Weeks 1, 6 and 13). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and stability over 8 days in the refrigerator in the dark were also determined (highest and lowest concentration, in week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Remarks:
Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 28-day oral study with the test substance by daily gavage in the rat (information provided by the sponsor)
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: at pretest: all animals (including spare animals), at Week 13: Groups 1 and 4

ESTROUS CYCLE DETERMINATION
- Time schedule for examinations: Day 69 up to and including Day 90 of treatment.
- How many animals: All females, except for one female at 0 mg/kg (no vaginal lavage was possible, since the vagina was not open)

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines .

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: during Week 12/13 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied 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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
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
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
No mortality occurred that was considered to be related to treatment with the test substance. One animal at 100 mg/kg died at blood sampling at the end of treatment. This was considered not to be related to treatment.

CLINICAL SIGNS
Treatment at 100 mg/kg resulted in the following clinical signs:
- Rales in 5/10 males from Week 8 onwards and in 4/10 females from Week 4 onwards,
- Hunched posture in 1/10 males in Weeks 8 to 10, in 2/10 females in Weeks 7 to 10 and in all females from Week 13 onwards.
Lethargy, flat posture, shallow respiration and ptosis shown by one male at 100 mg/kg on a single day only in Week 4 of treatment was considered unrelated to the test substance, given the incidental occurrence and absence of similar findings among all other animals of this dose group.
Salivation seen after dosing among animals of the 100 mg/kg dose group from Week 3 of the treatment period onwards 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). This sign may be related to irritancy/taste of the test substance and/or may be related to the microscopic observation of prominent intercalated ducts in the mandibular salivary glands.
No other treatment related clinical signs were observed. Incidental findings that were noted included scabs (neck), chromodacryorrhoea, rales (females at 30 mg/kg) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
At 100 mg/kg, body weights and body weight gain were lower than controls in males and females achieving a level of statistical significance on most occasions. Body weight gain was lower from Week 2 onwards and in females from Week 4 onwards. Group mean body weight after 13 weeks of treatment was approximately 20 and 12% lower than control mean weight for males and females, respectively. At 10 and 30 mg/kg, body weights were similar to controls throughout treatment.

FOOD CONSUMPTION
At 100 mg/kg, a trend towards slightly lower food consumption was noted for males. When corrected for body weight, food intake values were similar to control levels. Food consumption of males and females at 100 mg/kg remained within normal levels for rats of this age and strain. At 10 and 30 mg/kg, food intake was similar to control levels.

OPHTHALMOSCOPIC EXAMINATION
There were no toxicologically relevant ophthalmology findings at pre-dose and in Week 13. Incidental ophthalmology findings consisted of (focal) corneal edema or opacity, pinpoint corneal opacities, and haemorrhage from hyaloid vessel or iris. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.

ESTROUS CYCLE DETERMINATION
At 100 mg/kg, two females showed an acyclic estrous cycle of 11 or 14 days. All other females at 100 mg/kg, and all females of the control group and the 10 and 30 mg/kg groups showed a normal (regular) estrous cycle of 4 to 5 days during the period in which estrous cycle length was determined (Day 76 up to and including Day 90).

HAEMATOLOGY
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
- Lower relative reticulocyte counts in males at 30 and 100 mg/kg and females at 100 mg/kg,
- Lower haemoglobin and haematocrit level in females at 100 mg/kg.
The slightly higher relative neutrophils and lower relative lymphocyte counts in females at 100 mg/kg and the lower white blood cell counts (WBC) in both sexes at 10 mg/kg remained within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females at 100 mg/kg and in males at 30 mg/kg,
- Lower albumin and total protein level in males and females at 100 mg/kg,
- Higher urea level in males at 100 mg/kg,
- Higher glucose level in females at 100 mg/kg,
- Lower potassium level in males at 100 mg/kg,
- Lower chloride and calcium level in females at 100 mg/kg,
- Higher inorganic phosphatase level in males and females at 100 mg/kg (not statistically significant in females).
The higher alanine aminotransferase activities and lower albumin and total protein levels were generally outside the normal range for rats of this age and strain, while other changes remained within this range.
The higher sodium level in males at 10, 30 and 100 mg/kg was considered to be of no toxicological significance as these changes occurred in the absence of a dose-related trend, and remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Males and females at 100 mg/kg showed a reduced motor activity (both total movements and ambulations; only statistically significant for females).
One animal at 30 mg/kg showed an abnormal pupillary reflex in the right eye. As this was only one animal, this finding was considered to be of no toxicological relevance. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all other examined animals. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Slightly increased spleen weight and spleen to body weight ratio in males and females at 100 mg/kg (not statistically significant for absolute weights),
- Slightly lower seminal vesicles weights in males at 100 mg/kg.
Other statistically significant changes in organ weights at 100 mg/kg were attributed to the lower terminal body weight and occurred without correlating morphological findings. These changes consisted of higher brain to body weight ratio in males and females, lower heart weight and higher heart to body weight ratio in males and/or females, lower liver weight (not statistically significant in females) and higher liver to body weight ratio in males and females lower kidney weight in males and females, lower prostate weight, lower adrenal to body weight ratio in males, , higher epididymides and testes to body weight ratio, lower ovary weight and higher uterus to body weight ratio , lower thyroid and thymus weight in males .

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, anddid not show a clear dose-related incidence trend.


HISTOPATHOLOGY
Treatment-related microscopic findings were present in:
Trachea:
- Loss of cilia/loss of mucous cells was recorded at 100 mg/kg (Group 4) in 4/10 males (2: minimal, 1: moderate, 1: marked) and in 4/10 females (1: minimal, 1: slight, 2: moderate) and in females at 30 mg/kg (Group 3) in 3/10 animals (minimal).
- Hypertrophy/hyperplasia of the epithelium was recorded at 100 mg/kg in 2/10 males at a minimal degree and in 3/10 females (2: minimal, 1: slight) and in females at 30 mg/kg in 3/10 animals (2: minimal, 1: slight).
Lung (males):
- Alveolar macrophage foci (in some animals with a foamy appearance) were recorded at an increased incidence and/or severity at 100 mg/kg (Group 4) in 9/10 males (4: minimal, 5: slight) and at 30 mg/kg (Group 3) in 1/10 males (moderate). A background level of this finding was recorded in 1/10 males and 2/10 females of the control Group 1 and in 4/10 females at 100 mg/kg (Group 4), all at a minimal degree.
Duodenum:
- Hypertrophy of the duodenal villi was recorded at 100 mg in 2/10 males (1: minimal, 1: slight) and in 2/10 females (minimal).
Seminal vesicles:
- Vacuolar degeneration of the epithelium was recorded at 100 mg/kg (Group 4) in 10/10 males (5: minimal, 5: slight) and at 30 mg/kg (Group 3) in 2/10 males (minimal).
Thyroid gland (females):
- Minimal vacuolation of the follicular epithelium was recorded in 5/10 females at 100 mg/kg (Group 4).
Adrenal glands:
- Vacuolation of the zona glomerulosa was recorded at 100 mg/kg in 2/10 males (1: minimal, 1: slight) and in 7/10 females (4: minimal, 3: slight).
Mesenteric lymph node:
- Macrophage foci were recorded at an increased incidence and severity at 100 mg/kg (Group 4). This was recorded in 10/10 males (7: slight, 3: moderate) and in 10/10 females (1: minimal, 6: slight, 3: moderate). The incidences and severities of this finding recorded in the remaining dose Groups were considered to be within background: Control Group 1: 4/10 males and 4/10 females (minimal), 10 mg/kg (Group 2): 3/10 males (1: minimal, 2: slight) and 4/10 females (minimal) and at 30 mg/kg (Group 3): 6/10 males (5: minimal, 1: slight) and 5/10 females (4: minimal, 1: slight).
Salivary glands (mandibular):
- Prominent intercalated ducts were recorded at 100 mg/kg in 5/10 males (3: minimal, 2: slight) and in 6/10 females (4: minimal, 2: slight).
Spleen:
- Congestion was recorded at an increased incidence and severity at 100 mg/kg (Group 4) in 8/10 males (4: minimal, 3: slight, 1: moderate) and in 8/10 females (3: minimal, 4: slight, 1: moderate). A background level of this finding was recorded in 1/10 males of control Group 1 and in 1/10 males and 1/10 females at 30 mg/kg (Group 3) ), all at a minimal degree.
Bone marrow (sternum):
- Increased adipocytes in the sternal bone marrow at a slightly higher incidence and/or severity was recorded at 100 mg/kg (Group 4) in 5/10 males (minimal) and 4/10 females (2: minimal, 2: slight) and at 30 mg/kg (Group 3) in 5/10 females (3: minimal, 2: slight). A background level of this finding was recorded in 2/10 females of the control Group 1, 1/10 males and 3/10 females at 10 mg/kg (Group 2) and 1/10 males at 30 mg/kg (Group 3), all at a minimal degree.
Harderian glands (females):
-An increased amount of brown (porphyrin) pigment was recorded in females at 100 mg/kg (Group 4). This was seen in 7/10 animals (5: minimal, 2: slight). A background level of this finding was recorded in 2/10 males of the control Group 1, 3/10 females at 10 mg/kg (Group 2), 3/10 females at 30 mg/kg (Group 3) and 4/10 males at 100 mg/kg (Group 4), all at a minimal degree.

The findings recorded in trachea, lung (males), seminal vesicles and mesenteric lymph nodes are considered to be adverse microscopic findings at the incidences and severities recorded in this study. For the findings in duodenum, thyroid gland (females), adrenal glands and salivary glands adversity can’t be excluded.
The findings recorded in the spleen, sternal bone marrow and Harderian glands (females) can also be present as normal background findings in rats of this age and strain. Therefore, these findings, at these incidences/severities and in absence of any other indicators of toxicity in these organs, were not considered to be adverse.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.
Spermatogenic staging profiles were normal for all males examined.
Dose descriptor:
NOAEL
Effect level:
<= 10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 in Weeks 1 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The concentrations analysed in the formulations of Group 2, 3 and 4 in Week 13 showed slightly higher values than the target concentrations (i.e. the mean accuracies for Group 2, 3 and 4 were 115, 110 and 117%, respectively). No explanation was found for this. The formulations of Group 2 and 4 in Weeks 1, 6 and 13 were homogeneous (i.e. coefficient of variation ≤10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours and were found to be stable in the refrigerator in the dark for at least 8 days. The long term storage samples were stable at ≤-70°C for at least 41 days.

Conclusions:
In a 90-day oral repeated dose toxicity study with rats, the NOAEL was determined to be 10 mg/kg bw/day.

Executive summary:

The repeated dose toxicity of substance N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free, was evaluated in a 90-day oral gavage study in the rat, according to OECD guideline 408.

Dose levels were selected by the sponsor at 0, 10, 30 and 100 mg/kg, based on results of a 28-day study with the test substance.

The test substance, formulated in water, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability over 5 hours at room temperature and 8 days in the refrigerator.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12/13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Formulation analyses confirmed that formulations of test substance in propylene glycol in Weeks 1 and 6 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). However, the concentrations analysed in the formulations in Week 13 showed slightly higher values than the target concentrations (i.e. the mean accuracies for Group 2, 3 and 4 were 115, 110 and 117%, respectively). No explanation was found for this. Based on overall results, the analyzed accuracy of the preparations was considered acceptable for the purpose of this study, Formulations were prepared homogenously and were stable over at least 5 hours at room temperature and 8 days in the refrigerator in the dark under nitrogen.

No treatment-related mortality occurred in the study. Treatment at 100 mg/kg resulted in rales and/or hunched posture mainly at the end of treatment. Motor activity was also slightly decreased in animals at 100 mg/kg. A slightly lower food consumption and body weight was noted for primarily males (group mean body weight after 13 weeks of treatment was approximately 20% lower than control mean body weight).

Histopathological findings at 100 mg/kg consisted of loss of cilia/loss of mucous cells in the trachea, hypertrophy/hyperplasia of the epithelium in the trachea, hypertrophy of the villi of the duodenum, vacuolation of the follicular epithelium of the thyroid gland (females only), vacuolation of the zona glomerulosa of the adrenal gland, vacuolar degeneration of the epithelium of the seminal vesicles, increased incidence and severity of macrophage foci in the lungs (males only) and mesenteric lymph node and prominent intercalated ducts in the mandibular salivary glands (may correlate with the salivation observed at 100 mg/kg).

Changes in blood parameters at 100 mg/kg consisted of lower relative reticulocyte counts in both sexes, lower haemoglobin and haematocrit level in females, higher alanine aminotransferase activity and inorganic phosphatase level and lower albumin and total protein level in both sexes, higher urea and lower potassium level in males, and higher glucose and lower chloride and calcium level in females.

Histopathological findings at 30 mg/kg consisted of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles,, increased severity of macrophage foci in the lungs (in one male only).

Changes in blood parameters at 30 mg/kg were confined to lower relative reticulocyte counts and higher alanine aminotransferase activity in males.

Other treatment related microscopic findings consisted of increased incidence and severity of congestion of the spleen at 100 mg/kg (correlating with increased spleen weights), increased incidence/severity of adipocytes in the sternal bone marrow at 30 (females) and 100 mg/kg and increased incidence and severity of brown (porphyrin) pigment Harderian glands in females at 100 mg/kg. These findings can also be present as normal background findings in rats of this age and strain. Therefore, at these incidences/severities and in absence of any other indicators of toxicity in these organs, these findings were not considered to be adverse.

Two out of ten females at 100 mg/kg showed an abnormal (acyclic) estrous cycle, which is above the incidence reported in historical control data of about 4% with group incidences varying between 0 and 10%. Histopathological examination of the female reproductive organs did not show treatment-related lesions. The observation of an acyclic period was seen in two animals of the high dose group showing apparent toxicity, and within this group the two affected animals also showed the greatest effect on body weight. An indirect effect following non-specific systemic toxicity or malnutrition is the most likely cause for the observed acyclic estrous period in these animals.

The effects of lower seminal vesicle weight seen at 100 mg (only absolute, not relative, so linked to lower BW) and vacuolar degeneration of the epithelium of the seminal vesicles at 30 and 100 mg/kg are not considered to have reproductive effects considering that spermatogenic staging profiles were normal in all dose groups.

Conclusion

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for N,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free sample of 10 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality guideline study (OECD 408) in compliance to GLP

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 studies are available for the evaluation of repeated dose toxicity ofN,N,N’,N’,N’-Pentamethyl-N-C16-18 (even numbered) C18 unsat.-alkyl-1,3-propanediammonium chloride (Pure), solvent free, in which rats were dose by oral gavage for 28-days and 90-days respectively. Both studies were performed according to current OECD guidelines and under GLP.

 

The 28 day study was performed with test substance doses of 10, 30 and 100 mg/kg bodyweight administered per gavage to male and female rats. Additional dose groups (0 and 100 mg/kg bw) were allowed an additional 14 day recovery period. There were no deaths or treatment-related changes in clinical observations, FOB parameters, oestrus status, hematology and urine analysis parameters. There were also no macroscopic changes noted. In the highest dose group there was a reduction in food consumption, bodyweight gain and elevated ALAT activity. There were also slight changes in thymus weight (absolute and relative to brain weight) and microscopic findings in mesenteric lymph node, stomach (attributed to local irritation), thymus and lung (attributed to accidental aspiration). A NOAEL of 30 mg/kg bw/day was established based on these findings.Overall, the 28-day study indicated low toxicological activity. Evidence of recovery was observed during the recovery period as the bodyweight gain was higher than in the control animals.

 

The repeated dose toxicity was further evaluated in a 90-day oral gavage study in the rat, according to OECD guideline 408. Dose levels of 0, 10, 30 and 100 mg/kg were based on results of the 28-day study with the test substance. The test substance, formulated in water, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Results: No treatment-related mortality occurred in the study. Treatment at 100 mg/kg resulted in rales and/or hunched posture mainly at the end of treatment. Motor activity was also slightly decreased in animals at 100 mg/kg. A slightly lower food consumption and body weight was noted for primarily males (group mean body weight after 13 weeks of treatment was approximately 20% lower than control mean body weight).

A NOAEL of 10 mg/kg bw/day was established, based on histopathological findings at 30 mg/kg, that consisted of loss of cilia/loss of mucous and hypertrophy/hyperplasia of the epithelium in the trachea (females only), vacuolar degeneration of the epithelium of the seminal vesicles, increased severity of macrophage foci in the lungs (in one male only). Changes in blood parameters at 30 mg/kg were confined to lower relative reticulocyte counts and higher alanine aminotransferase activity in males.

The effects of lower seminal vesicle weight seen at 100 mg (only absolute, not relative, so linked to lower BW) and vacuolar degeneration of the epithelium of the seminal vesicles at 30 and 100 mg/kg are not considered to have reproductive effects considering that spermatogenic staging profiles were normal in all dose groups.

A curious observation is that in the 28-day study at the highest dose level of 100 mg/kg (in 10 mL water/kg) local effects in the stomach were reported, consisting of limited, reversible effects in forestomach in 4/5 males and 1/5 females, which were attributed to local irritation, but that in the 90-day study applying the same dose levels and formulations no such effects were reported.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study of longest duration. Only available sub-chronic study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Exposure of humans to Diamine quaternised C16-18, C18 unsaturated, via inhalation is not likely taking into account the low vapour pressure of the substance as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Substance is severely corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Because of the corrosive properties, exposures are likely to be limited.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Substance is (severely) corrosive.

Repeated dose toxicity: via oral route - systemic effects (target organ) respiratory: trachea

Justification for classification or non-classification

Dermal corrosive properties dominate the overall toxicity profile of the substance. The effects observed in the 28 -day and 90 -day toxicity studies by oral route are not severe enough to qualify for classification. According to the criteria laid down in EU regulation (EC) n° 1272 /2008/EC (CLP) and in EU directive67/548/EEC, the substance does not warrant a classification as Specific Target Organ Toxicant.