Registration Dossier

Administrative data

Description of key information

- Subacute (14 day dose-range finding study) repeated dose toxicity study oral (gavage), rat Crl:WI(Han)) m/f (similar to OECD TG 407, GLP), dose levels: 0, 50, 200, 500 mg/kg bw/d; no NOAEL derived from this dose range finding study; read-across from Stearic acid 3-(dimethylaminopropyl)amide
- Subchronic (90 day) repeated dose toxicity study, dermal, rabbit (New Zealand White) m/f (similar to OECD TG 411 GLP), dose levels: 5, 200 mg/kg bw/d: NOAEL = 200 mg/kg bw/d; read-across from Stearic acid 3-(dimethylaminopropyl)amide

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:
other: 14 d dose range finding study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For justification for read-across see endpoint summary "Repeated dose toxicity".
Reason / purpose:
read-across source
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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals at 500 mg/kg bw/d were sacrificed for humane reasons between Days 6 and 8.
No mortality occurred at 50 and 200 mg/kg bw/d.

500 mg/kg bw/d:
- lethargy, hunched posture, labored respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards

200 mg/kg bw/d:
- all animals showed piloerection on two days of week 2 only

50 mg/kg bw/d:
- no clinical signs were noted

Salivation seen after dosing among all animals at 200 and 500 mg/kg bw/d on a few days of treatment 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).

BODY WEIGHT AND WEIGHT GAIN
500 mg/kg bw/d
- two females showed weight loss between days 1 and 4 (2 or 5% compared to day 1), followed by a slight weight gain between days 4 and 7. One female and all males at 500 mg/kg bw/d showed a reduced weight gain throughout the treatment period.

At 50 and 200 mg/kg, body weights and body weight gain remained in the same range as controls over the study period (the animals used for dosing at 50 and 200 mg/kg bw/d were from a different batch of delivery, and hence the starting body weight on day 1 was different to that for control animals and animals at 500 mg/kg bw/d).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
500 mg/kg bw/d:
- food consumption before or after correction for body weight was reduced in both genders over days 1-4 and 4-7

200 mg/kg bw/d:
- food consumption before or after correction for body weight was similar to controls over the study period

50 mg/kg bw/d:
- food consumption before or after correction for body weight was similar to controls over the study period

HAEMATOLOGY
The following changes in haematology parameters distinguished treated from control animals and from normal range levels encountered for rats of this age and strain:
- Slightly lower red blood cell counts in males at 50 and 200 mg/kg bw/d (no clear dose related trend).
- Higher reticulocyte counts in males at 50 and 200 mg/kg bw/d (no clear dose related trend).

CLINICAL CHEMISTRY
The following changes in clinical biochemistry parameters distinguished treated animals from control animals and from normal range levels encountered for rats of this age and strain:
-higher alanine aminotransferase activity (ALAT) in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d
-Higher alkaline phosphatase activity (ALP) in one female at 200 mg/kg bw/d
-Higher potassium level in males at 50 and 200 mg/kg bw/d


ORGAN WEIGHTS
Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group.

The apparent lower liver, spleen and heart weight of males at 200 mg/kg bw/d was ascribed to a slightly lower terminal body weight as the ratio to body weight for these organs was similar to control levels.

GROSS PATHOLOGY
500 mg/kg bw/d:
- reduced size of seminal vesicles, prostate, epididymides, spleen and/or thymus, gelatinous contents in the gastro-intestinal tract, small intestines and/or caecum, gastro-intestinal tract distended with gas, and emaciated appearance

200 mg/kg bw/d:
- no abnormalities

50 mg/kg bw/d:
- no abnormalities

HISTOPATHOLOGY: NON-NEOPLASTIC
500 mg/kg bw/d:
-Thymus: Lymphoid atrophy in 2/2 females (moderate).
-Stomach: Hyperplasia of the forestomach in 3/3 males and 3/3 females at slight or moderate degree, inflammation of the forestomach in 3/3 males and 3/3 females up to moderate degree, ulceration of the forestomach in 1/3 males and 1/3 females at slight degree.
-Duodenum: Hyperplasia of the villi in 2/3 males and 2/3 females up to slight degree.
-Jejunum: Hyperplasia of the villi in 1/3 females at minimal degree.
-Mesenterial lymph node: Foamy macrophages and sinusoidal dilation in 3/3 males and 3/3 females at slight or moderate degree, and congestion/erythrophagocytosis in 1/3 males and 2/3 females at minimal degree.
-Testes: Absence of spermiation (massive degree) and degeneration of spermatids in 3/3 males up to slight degree.
-Epididymides: Oligospermia in 2/2 males at massive degree and seminiferous cell debris in 2/2 males at slight degree.
-Prostate: Reduced contents in 2/2 males at slight degree.
-Seminal vesicles: Reduced contents in 2/2 males at moderate degree.

No treatment-related microscopic abnormalities were noted at 50 and 200 mg/kg bw/d.
Dose descriptor:
other: based on the results, doses were selected for OECD TG 421 study
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified

All animals at 500 mg/kg bw/d were sacrificed for humane reasons between Days 6 and 8, and showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from Day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included:

-     Lymphoid atrophy of the thymus in 2/2 females examined, correlating to a reduced size of the thymus at necropsy.

-     Hyperplasia and inflammation (3/3 males and 3/3 females) and ulceration (1/3 males and 1/3 females) of the forestomach.

-     Hyperplasia of the villi in the duodenum (2/3 males and 2/3 females) and jejunum (1/3 females).

-     Foamy macrophages and sinusoidal dilation (3/3 males and 3/3 females) and congestion/ erythrophagocytosis (1/3 males and 2/3 females) in the mesenterial lymph node.

-     Absence of spermiation and degeneration of spermatids in the testes in 3/3 males, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles in 2/2 males examined, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

 

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of Week 2 only, whilst no clinical signs were noted at 50 mg/kgbw/d. Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

 

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

 

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d.

Conclusions:
In a 14 d dose range finding subacute toxicity study, the analogue source test substance Stearic acid 3-(dimethylaminopropyl)amide was administered to 3 Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.
All animals of the highest dose group were sacrificed for humane reasons based on clinical signs. The main cause for moribundity was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach.
In the 50 and 200 mg/kg bw/d dose groups, no mortality occurred, and no or only minor clinical signs were observed. No treatment related effects were observed at necropsy or histopathology.
Executive summary:

In a 14 d dose range finding study according to OECD guideline 407, adopted 03 October 2008, and EU method B.7, May 2008, the analogue source test substance Stearic acid 3-(dimethylaminopropyl)amide was administered to 3Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.

All animals in the 500 mg/kg bw/d dose group were sacrificed for humane reasons between days 6 and 8. They showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included: lymphoid atrophy of the thymus, correlating to a reduced size of the thymus at necropsy; hyperplasia and inflammation of the forestomach; hyperplasia of the villi in the duodenum and jejunum; foamy macrophages and sinusoidal dilation and congestion/ erythrophagocytosis in the mesenterial lymph node; absence of spermiation and degeneration of spermatids in the testes, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

 

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of week 2 only, whilst no clinical signs were noted at 50 mg/kg bw/d. Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

 

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

 

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d.

 

Based on these results, dose leves were selected for the reproduction/developmental toxicity screening test according to OECD guideline.

It can be expected that the study results are also applicable to the target substance Amides, C16-18 (even numbered), N-[(dimethylamino)propyl].

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
other: rat; animals in the highest dose group hat to be killed due to humane reasons; no NOAEL derived from this dose range finding study

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

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For justification for read-across see endpoint summary "Repeated dose toxicity".
Reason / purpose:
read-across source
Observations and examinations performed and frequency:


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily.
- Cage side observations included: Signs of ill health and abnormalities.

DETAILED CLINICAL OBSERVATIONS: Not reported

DERMAL IRRITATION: Yes, immediately prior to treatment each day (after Day 1), the skin sites were assessed on a numerical basis according to Draize skin reaction scoring system. The scoring scale for skin reactions is provided in the study report.

BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, blood samples were obtained from the central ear artery and were collected into tubes containing appropriate amounts of anticoagulant (EDTA).
- Time schedule for collection of blood: 7 days prior to the start of the study and at termination.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Complete blood counts including a differential white blood cell count.

CLINICAL CHEMISTRY: No
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortality or test related toxicity was observed throughout the study excepting slight conjunctivitis in one animal of control and two animals of Group 2 (5 mg/kg/day) animals. This was attributed to the continuous restraint which prevented the animals from grooming themselves.
The collars of one control animal, one Group 2 (5 mg/kg/day) animal and one Group 3 (200 mg/kg/day) animal were removed temporarily because of sores on the necks. Some animals removed their collars overnight and they were collared again the next day.

BODY WEIGHT AND WEIGHT GAIN: No treatment related changes in body weight and body weight gain were observed during the study.

HAEMATOLOGY: Statistically significant increases in the WBC values were noted in the Group 3 (200 mg/kg/day) animals. In addition, there was an increase in platelet values from baseline to necropsy of the Group 2 (5 mg/kg/day) animals. The changes in WBC of the Group 3 animals were attributed to the chronic stress of collaring and not considered to be test substance related. The significant increase in platelet values of Group 2 animals were a result of low baseline values.

ORGAN WEIGHTS: No test related biologically significant changes were noted in the absolute and relative liver, kidney and adrenal weight determinations.

GROSS & HISTO PATHOLOGY (NON-NEOPLASTIC):
Treated skin on the back of rabbits in both the treated groups had a dry hair coat with an accumulation of test material on the surface at necropsy. Except the treatment site skin, no test related gross effects were observed. Histopathological examinations revealed minimal acanthosis and hyperkeratosis in the treatment sites skin of all treated groups. The incidence and severity were similar in both groups. Incidental non treatment related histopathological changes were noted in several other tissues such as brain, liver, kidney, prostate and pancreas. However, these were considered to be incidental, and not treatment related.

OTHER FINDINGS: Local Dermal Reactions:
Treatment of animals with 0.25% test material (5 mg/kg/day) in 30/70% ethanol/water produced moderate or slight erythema, slight edema, slight desquamation and slight fissuring. At 10% (200 mg/kg/day), moderate erythema, slight edema, slight desquamation and slight fissuring were produced.


Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
>= 200 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
LOAEL
Remarks:
(local effects)
Effect level:
5 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: dermal irritation
Critical effects observed:
not specified
Conclusions:
Dermal application of the analogue source test substance Stearamidopropyldimethyl amine (SAPDMA) to hair clipped skin of New Zealand White rabbits at concentrations of 0 (distilled water), 0.25 and 10% w/v of the test substance in 30%/70% ethanol/water (equivalent to 0, 5 and 200 mg/kg bw/day) for 13 weeks revealed a systemic NOAEL of ≥10% w/v (equivalent to 200 mg/kg bw/day).
Executive summary:

The subchronic toxicity study of the analogue source test substance Stearamidopropyldimethyl amine (SAPDMA) was performed following methods comparable to the OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study).

Male and female New Zealand White rabbits, obtained from Hazleton Research Animals were used in the study. The body weight range of animals at study initiation was 1970 – 2593 g. Animals were acclimated to laboratory conditions for a period of 7 days prior to test initiation.

Test solutions were prepared fresh weekly in distilled, 30%/70% ethanol/water for each group. Post acclimation, animals were randomized into treatment/control groups (each consisting of 5 animals/sex).

The test solution was applied to intact skin of rabbits once daily, 5 days/week for thirteen consecutive weeks at the following dose levels:

Group 1(Control): Distilled water

Group 2: 0.25% w/v in 30%/70% ethanol/water (equivalent to 5 mg/kg/day)

Group 3: 10% w/v in 30%/70% ethanol/water (equivalent to 200 mg/kg/day) 

All animals (test and control) were dosed at a constant dosage volume of 2 mL/kg/day

Appropriate concentration of test material was applied evenly on the back of each animal, from shoulder to rump (approximately 15 cm wide) using a syringe.

Animals were observed for gross toxicity daily. The skin at the treatment sites was graded prior to each day’s treatment. Body weights were taken prior to initiation and then weekly until termination. Blood was taken prior to initiation and at termination for hematological examinations. After treatment, animals were sacrificed by an IV overdose of sodium pentobarbital. Adrenals, kidneys and liver were dissected free of fat and weighed. Tissue samples (including treated skin samples) were collected for histological examinations.

No mortality or test related gross toxicity was observed throughout the study except slight conjunctivitis in one control animal and two Group 2 (5 mg/kg/day) animals. This was attributed to the continuous restraint which prevented the animals from grooming themselves. The collars of one control animal, one Group 2 (5 mg/kg/day) animal and one Group 3 (200 mg/kg/day) animal were removed temporarily because of sores on the necks. Some animals removed their collars overnight and they were collared again the next day.

Treatment of animals with 0.25% test material (5 mg/kg/day) in 30/70% ethanol/water produced moderate or slight erythema, slight edema, slight desquamation and slight fissuring. At 10% (200 mg/kg/day), moderate erythema, slight edema, slight desquamation and slight fissuring were produced. No treatment related changes in body weight and body weight gain were observed during the study. No test related biologically significant changes were noted in the absolute and relative liver, kidney and adrenal weight determinations.

Statistically significant increases in the WBC values were noted in the Group 3 (200 mg/kg/day) animals. In addition, there was an increase in platelet values from baseline to necropsy of the Group 2 (5 mg/kg/day) animals. The changes in WBC of the Group 3 animals was attributed to the chronic stress of collaring and not considered to be test substance related. The significant increase in platelet values of Group 2 animals were a result of low baseline values.

Treated skin on the backs of rabbits in both treated groups had a dry hair coat with an accumulation of test material on the surface at necropsy. Histopathological examinations revealed minimal acanthosis and hyperkeratosis at the treatment sites of all treated groups. The incidence and severity were similar in both groups. Incidental non treatment related histopathological changes were noted in several other tissues such as brain, liver, kidney, prostate and pancreas. However, these were considered to be incidental, and not treatment related.

Based on above it can be concluded that, no systemic toxicity associated with the sub chronic percutaneous exposure to test material was observed at any dose levels.

Dermal application of Stearamidopropyldimethyl amine (SAPDMA) to hair clipped skin of New Zealand White rabbits at concentrations of 0 (distilled water), 0.25 and 10% w/v of the test substance in 30%/70% ethanol/water (equivalent to 0, 5 and 200 mg/kg bw/day)for 13 weeks revealed a systemic NOAEL of ≥10% w/v (equivalent to 200 mg/kg bw/day).

It can be expected that the study results are also applicable to the target substance Amides, C16-18 (even numbered), N-[(dimethylamino)propyl].

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
other: rabbit; highest dose tested

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For justification for read-across see endpoint summary "Repeated dose toxicity".
Reason / purpose:
read-across source
Observations and examinations performed and frequency:


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily.
- Cage side observations included: Signs of ill health and abnormalities.

DETAILED CLINICAL OBSERVATIONS: Not reported

DERMAL IRRITATION: Yes, immediately prior to treatment each day (after Day 1), the skin sites were assessed on a numerical basis according to Draize skin reaction scoring system. The scoring scale for skin reactions is provided in the study report.

BODY WEIGHT: Yes
- Time schedule for examinations: All rabbits were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, blood samples were obtained from the central ear artery and were collected into tubes containing appropriate amounts of anticoagulant (EDTA).
- Time schedule for collection of blood: 7 days prior to the start of the study and at termination.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Complete blood counts including a differential white blood cell count.

CLINICAL CHEMISTRY: No
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortality or test related toxicity was observed throughout the study excepting slight conjunctivitis in one animal of control and two animals of Group 2 (5 mg/kg/day) animals. This was attributed to the continuous restraint which prevented the animals from grooming themselves.
The collars of one control animal, one Group 2 (5 mg/kg/day) animal and one Group 3 (200 mg/kg/day) animal were removed temporarily because of sores on the necks. Some animals removed their collars overnight and they were collared again the next day.

BODY WEIGHT AND WEIGHT GAIN: No treatment related changes in body weight and body weight gain were observed during the study.

HAEMATOLOGY: Statistically significant increases in the WBC values were noted in the Group 3 (200 mg/kg/day) animals. In addition, there was an increase in platelet values from baseline to necropsy of the Group 2 (5 mg/kg/day) animals. The changes in WBC of the Group 3 animals were attributed to the chronic stress of collaring and not considered to be test substance related. The significant increase in platelet values of Group 2 animals were a result of low baseline values.

ORGAN WEIGHTS: No test related biologically significant changes were noted in the absolute and relative liver, kidney and adrenal weight determinations.

GROSS & HISTO PATHOLOGY (NON-NEOPLASTIC):
Treated skin on the back of rabbits in both the treated groups had a dry hair coat with an accumulation of test material on the surface at necropsy. Except the treatment site skin, no test related gross effects were observed. Histopathological examinations revealed minimal acanthosis and hyperkeratosis in the treatment sites skin of all treated groups. The incidence and severity were similar in both groups. Incidental non treatment related histopathological changes were noted in several other tissues such as brain, liver, kidney, prostate and pancreas. However, these were considered to be incidental, and not treatment related.

OTHER FINDINGS: Local Dermal Reactions:
Treatment of animals with 0.25% test material (5 mg/kg/day) in 30/70% ethanol/water produced moderate or slight erythema, slight edema, slight desquamation and slight fissuring. At 10% (200 mg/kg/day), moderate erythema, slight edema, slight desquamation and slight fissuring were produced.


Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
>= 200 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
LOAEL
Remarks:
(local effects)
Effect level:
5 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: dermal irritation
Critical effects observed:
not specified
Conclusions:
Dermal application of the analogue source test substance Stearamidopropyldimethyl amine (SAPDMA) to hair clipped skin of New Zealand White rabbits at concentrations of 0 (distilled water), 0.25 and 10% w/v of the test substance in 30%/70% ethanol/water (equivalent to 0, 5 and 200 mg/kg bw/day) for 13 weeks revealed a systemic NOAEL of ≥10% w/v (equivalent to 200 mg/kg bw/day).
Executive summary:

The subchronic toxicity study of the analogue source test substance Stearamidopropyldimethyl amine (SAPDMA) was performed following methods comparable to the OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study).

Male and female New Zealand White rabbits, obtained from Hazleton Research Animals were used in the study. The body weight range of animals at study initiation was 1970 – 2593 g. Animals were acclimated to laboratory conditions for a period of 7 days prior to test initiation.

Test solutions were prepared fresh weekly in distilled, 30%/70% ethanol/water for each group. Post acclimation, animals were randomized into treatment/control groups (each consisting of 5 animals/sex).

The test solution was applied to intact skin of rabbits once daily, 5 days/week for thirteen consecutive weeks at the following dose levels:

Group 1(Control): Distilled water

Group 2: 0.25% w/v in 30%/70% ethanol/water (equivalent to 5 mg/kg/day)

Group 3: 10% w/v in 30%/70% ethanol/water (equivalent to 200 mg/kg/day) 

All animals (test and control) were dosed at a constant dosage volume of 2 mL/kg/day

Appropriate concentration of test material was applied evenly on the back of each animal, from shoulder to rump (approximately 15 cm wide) using a syringe.

Animals were observed for gross toxicity daily. The skin at the treatment sites was graded prior to each day’s treatment. Body weights were taken prior to initiation and then weekly until termination. Blood was taken prior to initiation and at termination for hematological examinations. After treatment, animals were sacrificed by an IV overdose of sodium pentobarbital. Adrenals, kidneys and liver were dissected free of fat and weighed. Tissue samples (including treated skin samples) were collected for histological examinations.

No mortality or test related gross toxicity was observed throughout the study except slight conjunctivitis in one control animal and two Group 2 (5 mg/kg/day) animals. This was attributed to the continuous restraint which prevented the animals from grooming themselves. The collars of one control animal, one Group 2 (5 mg/kg/day) animal and one Group 3 (200 mg/kg/day) animal were removed temporarily because of sores on the necks. Some animals removed their collars overnight and they were collared again the next day.

Treatment of animals with 0.25% test material (5 mg/kg/day) in 30/70% ethanol/water produced moderate or slight erythema, slight edema, slight desquamation and slight fissuring. At 10% (200 mg/kg/day), moderate erythema, slight edema, slight desquamation and slight fissuring were produced. No treatment related changes in body weight and body weight gain were observed during the study. No test related biologically significant changes were noted in the absolute and relative liver, kidney and adrenal weight determinations.

Statistically significant increases in the WBC values were noted in the Group 3 (200 mg/kg/day) animals. In addition, there was an increase in platelet values from baseline to necropsy of the Group 2 (5 mg/kg/day) animals. The changes in WBC of the Group 3 animals was attributed to the chronic stress of collaring and not considered to be test substance related. The significant increase in platelet values of Group 2 animals were a result of low baseline values.

Treated skin on the backs of rabbits in both treated groups had a dry hair coat with an accumulation of test material on the surface at necropsy. Histopathological examinations revealed minimal acanthosis and hyperkeratosis at the treatment sites of all treated groups. The incidence and severity were similar in both groups. Incidental non treatment related histopathological changes were noted in several other tissues such as brain, liver, kidney, prostate and pancreas. However, these were considered to be incidental, and not treatment related.

Based on above it can be concluded that, no systemic toxicity associated with the sub chronic percutaneous exposure to test material was observed at any dose levels.

Dermal application of Stearamidopropyldimethyl amine (SAPDMA) to hair clipped skin of New Zealand White rabbits at concentrations of 0 (distilled water), 0.25 and 10% w/v of the test substance in 30%/70% ethanol/water (equivalent to 0, 5 and 200 mg/kg bw/day)for 13 weeks revealed a systemic NOAEL of ≥10% w/v (equivalent to 200 mg/kg bw/day).

It can be expected that the study results are also applicable to the target substance Amides, C16-18 (even numbered), N-[(dimethylamino)propyl].

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
5
Study duration:
subchronic
Species:
rabbit

Additional information

No repeated dose toxicity studies are available for C16-18 DMAPA amidoamine. However, an oral subacute repeated dose toxicity study (14 day dose-range finding study) as well as a dermal subchronic (90 day) repeated dose toxicity study with the structurally closely related source substance Stearic acid 3-(dimethylaminopropyl)amide are available. A justification for read-across is given below.

In a 14 d dose range finding study according to OECD guideline 407, adopted 03 October 2008, and EU method B.7, May 2008, Stearic acid 3-(dimethylaminopropyl)amide was administered to 3 Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.

All animals in the 500 mg/kg bw/d dose group were sacrificed for humane reasons between days 6 and 8. They showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included: lymphoid atrophy of the thymus, correlating to a reduced size of the thymus at necropsy; hyperplasia and inflammation of the forestomach; hyperplasia of the villi in the duodenum and jejunum; foamy macrophages and sinusoidal dilation and congestion/ erythrophagocytosis in the mesenterial lymph node; absence of spermiation and degeneration of spermatids in the testes, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of week 2 only, whilst no clinical signs were noted at 50 mg/kg bw/d. Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d. The results from this study were used to select the doses for the reproduction/developmental screening study.No reliable NOAEL could be derived from this dose-range-finding study. Due to the small number of animals, the relevance of the clinical biochemistry changes could not be fully justified.

 

A repeated dose 91-day dermal toxicity study comparable to OECD guideline 411 (reliable with restrictions), is available for Stearic acid 3-(dimethylaminopropyl)amide. The test substance was dermally administered to 5 New Zealand White rabbits/sex/group at dose levels of 0, 5 and 200 mg/kg bw/d for 4 h daily followed by rinsing, 5 times weekly. The animals were collared throughout the study to prevent oral ingestion of the test substance.

No test item related gross toxicity was observed throughout the study except slight conjunctivitis in 2/10 animals in the 5 mg/kg bw/d dose group. Animals of the 5 mg/kg bw/d dose group showed moderate or slight erythema, slight edema, slight desquamation and slight fissuring.

At 200 mg/kg bw/d moderate erythema, slight edema, slight desquamation and slight fissuring were noted. Body weight was not influenced by the treatment.

Statistically significant increases in white blood cell counts were noted in animals treated with 200 mg/kg bw/d. However, this was attributed to the chronic stress of collaring and not considered to be test item related.

An increase in platelet values at necropsy compared to baseline values was observed in animals treated with 5 mg/kg bw/d, which was considered to be the result of low baseline values.

No test item related changes in organ weights were observed. Histopathologic examination showed no test item related findings except from acanthosis and hyperkeratosis of the skin at the treatment sites.

In conclusion, Stearic acid 3-(dimethylaminopropyl)amide did not show systemic toxicity up to and including 200 mg/kg bw/d (highest dose administered). The dermal 90 d NOAEL in rabbit was 200 mg/kg bw/d in this study.

This dermal toxicity study in the rabbit is acceptable and is considered to be equivalent to the corresponding OECD guideline although minor deviations are reported: Only 2 dose levels were tested while the guideline recommends at least 3 doses; only 5 animals/sex/dose were used instead of 10 as recommended in the later implemented guideline; no vehicle control (30/70 EtOH/water) was tested, and no clinical chemistry was performed. These deviations are reflected by setting the reliability to 2.

This study used the appropriate route (dermal) and duration (subchronic) compared to the expected most relevant route of exposure in humans (dermal). Thus, it is considered to be relevant for hazard assessment.

 

There are no data gaps for the endpoint repeated dose toxicity. No human data are available. However, there is no reason to believe that these results from rat and rabbits would not be applicable to humans.

 

 

Justification for read-across

1. Read-across hypothesis and justification

This read-across is based on the hypothesis that source and target substances have similar toxicological properties because

·        they are manufactured from similar resp. identical precursors under similar conditions

·        the metabolism pathway leads to comparable products (amine backbone and long chain fatty acids) and non-common products predicted to have no toxicological effects (long chain fatty acids).

 

Therefore, read-across from the existing repeated dose toxicity studies on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VIII 8.6.1 and Annex VIX 8.6.2 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

The justification of the proposed read-across approach is elaborated in the next chapters.

 

 

2. Justification for read-across

2.1 Substance Identity

 

Table 1: Substance identities

 

Source substance

Target substance

Stearic acid 3-(dimethylaminopropyl)amide

C16-18 DMAPA amidoamine

mono constituent substance

UVCBsubstance

CAS number

7651-02-7

Chain length distribution

< C16: < 1.6%

C16: < 7%

C18: > 89.8%

> C18: < 1.6%

C14: <= 5 %

C16: 25-35 %

C18: >= 61 %

DMAPA

<0.002%

<=0.01%

 

2.1 Substance Identity

Substance descriptions

The target substanceC16-18 DMAPA amidoamine is a UVCB substance manufactured from saturated C16-18 fatty acids and N, N-dimethylpropylenediamine (DMAPA). It is composed of C16 and C18 amides of DMAPA, with C18 being the larger part (>/= 61%)

 

The source substanceStearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid andN, N-dimethylpropylenediamine. It is composed of mainly C18 amides (> 89.8%) of DMAPA and small amounts of the C16 amide (<7%).

 

2.2 Common breakdown products

The source substance Stearic acid 3-(dimethylaminopropyl)amide is the main component of the UVCB target substanceC16-18 DMAPA amidoamine. The only difference is the chain length distribution: the target substance also contains a significant amount of the C16 amide.

This is not considered to be of relevance for metabolism.Both substances are amides which after resorption may be hydrolysed by amidases resulting in free fatty acids and DMAPA. The carboxylic acids then are further degraded by the mitochondrial beta-oxidation process (for details see common text books on biochemistry). The fatty acids enter normal metabolic pathways and are therefore indistinguishable from fatty acids from other sources including diet. The amine compounds are not expected to be further metabolised, but excreted via the urine mainly unchanged. 

 

2.3 Differences

The slight differences in fatty acid chain length (higher percentage of C16 in the target substance vs. corresponding higher percentage C18 in the source substance) are not considered to be of relevance for systemic toxicity.

 

3. Physicochemical properties:

Table 2: Physicochemical properties

Endpoints

Source substanceStearic acid 3-(dimethylaminopropyl)amide

Target substanceC16-18 DMAPA amidoamine

Molecular weight

368.64 g/mol

340.59 - 368.64

Physical state at 20°C / 1013 hPa

Solid (paste)

Solid (waxy)

Melting point

OECD TG 102, RL1, non-GLP

67.4°C

OECD TG 102, RL1, non-GLP

41.8°C

Boiling point

OECD TG 103, RL1, non-GLP

412.3°C

OECD TG 103, RL1, non-GLP

320.5°C

Surface tension

ISO 4311, plate method, RL1, non-GLP

37.86 mN/m at 0.22 g/L

OECD TG 115, ring method, RL1, non-GLP

26.7 mN/m at 2.7 mg/L

Water solubility

OECD TG 105, RL1, non-GLP

10 mg/L at 20°C

OECD guideline 105/EU method A.6, slow stirring method/HPLC, RL1, non-GLP

3.65 mg/L at 23°C

Log Kow

---

Calculation (ACD/Labs Release 12.00, Product version 12.01 )

>6.6

EU method A.8, calculation based on solubility in n-Octanol and water; RL2, non-GLP

2.01 at 20°C, pH7

Read-across from Stearic acid 3-(dimethylaminopropyl)amide

Vapour pressure

OECD TG 104, RL1, ISO17025 compliance

3.4E-08 Pa at 20°C

Read-across from Stearic acid 3-(dimethylaminopropyl)amide

 

4. Toxicokinetics

Based on the very close structural relationship no relevant differences in toxicokinetics are expected.The source substance Stearic acid 3-(dimethylaminopropyl)amide is the main component of the UVCB target substanceC16-18 DMAPA amidoamine. The only difference is the chain length distribution: the target substance also contains a significant amount of the C16 amide.

Experimental toxicokinetic studies are not available for either the source substanceStearic acid 3-(dimethylaminopropyl)amide or the target substanceC16-18 DMAPA amidoamine. Based on physicochemical data and molecular weight,an oral absorption rate of 100% is assumed as a worst case default value in the absence of other data.

In a study according to OECD guideline 414 with dermal application of the source substance Stearic acid 3-(dimethylaminopropyl)amide systemic effects (lower body weight and lower food consumption) were noted. Thus, in the absence of detailed dermal penetration data it has to be assumed that dermal penetration may occur, and a dermal absorption rate of 100% is assumed as a worst case default value based on the physicochemical properties and on experimental toxicological data.

Based on the structure, both substances are likely to undergo hydrolysis by amidases, which in general have a broad substrate specificity. Hydrolysis of Stearic acid 3-(dimethylaminopropyl)amide would result in Stearic acid and 3-Aminopropyldimethylamine. Hydrolysis of the target substance C16-18 DMAPA amidoamine would additionally produce Palmitic acid.Stearic acid as well as palmitic acid are likely to enter the normal fatty acid metabolism and may be broken down to carbon dioxide or two carbon fragments, or be re-esterified to triacylglycerols and either metabolised for energy or stored in adipose tissue.

In general, lower primary aliphatic amines are metabolised to the corresponding carboxylic acid and urea. The tertiary site would be expected to undergo oxidation mediated bycytochrome P-450 or mixed function amine oxidases.

5. Comparison of data from human health endpoints

5.1 Toxicity data of the target and source substances

Table 3: General toxicological profiles forStearic acid 3-(dimethylaminopropyl)amide andC16-18 DMAPA amidoamine

Endpoints

Source substance

Stearic acid 3-(dimethylaminopropyl)amide

Target substanceC16-18 DMAPA amidoamine

Acute toxicity oral

OECD TG 423, RL1,GLP

 

LD50(rat) > 2000 mg/kg bw

OECD TG 423, RL1,GLP

 

LD50(rat) > 2000 mg/kg bw

Eye irritation

OECD TG 405, RL1, GLP

 

Category 1 (irreversible effects on the eye)

No data; read-across

OECD TG 437, RL1, GLP

 

not severely irritating /not corrosive

No data; read-across

Skin irritation

OECD TG 439, RL1, GLP

 

not irritating

OECD TG 439, RL1, GLP

 

not irritating

OECD TG 404, RL1, GLP

 

not irritating

No data; read-across

Sensitisation

 

OECD TG 406 (GPMT), RL1, GLP

 

not sensitising

No data; read-across

Genotoxicity

OECD TG 471 (Ames test), RL1, GLP

 

Negative

OECD TG 471, RL1, GLP

 

Negative

OECD TG 467 (MLY), RL1, GLP

 

Negative

No data; read-across

OECD TG 473 (Chromosome aberrations), RL1, GLP

 

Negative

No data; read-across

Repeated dose toxicity oral

Similar to OECD TG 407 (14 d DRF), rat, RL1, GLP

 

clinical signs/mortality (all animals at 500 mg/kg bw/d were sacrificed for humane reasons); haematology (slightly lower red blood cell and higher reticulocyte counts in males at 50 and 200 mg/kg bw/d); clinical biochemistry (higher ALAT activity in 2 males at 50 mg/kg bw/d, 2 males and 1 female at 200 mg/kg bw/d, higher alkaline phosphatase activity in 1 female at 200 mg/kg bw/d, higher potassium level in males at 50 and 200 mg/kg bw/d)

No data; read-across

Repeated dose toxicity dermal

Similar to OECD TG 411, rabbit, RL2, GLP

 

NOAEL(systemic) = 200 mg/kg bw/d (highest dose administred)

No data; read-across

Reproduction / Developmental Toxicity Screening Test

OECD TG 421, rat, RL1, GLP

 

NOAEL(parental)= 70 mg/kg bw/d;

NOAEL(fertility females)= 70 mg/kg bw/d;

NOAEL (fertility males) = 200 mg/kg bw/d;

NOAEL(development)= 200 mg/kg bw/d

No data; read-across

Prenatal developmental toxicity

Similar to OECD TG 414, rabbit, RL2, GLP

 

NOAEL(development)=200 mg/kg bw/d (highest dose administered)

No data; read-across

 

Experimental data for the target substance C16-18 DMAPA amidoamine are available for acute oral toxicity, skin irritation (in vitro) and genotoxicity (bacterial reverse mutation assay).

No experimental data are available for the target substance C16-18 DMAPA amidoamine concerning the endpoints eye irritation, sensitisation, repeated dose toxicity, and reproductive/developmental toxicity. However, as demonstrated above, the source substanceStearic acid 3-(dimethylaminopropyl)amide is the main constituent of the target substance. The additional minor constituents with differing fatty acid chain lengths are not considered to influence the outcome of the toxicological studies.

This is supported by the similar results observed in acute oral toxicity studies: Both, the target substance C16-18 DMAPA amidoamine and the source substance Stearic acid 3-(dimethylaminopropyl)amide are of low acute toxicity when administered orally. The LD50 for both substances was > 2000 mg/kg bw. In both studies, 2/6 animals died during the observation period.

Both, the target substance C16-18 DMAPA amidoamine and the source substance Stearic acid 3-(dimethylaminopropyl)amide were not irritating to skin in an in vitro skin irritation test.

Both,the target substance C16-18 DMAPA amidoamine and the source substance Stearic acid 3-(dimethylaminopropyl)amide were not mutagenic in the bacterial reverse mutation assay when tested up to cytotoxic concentrations.

 

5.2 Quality of the experimental data of the analogues:

The source substance Stearic acid 3-(dimethylaminopropyl)amide has been tested in reliable (RL1 and 2, respectively) GLP-compliant studies similar to OECD Guidelines 407 and 411. The subacute toxicity study was performed as dose range finding study using only 3 animals/sex/dose and including the evaluation of male reproductive parameters.

The dermal toxicity study in rabbit is acceptable and is considered to be equivalent to the corresponding OECD guideline although minor deviations are reported: Only 2 dose levels were tested while the guideline recommends at least 3 doses; only 5 animals/sex/dose were used instead of 10 as recommended in the later implemented guideline; no vehicle control (30/70 EtOH/water) was tested, and no clinical chemistry was performed.

These deviations are reflected by setting the reliability to 2.

Despite the deviations the available data are sufficiently reliable and can be used in an analogue approach.

 

5.4 Classification and labelling

Concerning human health hazards, the source substance Stearic acid 3-(dimethylaminopropyl)amide is classified for irreversible effects on the eye (Eye Damage, Category 1, H318: Causes serious eye damage. / Xi; R41 Risk of serious damage to eyes). Based on the read-across, the target substance C16-18 DMAPA amidoamine will be classified accordingly.

 

6. Conclusion

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles.

The dose descriptor obtained from the existing subacute and subchronic repeated dose toxicity studies performed with the source substance Stearic acid 3-(dimethylaminopropyl)amide is considered as an appropriate starting point for deriving a DNEL for the target substance C16-18 DMAPA amidoamine. As the remaining uncertainty associated with this read-across approach is considered to be low based on the close relationship of source and target substance, no additional assessment factors are required.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
dose range finding study similar to OECD guideline, GLP

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
similar to OECD guideline study, GLP

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
similar to OECD guideline study, GLP

Justification for classification or non-classification

Based on the available data - an oral 14 day dose range finding study as well as a dermal 90 day repeated dose toxicity study, both conducted with the closely related read-across substance Stearic acid 3-(dimethylaminopropyl)amide -, the target substance C16 -18 DMAPA amidoamine does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC with respect to repeated dose toxicity.