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Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-20 to 2012-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 11 wks
- Fasting period before study: no
- Housing:
Pre-mating: in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).

- Diet (e.g. ad libitum): pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations
- Amount of vehicle (if gavage): 5 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: max 14 d; females who had not shown evidence of mating were separated from their males
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
One female was mated with a proven male of the same dose group since the male that she was intended to be mated with was sacrificed before mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
Analysis with LC-MS/MS (lower limit of quantitation = 0.996 mg/g; calibration curve ranged from 1.00 to 25.0 mg/L)
The concentrations analysed in the formulations were in agreement with the target concentrations (mean accuracies between 85% and 115%).
Duration of treatment / exposure:
Males: 28 days (2 weeks prior to mating, during mating, up to the day prior to scheduled necropsy)
Females: 41 - 54 days (2 weeks prior to mating, during mating, during post-coitum, during at least 4 days of lactation (up to the day prior to scheduled necropsy)); one female of the control group was not dosed during littering
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated (screening study)
- Parturition: females were allowed to litter normally; day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Remarks:
Doses / Concentrations:
0, 20, 70, 200 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 14 day dose-range finding study with dose levels of 50, 200 and 500 mg/kg bw/d; considering the significant toxicity at 500 mg/kg bw/d, it was considered that dose levels for a subsequent study of longer duration should not exceed 200 mg/kg bw/d
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean
Positive control:
no
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily; mortality/viability: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed on the first day of exposure and weekly thereafter.
Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. High dose group males were weighed daily from 02-05 October 2012 (Days 11-14 of the premating period) in order to correct the actual dose volume for lower body weights recorded for these animals on a daily basis. Body weights determined daily between the regular body weight determinations (i.e. on Days 1, 8, 15, 22 and 28) are not reported since these were intended for calculation of the actual dose volumes only.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

OTHER:
General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, other: staging of spermatogenesis, histopathology of testes and epididymides in control + high dose group, histopathology of reproductive organs (coagulation gland, epididymides, prostate gland, seminal vesicles, testes) of males suspected to be infertile
Litter observations:
STANDARDISATION OF LITTERS
no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain
Mortality / Viability: numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, Following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals, females which delivered: lactation days 5-7; female no. 74 which failed to deliver (no evidence of mating): 21 days after the last day of the mating period
Euthanized in extremis : When pain, distress or discomfort was considered not transient in nature or was likely to become more severe

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed
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 (Ref. 2; 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 (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Ref. 4) was applied to frequency data.

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. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Mating index (Number of females mated/Number of females paired x 100),
Fertility index (Number of pregnant females/Number of females paired x 100),
Conception index (Number of pregnant females/Number of females mated x 100),
Gestation index (Number of females bearing live pups/Number of pregnant females x 100)
Offspring viability indices:
Percentage live males at First Litter Check (Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100),
Percentage of postnatal loss Days 0-4 of lactation (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100),
Viability index (Number of live pups on Day 4 post partum/Number of pups born alive x100)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
obserevd effects were due to gavage trauma, not substance-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No test substance-related mortality occurred during the study period.
Two males at 200 mg/kg bw/d (nos. 31 and 37) and one female at 70 mg/kg bw/d(no. 66) were euthanized in extremis on Days 11 (nos. 37 and 66) or Day 20 (no. 31): macroscopic and microscopic examinations suggested that gavage trauma was the cause of morbidity for these animals; the deaths were not substance-related

- no toxicologically relevant clinical signs were noted
- 200 mg/kg bw/d: hunched posture was noted among all males primarily during the second week of treatment, and at lower incidence, rales, piloerection and lean appearance were noted among some males. These findings had resolved for most animals as treatment progressed.
- clinical signs noted for the animals euthanized in extremis (nos. 31, 37 and 66) included (but were not limited to) hunched posture, rales, gasping, abdominal swelling, piloerection, lethargy and laboured respiration and chromodacryorrhoea, and were considered to be due to gavage trauma.
- salivation noted at 70 and 200 mg/kg bw/d 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 the taste of the test substance. No toxicological relevance was ascribed to these changes.

Incidental findings: included rales, alopecia and scabs; the incidence remained within the range of background findings to be expected for rats of this age and strain housed and treated under the conditions in this study; these were not considered to be toxicologically relevant


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
200 mg/kg bw/d
- males showed weight loss up to 15% of day 1 weight during the first 2 weeks of treatment, which largely recovered during the treatment period
- mean body weight and body weight gain remained statistically significantly lower throughout treatment, but body weight gain exceeded that of controls during the mating period
- females showed minor (statistically significant) reduced body weight gain during the first two weeks of treatment
- at start of post-coitum, mean body weight was similar to control levels, but body weight (gain) was lower during the post coitum phase
- absolute and relative food consumption was reduced for males during the premating period, and for females during the first week of the premating period
- for males, food consumption had recovered to control levels during the mating period, while for females food consumption remained slightly lower throughout the post-coitum and lactation period

70 mg/kg bw/d
- slightly lower (but statistically significant) body weight gain was noted for females during the last week of the post coitum phase
- lower absolute and relative food consumption for females at 70 mg/kg bw/d throughout the post-coitum and lactation period (statistically significant on most occasions)


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- spermatogenic staging profiles were normal for males examined
- testes and epididymides weights were unaffected by treatment


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- significantly lower number of implantation sites at 200 mg/kg bw/d
- attributable to low numbers for female nos. 79 and 80; upon exclusion of the values for these two females, the mean was similar to that of the 70 mg/kg bw/d group

- mating, fertility and conception indices, precoital time, and number of corpora lutea were unaffected by treatment
- in one female the number of pups born was slightly higher than the number of implantations and corpora lutea recorded; this was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 7 of lactation

- No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy)

- significantly lower mean number of living pups at first litter check at 70 and 200 mg/kg bw/d
- at 200 mg/kg bw/d, 4/9 females had litter sizes of 3-9 pups (the lowest litter size was found for the two females with a low number of implantation sites)
- at 70 mg/kg bw/d, 5/9 females had litter sizes of 7-9 pups
- in the control group, 1/10 female had a litter size of 8 pups, while all other females had litter sizes of 12-15
- the historical control mean values for litter size in this lab is 11.8 (st.dev.= 2.47), n=588 litters (min=2, max=18), 95% confidence interval: 7-15.


ORGAN WEIGHTS (PARENTAL ANIMALS)
- Testes and epididymides weights were unaffected by treatment.
- lower terminal body weights for males at 200 mg/kg bw/d were in line with the lower in-life body weight noted for these animals


GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were attributable to treatment with the test substance.

Perforation of the esophagus was noted for two animals (male no. 37 (200 mg/kg bw/d) and female no. 66 (20 mg/kg bw/d)) that were euthanised in extremis. This was considered direct evidence that these deaths were considered due to gavage trauma. For the other male at 200 mg/kg bw/d euthanised in extremis (no. 31) macroscopic findings were not directly indicative of gavage trauma, but based on histopathological assessment this death was also ascribed to a gavage-related incident. Other macroscopic findings noted for these deaths included emaciated appearance, gastro-intestinal tract distended with gas, red foci on the lungs, irregular surface of the forestomach, reddish discoloration of the mesenteric lymph node or the stomach glandular mucosa, reduced size of the spleen and/or thymus.

The incidence of other necropsy findings noted for control and/or treated animals remained within the background range of findings encountered among rats of this age and strain, and did not show a dose-related trend. They were not considered to be toxicologically relevant.


HISTOPATHOLOGY (PARENTAL ANIMALS)
- no test item related microscopic findings
- no findings in the reproductive organs for animals that failed to sire or deliver healthy offspring that were outside the range of normal background pathology
- spermatogenic staging profiles were normal for males examined
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
70 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: body weight; food consumption
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
70 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: lower number of implantation sites
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: overall effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
One pup in the control, 70 and 200 mg/kg bw/d groups went missing during lactation. These pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. No pups died or went missing at 20 mg/kg bw/d.

CLINICAL SIGNS (OFFSPRING)
Scabs on the left foreleg were noted for two pups at 200 mg/kg bw/d. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 200 mg/kg bw/d.

GROSS PATHOLOGY (OFFSPRING)
Scabs on the left foreleg were noted for two pups at 200 mg/kg bw/d were incidental in nature. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
other: overall effects
Reproductive effects observed:
not specified

Reproduction data

The number of implantation sites was statistically significantly lower at 200 mg/kg bw/d. This was attributable to low numbers for female nos. 79 and 80. Upon exclusion of the values for these two females, the mean was similar to that of the 70 mg/kg group bw/d.

The mating, fertility and conception indices, precoital time, and number of corpora lutea were unaffected by treatment.

For female no 42, the number of pups born was slightly higher than the number of implantations and corpora lutea recorded. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 7 of lactation.

 

Developmental data

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

The gestation index and duration of gestation were unaffected by treatment up to 200 mg/kg bw/d.

No signs of difficult or prolonged parturition were noted among the pregnant females.

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

The number of dead pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

The mean number of living pups at first litter check was significantly lower than controls at both 70 and 200 mg/kg bw/d. At 200 mg/kg bw/d, four of the nine females had litter sizes of 3-9 pups (the lowest litter size was found for the two females with a low number of implantation sites) and five of nine females at 70 mg/kg bw/d also had litter sizes of 7-9 pups. In contrast, only one female in the control group had a litter size of 8 pups, while all other females had litter sizes of 12-15. The historical control mean values for litter size in this lab is 11.8 (st.dev.= 2.47), n=588 litters (min=2, max=18), 95% confidence interval: 7-15.

 

Mortality

One pup in the control, 70 and 200 mg/kg bw/d groups went missing during lactation. These pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. No pups died or went missing at 20 mg/kg bw/d.

Clinical signs

Scabs on the left foreleg were noted for two pups at 200 mg/kg bw/d.The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.

 

Body weights

Body weights of pups were unaffected by treatment up to 200 mg/kg bw/d.

 

Macroscopy

Scabs on the left foreleg were noted for two pups at 200 mg/kg bw/d were incidental in nature.The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.

 

 

Body weights of parental animals

At 200 mg/kg bw/d, most males showed weight loss (up to 15% of Day 1 values) during the first two weeks of treatment. Body weights largely recovered as treatment progressed. Although mean body weight and body weight gain remained statistically significantly lower throughout treatment, body weight gain exceeded that of controls during the mating period (by comparison of mean body weight increase over Days 1-15 of the mating period).

For females at 200 mg/kg bw/d, statistically significant reduced body weight gain was noted during the first two weeks of treatment. At start of post-coitum, mean body weight was similar to control levels, but body weight (gain) was lower during the post coitum phase.

 

At 70 mg/kg bw/d, a slightly lower (but statistically significant) body weight gain was noted for females during the last week of the post coitum phase.

   

Conclusions:
Based on the results of this Reproduction/Developmental Toxicity Screening Test, the following NOAELs were derived for Stearic acid 3-(dimethylaminopropyl)amide:
parental NOAEL: 70 mg/kg bw/d
fertility NOAEL, females: 70 mg/kg bw/d
fertility NOAEL, males: 200 mg/kg bw/d
developmental NOAEL: 200 mg/kg bw/d
Executive summary:

In a Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421 (July 1995) Stearic acid 3-(dimethylaminopropyl)amide (100% a.i.) was administered to groups of 10 Wistar rats/sex/dose inby gavageat dose levels of 0, 20, 70 and 200 mg/kg bw/d. 

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 – 54 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

At 200 mg/kg bw/d, males showed weight loss up to 15% of day 1 weight during the first 2 weeks of treatment, which largely recovered during the treatment period. The mean body weight and body weight gain remained statistically significantly lower throughout treatment. Females of the same dose group showed statistically significant reduced body weight gain during the first two weeks of treatment, as well as during pregnancy. Food intake was reduced for males during the premating period, and for females during the first week of the premating period; for females food intake remained slightly lower throughout pregnancy and lactation.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. macroscopic examination, organ weights, and microscopic examination).

 

The mean number of corpora lutea was slightly lower in the 70 and 200 mg/kg bw/d dose groups compared with the control animals, however, this was not statistically significant.

A statistically significant lower number of implantation sites were noted for females at 200 mg/kg bw/d. This wasattributable to extremely low numbers of implantation sites in two females (3 and 6, respectively); upon exclusion of the values for these two females, the mean number of implantation sites was similar to that of the 70 mg/kg bw/d group, which showed also a slight, but not statistically significant reduction of implantations when compared to control animals.

A statistically significant lower number of living pups was noted in the 70 and 200 mg/kg bw/d dose groups. However, as the lower litter size correlated with lower number of implantation sites also when regarding single animals, this was considered to be a consequence of the reduced number of implantation sites.

No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices and precoital time, testes and epididymides weights, spermatogenic staging profiles).

 

Due to the remarkable effects on body weight /body weight gain and food consumption, the observed fertility effects – reduced number of implantation sites and subsequently lower litter size – are considered to be a consequence of general parental toxicity.

 

Based on these results, the following NOAELs were derived:

parental NOAEL: 70 mg/kg bw/d

fertility NOAEL, females: 70 mg/kg bw/d

fertility NOAEL, males: 200 mg/kg bw/d

developmental NOAEL: 200 mg/kg bw/d

Endpoint:
toxicity to reproduction
Remarks:
other: 90 d repeated dose toxicity study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From March 14, 1984 to June 13, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: comparable to guidelines with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Only 2 dose levels tested while guideline recommends at least 3 doses; 5 animals/sex/group against standard 10 animals/sex/group; vehicle control (30/70 EtOH/water) not tested; clinical chemistry not performed. Test material was applied only for 4 h
Principles of method if other than guideline:
RSS for same study has been compiled in more detail under 7.5.1 Repeated dose toxicity: dermal
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: 1970 – 2593 g
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
No information on environmental conditions is provided by the study report. However, standard operating procedure of the test facility was followed for environmental conditions.

IN-LIFE DATES: From: March 14, 1984 To: June 13, 1984
Route of administration:
dermal
Vehicle:
other: 30/70 Ethanol/water
Details on exposure:
TEST SITE
- Area of exposure: Area on the back of each animals, from shoulder to rump, approximately 15 cm wide (intact skin)
- % coverage: Not reported
- Time intervals for shavings or clippings: The animals were clipped as needed while the test is in progress.

METHOD OF APPLICATIONS: Appropriate concentration of test material or control substance was applied evenly over the clipped area using a syringe.

REMOVAL OF TEST SUBSTANCE
- Washing: The treated skin was washed with tepid water and gently blotted dry with disposable paper towels or equivalent.
- Time after start of exposure: 4 hours

TEST MATERIAL:
- Amount(s) applied: 2.0 mL/kg/day (each animal received a constant dosage volume based upon its most recent bodyweight).
- Concentration: 0 (distilled water), 0.25 and 10% w/v of the test substance in 30%/70% ethanol/water.
- Constant concentration used: Yes
- Rate of preparation of dosing solution: Dosing solutions were prepared fresh weekly.
- Storage temperature of dosing solution: Stock solutions were stored at ambient temperature and humidity upon receipt and during the entire length of the study. Test solutions after preparation were stored in refrigerator. Each day’s dosing solution was equilibrated to room temperature prior to dosing.

VEHICLE: 30%/70% ethanol/water

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, collars were used to restrain animals from oral ingestion.
Details on mating procedure:
animals were not mated
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily (5 days/week)
Details on study schedule:
animals were not mated
Remarks:
Doses / Concentrations:
5, 200 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
other: yes, distilled water
Parental animals: Observations and examinations:
Mortality/Morbidity: Yes
- Time schedule: Once daily

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
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
reprodutive organs histopathologic examinations conducted on prostate, seminal vesicles, testis, epididymis
Litter observations:
n/a
Postmortem examinations (parental animals):
SACRIFICE: Yes, all the surviving animals were sacrificed with an IV overdose of sodium pentobarbital.

GROSS PATHOLOGY: Yes, complete necropsy was performed on all animals and macroscopic appearance of the tissues was recorded.

HISTOPATHOLOGY: Yes, tissues collected at necropsy were processed for microscopic evaluations.
-Samples of the following tissues from all rabbits collected for histological examination: Lung, heart, aorta, tongue, esophagus, thyroid (parathyroid), submandibular lymph node, ileocecocolic lymph node, anterior mesenteric lymph node, stomach, liver, gallbladder, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, kidneys, reproductive tracts, adrenal glands, thymus, spleen, pancreas, bone marrow, skin, brain, spinal cord, sciatic nerve, submandibular salivary gland, pituitary, eye. For paired tissues (except kidney and lungs), the left side was taken for processing and the right side was placed into a save jar for possible later histology.
All tissues were fixed in 10% neutral buffered formalin with the exception of the following tissues which were fixed in a glutaraldehyde/formalin fixative:
Anterior mesenteric lymph nodes, ileocecocolic lymph node, submandibular lymph node, spleen, adrenals, duodenum, jejunum, ileum, cecum, colon, rectum.
Postmortem examinations (offspring):
n/a
Statistics:
Initial body weights, body weight changes, hematology, and organ/body weight ratios were statistically analyzed by R D Bruce and B Stinson of Statistical resources using Procter & Gamble’s computer program B8944.
Reproductive indices:
n/a
Offspring viability indices:
n/a
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
dermal irritation
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
- no effects on reproductive organs
For other details see endpoint study record "Repeated dose toxicity (dermal)_30365_C7651-02-7_EC231-609-1 _QCE13Mar13"
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
200 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
n/a
Reproductive effects observed:
not specified
Conclusions:
Dermal application of Stearamidopropyldimethyl amine 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). No effects on reproductive organs were noted in this study.
Executive summary:

In a 90 day repeated dose toxicity study similar to OECD guideline 411 Stearic acid 3-(dimethylaminopropyl)amide 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.

No test item related findings concerning the reproductive organs (reprodutive organs examinations conducted for males on prostate, seminal vesicles, testis, epididymis; for females on ovary, uterus, vagina) and no other signs of systemic toxicity were noted. The dermal 90 d NOAEL in rabbit was 200 mg/kg bw/d in this study.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
other: rat / NOAEL(female fertility) = 70 mg/kg bw/d, NOAEL(male fertility) = 200 mg/kg bw/d
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
other: rabbit; highest dose tested
Additional information

For the assessment of effects of C16-18 DMAPA amidoamine to fertility results from the following studies are taken into consideration:

- an oral 14 day dose-range finding study in rats similar to OECD guideline 407

- a dermal 90 day study in rabbit

- an oral reproduction / developmental toxicity screening test in rats according to OECD Guideline 421

All available studies were conducted with the read-across substanceread-across from Stearic acid 3-(dimethylaminopropyl)amide. A justification for read-across is given below.

 

The repeated dose toxicity studies are described in full detail in section “Repeated dose toxicity”; in this section only the aspects relevant for fertility are discussed.

 

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 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. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach.

The 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 of these animals was attributed to considerable general toxicity. At the lower dose levels no fertility-related effects (or other adverse effects) were noted.

 

In the 90 day study Stearic acid 3-(dimethylaminopropyl)amide 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.

No test item related findings concerning the reproductive organs (reproductive organs examinations conducted for males on prostate, seminal vesicles, testis, epididymis; for females on ovary, uterus, vagina) and no other signs of systemic toxicity were noted. The dermal 90 d NOAEL in rabbit was 200 mg/kg bw/d in this study.

In a Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421 (July 1995) Stearic acid 3-(dimethylaminopropyl)amide (100% a.i.) was administered to groups of 10 Wistar rats/sex/doseby gavageat dose levels of 0, 20, 70 and 200 mg/kg bw/d. 

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 – 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

At 200 mg/kg bw/d, males showed weight loss up to 15% of day 1 weight during the first 2 weeks of treatment, which largely recovered during the treatment period. The mean body weight and body weight gain remained statistically significantly lower throughout treatment. Females of the same dose group showed statistically significant reduced body weight gain during the first two weeks of treatment, as well as during pregnancy. Food intake was reduced for males during the premating period, and for females during the first week of the premating period; for females food intake remained slightly lower throughout pregnancy and lactation.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. macroscopic examination, organ weights, and microscopic examination).

The mean number of corpora lutea was slightly lower in the 70 and 200 mg/kg bw/d dose groups compared with the control animals, however, this was not statistically significant.

A statistically significant lower number of implantation sites were noted for females at 200 mg/kg bw/d. This was attributable to extremely low numbers of implantation sites in two females (3 and 6, respectively); upon exclusion of the values for these two females, the mean number of implantation sites was similar to that of the 70 mg/kg bw/d group, which showed also a slight, but not statistically significant reduction of implantations when compared to control animals.

A statistically significant lower number of living pups was noted in the 70 and 200 mg/kg bw/d dose groups. However, as the lower litter size correlated with lower number of implantation sites also when regarding single animals, this was considered to be a consequence of the reduced number of implantation sites.

No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices and precoital time, testes and epididymides weights, spermatogenic staging profiles).

Due to the remarkable effects on body weight /body weight gain and food consumption, the observed fertility effects – reduced number of implantation sites and subsequently lower litter size – are considered to be a consequence of general parental toxicity.

Based on these results, the following NOAELs were derived from this study:

parental NOAEL: 70 mg/kg

fertility NOAEL, females: 70 mg/kg

fertility NOAEL, males: 200 mg/kg

developmental NOAEL: 200 mg/kg

 

Discussion of effects concerning fertility

Male fertility (dose range finding study)

The effects on spermiation noted in the 14 day dose range finding study were noted only in the high dose group animals which had to be sacrificed between day 6 and 8 due to severe signs of general toxicity. No such effects were noted in the animals in the 200 mg/kg bw/d dose group. Thus, the effects on spermiation are considered to be secondary to general toxicity.

This is also supported the fact that in the OECD guideline 421 study spermatogenic staging was normal in males treated with 200 mg/kg bw/d for 28 days.

 

Reduced number of implantation sites (reproduction / developmental toxicity screening test)

In the reproduction / developmental toxicity screening test a reduction in the mean number of implantation sites was noted in females of the 200 mg/kg bw/d dose group (mean number of implantation sites 9.8). However, this reduction was attributed to only 2/9 animals which had very low numbers of implantation sites. When considering these two animals as outliers and not taking them into account for the calculations, the new calculated mean would be 11.3 (compared to 13.5, 13.2 and 11.4 in the control, 20 and 70 mg/kg bw/d groups).

 

The mean number of corpora lutea was also slightly lower in the 70 and 200 mg/kg bw/d dose groups compared with the control animals, however, this was not statistically significant. The two females of the high dose group with extremely low implantation sites had also the lowest numbers of corpora lutea (9 compared to 11-20 in the other 7 animals of the high dose group).

 

In this study the following signs of general toxicity were noted:

In females of the 200 mg/kg bw/d dose group, a statistically significant reduction in body weight gain was noted during the first two weeks of treatment, as well as during pregnancy. Also in males effects on body weight were noted in the high dose group: most males showed weight loss (up to 15% of day 1 values) during the first two weeks of treatment. Although body weights largely recovered as treatment progressed, this effect was considered to be of toxicological relevance.

The absolute and relative food consumption was reduced for males at 200 mg/kg bw/d during the premating period (31 g/kg bw/d vs. 61 g/kg bw/d for the control animals in week 1, 46 g/kg bw/d vs. 63 g/kg bw/d for the control animals in week 2), and for females at during the first week of the premating period (48 g/kg bw/d vs. 68 g/kg bw/d for the control animals). The relative food consumption of females of the 70 and 200 mg/kg bw/d dose groups were significantly lower throughout pregnancy.

 

This is also supported by the findings in the14 day dose range finding study, where all animals treated with 500 mg/kg bw/d were sacrificed for humane reasons between days 6 and 8. Animals showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. This shows, that there is a steep dose-response-curve for the tested substance and that the dose of 200 mg/kg bw/day is most probably the highest tolerable dose with already some signs of general toxicity.

Based on the remarkable effects on body weight /body weight gain and food consumption in the reproduction / developmental toxicity screening test as well the findings of the 14 day dose range finding study, the observed fertility effects – reduced number of implantation sites and subsequently lower litter size – may also be explained as a consequence of general parental toxicity.

 

Reduced litter size (reproduction / developmental toxicity screening test)

A statistically significant lower number of living pups was noted in the 70 and 200 mg/kg bw/d dose groups (mean numbers of living pups 12.9, 12.5, 10.0* and 8.8** at 0, 20, 70 and 200 mg/kg bw/d, respectively; * p<0.05, ** p<0.01).

As the lower litter size of the 200 mg/kg bw/d dose group was predominantly the consequence of the lower number of implantation sites, the two dams with the extremely low implantation sites were not taken into account for calculation, resulting in mean number of living pups of 10.0 for the 200 mg/kg bw/d dose group. This was still lower than the number of living pups of the control animal, but within the range of historical control data of the laboratory for animals of that strain. It has also to be taken into account that the mean litter size of the control group in this experiment was rather high in comparison to historical control values of the performing laboratory (mean = 11.8, SD = 2.47, n = 588 litters, min=2, max=18, 95% confidence interval: 7-15).

 

Nature of the effect

A reduced litter size may be a developmental effect. However, in this study the lower mean litter size is accompanied by a lower number of implantation sites. This can also be observed at the level of individual animals as demonstrated in the table below.

In the mid dose group the animals with relatively low litter size (7 to 9) had in general also lower implantation sites (9 to 11). In the high dose group the animals which delivered a very low number of living pups (6 or 3) had the same number of implantation sites.

 

Group

Animal number

Corpora lutea

Implantations

Living pups

% prenatal loss

 

Control

41

13

12

12

7.7

42

13

13

15(*)

-15.4

43

12

12

12

0.0

44

13

13

13

0.0

45

15

14

13

13.3

46

15

15

15

0.0

47

17

14

13

23.5

48

16

16

15

6.3

49

14

14

13

7.1

50

13

12

8

38.5

20 mg/kg bw/d

51

16

14

12

25.0

52

14

13

13

7.1

53

12

11

11

8.3

54

16

16

14

12.5

55

13

10

9

30.8

56

16

13

12

25.0

57

16

16

16

0.0

58

16

12

12

25.0

59

14

14

14

0.0

60

13

13

12

7.7

70 mg/kg bw/d

61

12

12

9

25.0

62

15

14

13

13.3

63

15

9

9

40.0

64

12

9

7

41.7

65

13

11

8

38.5

67

9

9

9

0.0

68

13

13

12

7.7

69

15

15

13

13.3

70

13

11

10

23.1

200 mg/kg bw/d

71

11

11

11

0.0

72

15

13

12

20.0

73

20

10

8

60.0

75

14

13

10

28.6

76

12

11

10

16.7

77

12

11

9

25.0

78

13

10

10

23.1

79

9

6

6

33.3

80

9

3

3

66.7

(*) in this female the number of pups born was slightly higher than the number of implantations and corpora lutea recorded. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 7 of lactation.

 

Based on these data, the lower litter size is considered to be secondary to the reduction of implantation sites. Therefore, the reduced litter size is judged to be not a developmental effect.

Thus, in this study the NO(A)EL fertility was 70 mg/kg bw/d (based on reduced implantation sites at 200 mg/kg bw/d) and the NO(A)EL development was 200 mg/kg bw/d.

 

Also from the available data on DMAPA no concern on reproductive toxicity was recognised. A reproduction/developmental toxicity screening test cited by OECD SIDS and NTP resulted in a NOAEL of 200 mg/kg bw/d (highest dose administered) for reproductive toxicity (NTP, OECD SIDS).

 

There are no data gaps for effects on fertility. No human data are available. However, there is no reason to believe that these results from rat 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 and reproductive toxicity studies on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex VIII 8.6.1, 8.7.1 and Annex IX 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 substance C16-18 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 substance Stearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid and N, 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 substance C16-18 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 substance C16-18 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 substance Stearic acid 3-(dimethylaminopropyl)amide or the target substance C16-18 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 by cytochrome 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 substance Stearic 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.

OECD SIDS concluded that there is also no concern of DMAPA having any adverse effects to reproduction based on an OECD TG 421 study (OECD SIDS).

The read-across is supported by the similar results observed in the available 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.

 

A GLP compliant reproduction / developmental toxicity screening test according to OECD Guideline 421 (RL1) is available for thesource substance Stearic acid 3-(dimethylaminopropyl)amide.

 

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 as well as thereproduction / developmental toxicity screening testperformed 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.

 

References

NTP (National Toxicology Program),3-Dimethylaminopropylamine109-55-7; available online: http://ntp-server.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/Dimethylaminopropylamine_508.pdf

 

OECD SIDS,3-AMINOPROPYLDIMETHYLAMINE CAS N°: 109-55-7, available online: http://www.inchem.org/documents/sids/sids/109557.pdf


Short description of key information:
- Subacute (14 day dose-range finding study, with additional paramaters addressing male fertility) 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; NOAEL(fertility) = 200 mg/kg bw/d; 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(fertility) = 200 mg/kg bw/d; read-across from Stearic acid 3-(dimethylaminopropyl)amide

- Reproduction / developmental toxicity screening test, oral (gavage), rat (Crl:WI(Han)) m/f (OECD TG 421; GLP), dose levels: 0, 20, 70, 200 mg/kg bw/d: NOAEL(development) = 200 mg/kg; NOAEL(parental toxicity) = 70 mg/kg bw/d (reduced body weight gain/food consumption at 200 mg/kg bw/d); NOAEL(male fertility) = 200 mg/kg bw/d; NOAEL(female fertility) = 70 mg/kg bw/d (reduced number of implantation sites at 200 mg/kg bw/d); read-across from Stearic acid 3-(dimethylaminopropyl)amide

Justification for selection of Effect on fertility via oral route:
OECD guideline study, no deviations, GLP

Justification for selection of Effect on fertility via dermal route:
similar to OECD guideline study, GLP

Effects on developmental toxicity

Description of key information
- Prenatal developmental toxicity study, dermal, rabbit (New Zealand White) m/f (similar to OECD TG 414; GLP); dosing: day 7-18 of presumed gestation; dose levels: 0, 5, 100 and 200 mg/kg bw/day; NOAEL(development) = 200 mg/kg bw/d; read-across from Stearic acid 3-(dimethylaminopropyl)amide 
- Reproduction / developmental toxicity screening test, oral (gavage), rat (Crl:WI(Han)) m/f (OECD TG 421; GLP), dose levels: 0, 20, 70, 200 mg/kg bw/d: NOAEL(development) = 200 mg/kg; NOAEL(parental toxicity) = 70 mg/kg bw/d (reduced body weight gain/food consumption at 200 mg/kg bw/d); NOAEL(male fertility) = 200 mg/kg bw/d; NOAEL(female fertility) = 70 mg/kg bw/d (reduced number of implantation sites at 200 mg/kg bw/d); read-across from Stearic acid 3-(dimethylaminopropyl)amide
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
other: rat; highest dose tested
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
other: rabbit; highest dose tested
Additional information

For the assessment of effects of C16-18 DMAPA amidoamineto development results from the following studies are taken into consideration:

- a dermal prenatal developmental toxicity study in rabbit conducted with Stearic acid 3-(dimethylaminopropyl)amide

- an oral reproduction / developmental toxicity screening test in rats according to OECD Guideline 421 conducted with Stearic acid 3-(dimethylaminopropyl)amide

 

All available studies were conducted with the read-across substanceread-across from Stearic acid 3-(dimethylaminopropyl)amide. A justification for read-across is given below.

 

In a Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421 (July 1995) Stearic acid 3-(dimethylaminopropyl)amide (100% a.i.) was administered to groups of 10 Wistar rats/sex/doseby gavage at dose levels of 0, 20, 70 and 200 mg/kg bw/d. 

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 – 54 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

At 200 mg/kg bw/d, males showed weight loss up to 15% of day 1 weight during the first 2 weeks of treatment, which largely recovered during the treatment period. The mean body weight and body weight gain remained statistically significantly lower throughout treatment. Females of the same dose group showed statistically significant reduced body weight gain during the first two weeks of treatment, as well as during pregnancy. Food intake was reduced for males during the premating period, and for females during the first week of the premating period; for females food intake remained slightly lower throughout pregnancy and lactation.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. macroscopic examination, organ weights, and microscopic examination).

The mean number of corpora lutea was slightly lower in the 70 and 200 mg/kg bw/d dose groups compared with the control animals, however, this was not statistically significant.

A statistically significant lower number of implantation sites were noted for females at 200 mg/kg bw/d. This wasattributable to extremely low numbers of implantation sites in two females (3 and 6, respectively); upon exclusion of the values for these two females, the mean number of implantation sites was similar to that of the 70 mg/kg bw/d group, which showed also a slight, but not statistically significant reduction of implantations when compared to control animals.

A statistically significant lower number of living pups was noted in the 70 and 200 mg/kg bw/d dose groups. However, as the lower litter size correlated with lower number of implantation sites also when regarding single animals, this was considered to be a consequence of the reduced number of implantation sites.

No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices and precoital time, testes and epididymides weights, spermatogenic staging profiles).

Due to the remarkable effects on body weight /body weight gain and food consumption, the observed fertility effects – reduced number of implantation sites and subsequently lower litter size – are considered to be a consequence of general parental toxicity.

Based on these results, the following NOAELs were derived from this study:

parental NOAEL: 70 mg/kg (based on lower body weight and food consumption)

fertility NOAEL, females: 70 mg/kg (based on lower number of implantation sites at 200 mg/kg bw/d)

fertility NOAEL, males: 200 mg/kg (highest dose tested)

developmental NOAEL: 200 mg/kg (highest dose tested)

 

In a prenatal developmental toxicity study Stearic acid 3-(dimethylaminopropyl)amide in 30% isopropanol and 70% water (vehicle)wasadministered topically at dose levels of 0, 5, 100 and 200 mg/kg bw/day (dose volume 2 mL/kg bw) to 20 artificially inseminated rabbits/group for 12 consecutive days (during Days 7 through 18 of presumed gestation) for 2 h/day. Animals were collared during treatment to prevent oral ingestion of the test substance.

On Day 29 of presumed gestation all rabbits were sacrificed, and a complete gross necropsy was performed, including examination of the brain. The abdomen of each rabbit was opened, and the uterus was examined for pregnancy, number of implantations, live and dead fetuses and early and late resorptions. Corpora lutea were counted. Each fetus was weighed and subsequently examined for gross external variations and gender, prior to examination for soft tissue and skeletal variations.

No does died during the course of the study. Clinical signs attributed to administration of the test material included alopecia (5, 100, 200 mg/kg bw/d), excess lacrimation (100 and 200 mg/kg bw/d), ungroomed coat and greencolored matted fur around mouth and rump (200 mg/kg bw/d). Other signs (anorexia, soft or liquid feces, red exudates in cage pan) were not attributed to the test material;they were either were single events which were not dosage dependant or occurred in the absence of administration of the test material (red exudates in cage pan).

All skin reactions demonstrated dosage dependant onset, incidence and severity with recovery evident during the dosage and post dosage periods. Atonia, desquamation and fissuring was observed in mid and high dose groups. One high dose group rabbit had eschar present, attributed to the treatment.

Two low dosage group rabbits aborted on Day 21 of gestation. As the incidence of abortion was not dosage dependant, these events were not attributed to administration of the test material. One high dosage group doe delivered its litter on day 28 of gestation. As premature delivery frequently occurs in one rabbit in a population of 80 rabbits, this event was not attributed to administration of the test material.

As compared to control, body weight gain was significantly less for the high dose group rabbits. High dose group rabbits showed a decrease in average body weight during treatment, and continued to have lower average body weights than control rabbits during the post dosage period, this was, however, not statistically significant.

Maternal feed consumption was affected at mid and high dose levels, as compared with the control. The effect was slight for the mid dose group rabbits and moderate for the high dose group rabbits. As compared with control values, average daily feed consumption of the high dose group rabbits was generally significantly decreased from day 15 through day 21 of gestation. Thereafter, food consumption was increased over control.

All control rabbits were pregnant, 17 (85.0%) rabbits in each of the low and middle dosage groups were pregnant, and 16 (84.2%) of the high dosage group rabbits were pregnant. Two low dosage group rabbits aborted and one high dosage group rabbit delivered prior to scheduled Caesarean section. As a result, Caesarean delivery observations were based on 20 control, 15 low, 17 middle, and 15 high dosage group rabbits.

In the high dose group the average number of implantation sites per pregnant doe was slightly lower for rabbits in this dosage group, as compare to the control group. The effect was not statistically significant (p>0.05). As the result of the slightly lower implantation value, the average litter size for the high dosage group was also slightly decreased, as compared with the control values. All of the values were within expected historical control values.

Administration of these dosages of the test material did not adversely affect pregnancy incidence or average numbers or resorptions. Viable fetus(es) were present in 20, 14, 17, and 14 litters from control, low, middle, and high dosage groups, respectively.1 rabbit each from low and high dose group had all implantations resorbed. Total of 138, 105, 94, and 80 live Day 29 fetuses which were Caesarean delivered from 20, 14, 17, and 14 control, low, middle, and high dosage group litters, respectively, were examined for gross external, soft tissue and skeletal variations. Statistical analyses were based on these litters and specimens. No fetal variations at gross external, soft tissue or skeletal examination were attributed to the treatment with test substance. Incidence of variations observed either were not dosage dependant or were not significantly different among the four dosage groups.

In conclusion, percutaneous application of Stearamidpropyl dimethylamine to New Zealand White rabbits from Days 7 through 18 of presumed gestation at dose levels of 0, 5, 100 and 200 mg/kg bw/d did not produce evidence for developmental toxicity.

The NOAEL for developmental toxicity was determined to be 200 mg/kg bw/day (highest dose administered).

 

The observed reversible effects on maternal food consumption and body weight gain at dose levels of 100 and 200 mg/kg bw/d, which were statistically significant at 200 mg/kg bw/d, may well have been caused by general indisposition due to remarkable skin irritation effects. Based on the fact that the effect was very slight and not statistically significant at 100 mg/kg bw/d, in contrast to the study authors, we have considered 5 mg/kg bw/d to be the NOEL, and 100 mg/kg bw/d to be the NOAEL for maternal toxicity in this study.

 

Discussion of effects concerning development

Reduced litter size (reproduction / developmental toxicity screening test)

It has already been discussed, that the lower litter size observed in the 70 and 200 mg/kg bw/d dose groups is within the range of historical controls if the two animals in the high dose group which had a very low number of pups are not taken into account for calculation.

 

Group

Animal number

Corpora lutea

Implantations

Living pups

% prenatal loss

 

Control

41

13

12

12

7.7

42

13

13

15(*)

-15.4

43

12

12

12

0.0

44

13

13

13

0.0

45

15

14

13

13.3

46

15

15

15

0.0

47

17

14

13

23.5

48

16

16

15

6.3

49

14

14

13

7.1

50

13

12

8

38.5

20 mg/kg bw/d

51

16

14

12

25.0

52

14

13

13

7.1

53

12

11

11

8.3

54

16

16

14

12.5

55

13

10

9

30.8

56

16

13

12

25.0

57

16

16

16

0.0

58

16

12

12

25.0

59

14

14

14

0.0

60

13

13

12

7.7

70 mg/kg bw/d

61

12

12

9

25.0

62

15

14

13

13.3

63

15

9

9

40.0

64

12

9

7

41.7

65

13

11

8

38.5

67

9

9

9

0.0

68

13

13

12

7.7

69

15

15

13

13.3

70

13

11

10

23.1

200 mg/kg bw/d

71

11

11

11

0.0

72

15

13

12

20.0

73

20

10

8

60.0

75

14

13

10

28.6

76

12

11

10

16.7

77

12

11

9

25.0

78

13

10

10

23.1

79

9

6

6

33.3

80

9

3

3

66.7

(*) in this femalethe number of pups born was slightly higher than the number of implantations and corpora lutea recorded. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 7 of lactation.

 

 

Furthermore it has been discussed that this is not really an effect on development but rather an effect concerning fertility. The reduced litter size is considered secondary to a reduced number of implantations.

 

In discrepancy to the opinion of the study author, the observed reduced number of implantations and the subsequently smaller litter sizes were considered as a consequence of general toxicity.

According to the OECD definition in guideline 414 (below), within the context of the above screening study, an observation of developmental toxicity (rather than reproductive toxicity) implies post natal death of the conceptus.

“Developmental toxicology: the study of adverse effects on the developing organism that may result from exposure prior to conception, during prenatal development, or postnatally to the time of sexual maturation. The major manifestations of developmental toxicity include 1) death of the organism, 2) structural abnormality, 3) altered growth, and 4) functional deficiency. Developmental toxicology was formerly often referred to as teratology.”

No treatment related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and pup mortality, clinical signs, body weight and macroscopy).

A treatment-related reduction in both implantations and litter size at parturition in a rat reproduction study, with no corresponding morphological abnormalities or adverse effects on the pups after birth, could be taken as evidence of a lack of developmental toxicity.

The embryos that succeeded in implanting on the uterus continued to develop normally, even at dose levels high enough to cause effects on fertility. This argument is further strengthened by signs systemic toxicity for male and female adults:

At 200 mg/kg bw/d, most males showed weight loss (up to 15% of Day 1 values) during the first two weeks of treatment. However, body weights largely recovered as treatment progressed. Although mean body weight and body weight gain remained statistically significantly lower throughout treatment, body weight gain exceeded that of controls during the mating period (by comparison of mean body weight increase over Days 1-15 of the mating period).Since body weights of males had recovered to control levels during treatment, these changes were not considered to be adverse in nature (by the study director). In contrast to the opinion of the study director, the effect is considered adverse but reversible.

At 200 mg/kg bw/d, hunched posture was noted among all males primarily during the second week of treatment, and at lower incidence, rales, piloerection and lean appearance were noted among some males.

In females of the 200 mg/kg bw/d dose group, a statistically significant reduction in body weight gain was noted during the first two weeks of treatment, as well as during pregnancy. Although the study authors did notconsider these effects to be adverse in nature, we came to a differing conclusion. The effect on body weight (gain) is considered to be reversible, but nevertheless a sign of systemic toxicity.

 

This is also supported by the findings in the14 day dose range finding study, where all animals treated with 500 mg/kg bw/d were sacrificed for humane reasons between days 6 and 8. Animals showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. This shows, that there is a steep dose-response-curve for the tested substance and that the dose of 200 mg/kg bw/day is most probably the highest tolerable dose with already some signs of general toxicity.
Based on the remarkable effects on body weight /body weight gain and food consumption in the reproduction / developmental toxicity screening test as well the findings of the 14 day dose range finding study, the observed fertility effects – reduced number of implantation sites and subsequently lower litter size – may also be explained as a consequence of general parental toxicity.

 

Slightly reduced number of implantation sites (dermal developmental toxicity test)

As described in the study report, administration of 200 mg/kg bw/day of the test material may have slightly impaired implantation, as the average number of implantation sites per pregnant doe was slightly lower for rabbits in this dosage group, as compared to the control group. However, the effect was not statistically significant.

We do not follow this conclusion. In contrast to the recent OECD guideline, which recommends dosing through the entire period of gestation to the day before caesarean section, the substance was dermally administered from day 7 through 18 of presumed gestation only in accordance with former versions of the guideline. Implantation in the rabbit takes place on day 6 to 7 (according to OECD guideline 414, adopted 22ndJanuary 2001). Thus, an effect of the test substance on implantation is unlikely, since implantation is normally complete before GD7, i.e. the start of treatment in this study.

Moreover, the substance was administered dermally for 2 h/day, making it highly unlikely that relevant systemic levels could have been reached on the first day of administration.

Thus, the slight reduction in the number of implantation sites is considered to be incidential and not treatment-related.

As the result of the slightly lower implantation sites, the average litter size for the high dosage group was also slightly decreased, as compared with the control values. All of the values were within expected historical control values. However, as this effect was secondary to the incidentially reduced number of implantation sites, this effect is not treatment-related.

The lack of developmental toxicity is further supported by the fact that administration of test material did not adversely affect pregnancy incidence or average numbers of resorption.

 

Also from the available data on DMAPA no concern on developmental toxicity was recognised. Areproduction/developmental toxicity screening test cited by OECD SIDS and NTP resulted in a NOAEL of 200 mg/kg bw/d (highest dose administered) for developmental toxicity (NTP, OECD SIDS).

 

In conclusion, there is no concern for developmental toxicity.The results of the prenatal developmental toxicity study do not indicate a substance-related effect on the fetus up to the highest tested dose of 200 mg/kg bw/d. There are no data gaps. No human data are available. However, there is no reason to believe that these results from rat 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 prenatal developmental toxicity study on the source substance is considered as an appropriate adaptation to the standard information requirements of Annex IX 8.7.2 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 substance C16-18 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 substance Stearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid and N, 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 substance C16-18 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 substance C16-18 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 substance Stearic acid 3-(dimethylaminopropyl)amide or the target substance C16-18 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 by cytochrome 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 substance Stearic 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.

OECD SIDS concluded that there is also no concern of DMAPA having any adverse effects to reproduction based on an OECD TG 421 study (OECD SIDS).

The read-across is supported by the similar results observed in in teh available 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:

Prenatal Developmental Toxicity:

The source substance Stearic acid 3-(dimethylaminopropyl)amidehas been tested in a reliable with restrictions (RL2) GLP-compliant prenatal developmental toxicity study similar to OECD Guideline 414.

A GLP compliant reproduction / developmental toxicity screening test according to OECD Guideline 421 (RL1) is available for the source substance Stearic acid 3-(dimethylaminopropyl)amide.

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.

Thus, the absence of prenatal developmental toxicity for the source substance Stearic acid 3-(dimethylaminopropyl)amide is considered to be also relevant for the target substance C16-18 DMAPA amidoamine.

 

References

NTP (National Toxicology Program),3-Dimethylaminopropylamine109-55-7; available online: http://ntp-server.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/Dimethylaminopropylamine_508.pdf

 

OECD SIDS,3-AMINOPROPYLDIMETHYLAMINE CAS N°: 109-55-7, available online: http://www.inchem.org/documents/sids/sids/109557.pdf


Justification for selection of Effect on developmental toxicity: via oral route:
OECD guideline study (screening), no deviations, GLP

Justification for selection of Effect on developmental toxicity: via dermal route:
similar to OECD guideline study, GLP

Justification for classification or non-classification

Based on the available data for the closely related read-across substance Stearic acid 3-(dimethylaminopropyl)amide, the target substance C16-18 DMAPA amidoamine does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.