Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Subacute (14 day dose-range finding study) repeated dose toxicity study oral (gavage), rat Crl:WI(Han)) m/f (similar to OECD TG 407, GLP), dose levels: 0, 50, 200, 500 mg/kg bw/d; NOAEL(fertility) = 200 mg/kg bw/d; read-across: 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: Stearic acid 3-(dimethylaminopropyl)amide

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- 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 individuallyOne 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.
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
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 dataMale 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, non-treatment-related
Description (incidence and severity):
- 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
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- 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
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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

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
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/d
- 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
- 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)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- 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
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- spermatogenic staging profiles were normal for males examined
- testes and epididymides weights were unaffected by treatment
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- 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)
- 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.
- 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.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
70 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
70 mg/kg bw/day
Based on:
act. ingr.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment up to 200 mg/kg bw/d.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
Reproductive effects observed:
not specified
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/dfertility NOAEL, females: 70 mg/kg bw/dfertility NOAEL, males: 200 mg/kg bw/ddevelopmental 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

fertility NOAEL, females: 70 mg/kg

fertility NOAEL, males: 200 mg/kg

developmental NOAEL: 200 mg/kg

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
Quality of whole database:
The available studies were conducted according to guidelines and are of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No experimental data on toxicity to reproduction are available for C16 Alkylamidopropyltrimethylammonium Chloride. However, reliable data from an oral reproduction / developmental toxicity screening test in rats as well as from the corresponding 14 d dose range finding study conducted with the closely related source substance Stearic acid 3-(dimethylaminopropyl)amide is available. 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 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 by 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 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 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 

 

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 thereproduction / 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.

 

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

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across attached as pdf document to section 13 of the IUCLID file.

 

Structural similarity

a. Structural similarity and functional groups

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is manufactured from hexadecanoic acid and N,N-dimethyl-propylenediamine. Methyl chloride is used to quaternise the dimethylamino group of the fatty acid amidoamine.

The substance is composed of mainly C16 amides (ca. 92%) of DMAPA and small amounts of the C14 (ca. 2.5%) and C18 amide (ca. 5.5%).

 

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%).

 

b. Common breakdown products

The metabolism that is expected to occur is for the target substance C16 Alkylamidopropyltrimethylammonium Chloride and the source substance Stearic acid 3-(dimethylaminopropyl)amide the hydrolysis of the amide-bond by amidases. Metabolism would result in free fatty acids and di- or trimethylaminopropylamine. The free fatty acids enter normal metabolic pathways (e.g. degradation by the mitochondrial beta-oxidation process) 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. 

 

c. Differences

Chain length:

The slight differences in fatty acid chain length (higher percentage of C16 in the target substance vs. higher percentage C18 in the source substance Stearic acid 3-(dimethylaminopropyl)amide) are not considered to be of relevance for systemic toxicty.

 

Methylation/quaternation:

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is methylated during the manufacturing process resulting in the quaternised ammonium ion.

Stearic acid 3-(dimethylaminopropyl)amide)on the other hand is not methylated during the manufacturing process. But based on physicochemical data (pKa) it is concluded that at physiological relevant pH, the substance is mostly protonated similarly resulting in a positively charged ammonium ion. This difference isnot considered to be of relevance forsystemic toxicty.

 

Overall, the provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

Comparison of prenatal developmental toxicity data

Endpoint

Target substance

C16 Alkylamidopropyltrimethylammonium Chloride 

Source substance

Stearic acid 3-(dimethylaminopropyl)amide

Developmental Toxicity

 

No data, read-across

OECD guideline 421(Reproduction/Developmental Toxicity Screening Test)
RL 1, GLP)

10 Wistar rats/sex/dose exposedby gavageat dose levels of 0, 20, 70 and 200 mg/kg bw/d.

Males were exposed for 28 days; Females were exposed for 41 – 54 days

 

Fertility effects: reduced number of implantation sites and subsequently lower litter size at 200 mg/kg bw/day

 

Developmental effects: none

 

General toxicity: reduced body weight gain and food intake at 200 mg/kg bw/d

 

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

 

The physico-chemical properties of the target substance C16 Alkylamidopropyltrimethylammonium Chloride and the source substance Stearic acid 3-(dimethylaminopropyl)amide are very similar; thus, a comparable toxicokinetic behaviour can be expected. Based on the structural similarities with identical functional groups (quaternary amines, amide bonds) and saturated fatty acid chains with comparable length as well as very similar physicochemical properties, the read-across approach from the available Reproduction/Developmental Toxicity Screening Test is considered to be reliable.

 

Quality of the experimental data of the analogues:

The source substance Stearic acid 3-(dimethylaminopropyl)amide has been tested in a reliable study according to OECD TG 421. All tests have been conducted according to GLP criteria. Therefore these data have no uncertainties and can be used in an analogue approach. The available data from the source chemical is sufficiently reliable to justify the read-across approach.

 

Conclusion for read-across

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 under the experimental conditions in the considered studies for the endpoint reproductive toxicity.

Thus, the absence of toxicity to reproduction for the source substance Stearic acid 3-(dimethylaminopropyl)amide is considered to be also relevant for the target substance C16 Alkylamidopropyltrimethylammonium Chloride.


Effects on developmental toxicity

Description of key information
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
Link to relevant study records
Reference
Endpoint:
developmental toxicity
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:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system 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
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%).
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 individuallyOne 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.
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
Duration of test:
Males: 28 days
Females: 41 - 54 days
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
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
Maternal 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.
Ovaries and uterine content:
n/a
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.
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)
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:
no effects observed
Description (incidence and severity):
- 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
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- 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
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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

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
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
200 mg/kg bw/d
- 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
- 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)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- Testes and epididymides weights were unaffected by treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- 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
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
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.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
70 mg/kg bw (total dose)
Based on:
act. ingr.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Body weights of pups were unaffected by treatment up to 200 mg/kg bw/d.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
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.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects
Abnormalities:
no effects observed
Developmental effects observed:
no
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 fertility NOAEL, females: 70 mg/kg fertility NOAEL, males: 200 mg/kg developmental NOAEL: 200 mg/kg
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

fertility NOAEL, females: 70 mg/kg

fertility NOAEL, males: 200 mg/kg

developmental NOAEL: 200 mg/kg

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:
rat
Quality of whole database:
The available study was conducted according to guideline and is of high quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No experimental data on developmental toxicity are available for C16 Alkylamidopropyltrimethylammonium Chloride. However, reliable data from an oral reproduction / developmental toxicity screening test in rats conducted with the closely related source substance Stearic acid 3-(dimethylaminopropyl)amide is available. A justification for read-across is given in section "effects on fertility".

 

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 by 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 bw/d (based on lower body weight and food consumption)

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

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

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

 

Discussion of effects concerning development(reproduction/developmental toxicity screening test)

Reduced litter size

 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.

 

Conclusion

Based on the data of the reproduction/developmental toxicity screening test with the source substance Stearic acid 3-(dimethylaminopropyl)amide, 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. 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.

Based on the remarkable effects on body weight /body weight gain and food consumption in the reproduction / developmental toxicity screening test (general toxicity NOAEL = 70 mg/kg bw/day), 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.

 

Justification for classification or non-classification

Results of existing studies with structural very similar substances indicate that C16 Alkylamidopropyltrimethylammonium Chloride does not need to be classified for toxicity to reproduction, developmental toxicity and teratogenicity according to the criteria given in regulation (EC) 1272/2008. Therefore labelling is not necessary.

Additional information