Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
Reproduction/ developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2017 - 21 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: See "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Product name: Sodium cocopropylenediamine propionate
CAS no (old) : 97659-50-2
CAS no (new): 2136366-30-6
Batch no.: 48724
Date of Production: 18.05.2017
Best before Date: 17.05.2020
Purity (certified): 29.7% w/w (UVCB) - Total solids (activity) % w/w 29.7 Lower limit: 29.0, Upper limit: 30.0

Main active ingredients
Dodecylpropylenediamine tripropionate: 64% w/w
Tetradecylpropylenediamine tripropionate: 16% w/w
(considering the composition of the other constituents it is considered justified that the two main constituents represent the whole test item)

Water solubility: soluble
Appearance: yellow, clear
State:liquid
Stability under test conditions: not specified

Viscosity at 20°C Cps 34, Upper limit:150
Color (20% aq solution) Hu 100, Upper limit: 250
pH (20% aq solution) pH 6.3, Lower limit: 6.0, Upper limit: 7.0
Recommended storage
Store container tightly closed in a dry, well-ventilated place
Specific details on test material used for the study:
Purity/Composition correction factor: ~30% based on solid content;
pH (20% aq. solution): 6.3 (6.0 - 7.0);
Specific gravity / density: 1.047 kg/m3 at 20°C.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 14 weeks
- Weight at study initiation: 210 - 252 g
- Fasting period before study: No (during motor activity measurements the animals had no access to food)
- Housing: On arrival and following the pretest and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 8 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed. There were no known contaminants in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 32-57
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 Nov 2017 To: 31 Jan 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. From start until Day 24 of treatment, the dosing formulations were prepared daily as a solution and dosed within 5 hours after completion of the preparation of the formulation. From Day 25 of treatment, the dosing formulations were prepared weekly in bulk, filled out in daily portions and stored at room temperature for a maximum of 8 days from preparation. Prior to dosing, the formulations were stirred for at least 30 minutes. All formulations were used within the demonstrated stability period of 8 days of preparation. Adjustment was made for specific gravity of the test item. A factor of 3.4 was used to correct for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure. Dose formulation samples were collected in the first week of treatment for concentration analysis (all groups), and for homogeneity and stability analysis (low and high dose groups formulations). The homogeneity results obtained from the top, middle and bottom for the low and high dose group preparations were averaged and utilized as the concentration results. Duplicate sets of samples (approximately 500 mg) for each sampling time point were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target conc
entration. Stability of the test item in water was determined after 5 hours and 8 days storage at room temperature.
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Detection of mating was not confirmed in first instance for two females. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in these females. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 13-15 days of lactation (for 60-70 days). Females that failed to deliver pups were treated for 40-42 days.
Frequency of treatment:
Once daily
Duration of test:
Same as treatment
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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:
Initially, the dose range finder was started by treatment of two groups for 10 consecutive days, i.e. Group 1 at 500 mg/kg bw/day and Group 2 at 1000 mg/kg bw/day. As the results were considered to be inconclusive for selecting dose levels for the main study, an additional group was treated at 750 mg/kg bw/day for 25 consecutive days. Each group consisted of 3 females.
The following parameters were included:
Mortality: Twice daily throughout the study.
Clinical Observations: At least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body Weights: On Day 1 prior to dosing and on Days 5 and 10. For Group 3 the body weights were determined every 5 days from Day 1 onwards.
Food Consumption Over Days 1-5 and 5-10. For Group 3 food consumption was determined over each of these 5-day periods between body weight measurements.
All animals were subjected to an external, thoracic and abdominal examination on Day 10 for groups 1 and 2 and on day 26 for group 3 after the last observation of clinical signs (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight
were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for 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 during lactation on PND 1, 4, 7, and 13.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only (overnight with a maximum of 24 hours), females were not fasted)
- How many animals: All
- All parameters according to guidelines were examined and coagulation parameters (prothrombin time and activated partial thromboplastin time).
Ovaries and uterine content:
Ovaries were examined histopathologically. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Fetal examinations:
All surviving pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) 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 live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Historical control data:
Present at test facility and included in the report where relevant.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the arena observations in this study. Rales were observed in one female at 150 mg/kg bw/day and one female at 500 mg/kg bw/day. This clinical sign was considered to be of no toxicologically relevance as it was temporary observed in single animals per group and because of its equal distribution over the dose groups (including controls). Salivation seen after dosing among all females at 500 mg/kg bw/day was considered to be a physiological response (possibly due to the taste of test item) rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Any other clinical signs noted during the treatment period (i.e. scabs and alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. On day 2 of treatment, one female dosed at 50 mg/kg bw/day started showing breathing difficulties during dosing and died a few moments later. Macroscopic examination revealed liquid in the lungs . Based on these finding its death was considered the result of a dosing error and not related to the test item. After its death, this female was replaced by one of the reserve females. Initiation of treatment of the replacement female was on Day 2.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight gain of treated male and female rats remained in the same range as concurrent controls over the treatment period. It should be noted that body weight gain in one control male and one female at 500 mg/kg bw/day was deviating from normal over (part of) the treatment period. For the male, a normal weight gain was observed for the first two weeks of the study, followed by a minimal body weight loss (of approximately 3%) over the remaining two week study period. For the female, a low level of body weight gain was observed during the last part of its gestation period. Nevertheless it delivered a normal litter, and its growth during lactation was normal. Based on the single incidences of deviating growth, these findings were considered of no toxicological relevance and not related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were noted over the treatment period. It should be noted that a low food consumption was observed for one female at 500 mg/kg bw/day during the last part of its gestation period, which was concurrent to its low body weight gain.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day, increased counts for neutrophils, and a concurrent decreased count for lymphocytes were observed in plasma of female animals, achieving levels of statistical significance for neutrophils (as percentage of whole blood count) and for percentage of lymphocytes in females when compared to controls. As a consequence, increased neutrophils-to-lymphocytes ratios were observed for females at 500 mg/kg bw/day when compared to controls and the animals treated at lower doses (neutrophilsto-lymphocytes ratio's of 0.42, 0.41, 0.38 and 0.79 for control rats and rats dosed at 50, 150 and 500 mg/kg bw/day, respectively). Since an increased neutrophilsto-lymphocytes ratio is considered a marker for (systemic) inflammation, a relation of the changes in these ratios to the findings in the mesenteric lymph nodes could not be ruled out, but
could not be established in the current study. Coagulation parameters of treated rats were considered not to be affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment. Any other statistically significant changes in clinical biochemistry parameters, i.e. alanine aminotransferase in females at 50 mg/kg bw/day, were considered not to be related to treatment as these occurred in the absence of a dose-related trend.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. Motor activity was considered similar between treated and control groups. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. For several treated females, a pale discoloured thyroid gland was observed. The incidence of this observation was equally distributed over the treated groups, i.e. 2/10, 3/10 and 2/10 in the 50, 150 and 500 mg/kg bw/day treated females respective ly. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Foamy macrophage foci in the mesenteric lymph nodes were present at 150 mg/kg bw/day in females at minimal degree and at 500 mg/kg bw/day in males at minimal degree and in females up to mode rate degree and multifocal necrosis was present at 500 mg/kg bw/day in a single female at moderate degree. There were no other test item-related histologic changes.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were not considered to be affected by treatment. The gestation index was 100% for all groups. All pregnant females delivered their offspring after 21 to 22 days of gestation. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The post-implantation survival indices were 90, 90, 94 and 91% for the control, 50, 150 and 500 mg/kg bw/day, respectively. For one female dosed at 50 mg/kg bw/day and one female dosed at 500 mg/kg bw/day, the number of pups were higher than the number of implantations, i.e. 11 vs 10 and 10 vs 9, respectively. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. The live birth index was 100% for all groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Duration of gestation were not considered to be affected by treatment.
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
No signs of difficult or prolonged parturition were noted among the pregnant females.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Multi focal necrosis in the mesenteric lymph nodes in 1/10 females at 500 mg/kg bw/day

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment. The variation in mean body weights of male and female pups on PND 1 between the dose groups corresponded with the variation in litter size in each group. The statistical significance apparent for the body weight of the male and female pups at 50 mg/kg bw/day was therefore considered to have occurred by chance and not related to treatment. As the growth of the pups in litters of all groups was comparable no toxicological significance was attached to the statistical significance.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. The viability indices were 99% at 50 mg/kg bw/day and 100% for the other groups. One pup at 50 mg/kg bw/day had to be sacrificed in extremis on PND 1 since it presented with pale skin and laboured breathing. Because of this single incidence, it was considered not related to treatment and of no toxicological relevance.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was not considered to be affected by treatment. The male/female ratios were 45/55, 52/48, 51/49 and 43/57 for the control, 50, 150 and 500 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was not considered to be affected by treatment. The mean number of pups per litter per dose group were 10.4, 12.3 12.4 and 11.4 for the control, 50, 150 and 500 mg/kg bw/day, respectively. Body weights of pups were not considered to be affected by treatment.The variation in mean body weights of male and female pups on PND 1 between the dose groups corresponded with the variation in litter size in each group. The statistical significance apparent for the body weight of the male and female pups at 50 mg/kg was therefore considered to have occurred by chance and not related to treatment. As the growth of the pups in litters of all groups was comparable no toxicological significance was attached to the statistical significance.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. The lactation indices were 98% for the control group and 100% for the treated groups. One pup of the control group (dam no. 43) had to be sacrificed in extremis on PND 13 since it presented with gasping and haemorrhage in the nose region. Because of this single incidence, it was considered not related to treatment and of no toxicological relevance.
External malformations:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. Treatment up to and including 500 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed on PND 13. No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age and were, therefore, not considered to be related to treatment. For one pup dosed at 50 mg/kg bw/day that had to be sacrificed in extremis on PND 1, absence of milk in the stomach was noted at necropsy.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at highest dose tested (500 mg/kg bw/day)

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of a 28 day repeated dose study with screening for reproduction and developmental effects, the maternal NOAEL was found to be 150 mg/kg bw/day. The NOAEL for development was established to be at least 500 mg/kg bw/day.
Executive summary:

A combined 28 day repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Sodium Cocopropylenediamine Propionate was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 60-70 days). Females that failed to deliver pups were treated for 40-42 days. In females at 150 mg/kg bw/day and in both females and males at 500 mg/kg bw/day , test item-related microscopic findings were observed in the mesenteric lymph node. These findings consisted of foamy macrophage foci at 150 mg/kg bw/day in females and at 500 mg/kg bw/day in males and females which were considered non-adverse, and of multifocal necrosis in a single female at 500 mg/kg bw/day which was considered adverse.

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 14-16) and macroscopic examination).

Based on the adverse effects on mesenteric lymph nodes (seen in 1/10 females) at 500 mg/ kg bw/ day, a No Observed Adverse Effect Level (NOAEL) for Sodium cocopropylenediamine propionate of 150 mg/kg bw/ day was established. The NOAEL for development was established to be at least 500 mg/kg bw/day, as no adverse effects were seen up to and at this dose level.