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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 2020 - 7May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Principles of method if other than guideline:
The purpose of this study was to provide information on toxicity of C12 AAPB in rats after daily oral administration for at least 14 days. This study was performed with the objective to select dose levels for a subsequent Reproduction/Developmental toxicity screening test in rats (OECD 421).
GLP compliance:
no
Remarks:
This study is a preliminary study and is exempt from compliance with the Principles on Good Laboratory Practice of the OECD. However, it will be carried out in a GLP compliant facility.

Test material

Constituent 1
Chemical structure
Reference substance name:
(carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide
EC Number:
224-292-6
EC Name:
(carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide
Cas Number:
4292-10-8
Molecular formula:
C19H38N2O3
IUPAC Name:
(carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide
Test material form:
solid - liquid: aqueous solution

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: fromCharles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
- Weight at study initiation:
- Housing: 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese)
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of C12 AAPB was dissolved in the vehicle. The preparations were made daily (concentrations of 10 and 30mg/mL for the entire treatment period, and 100mg/mL for Days 1 and 2). Concentrations were calculated and expressed in terms of test item corrected for purity (30.43%).
Details on mating procedure:
Due to unexpected mortality in the high dose group after 2 days of treatment, it seemed evident that the high dose level could not be applied to pregnant animals and consequently the treatment at 1000 mg/kg/day was stopped in agreement with the Sponsor, and the study design was changed to a 2-week toxicity study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study to confirm that the proposed preparation procedure was acceptable and that the stability of the preparations was satisfactory. The analytical method was validated in ERBC Study no. A3723 in the range from 1 to 200 mg/mL.
A 28 hour stability at room temperature and an 8 days stability at 2-8°C were verified in the range from 1 to 200 mg/mL.
In this study, samples of the preparations prepared on Day 1 were analysed to check the concentration and the results of the analyses were within the acceptability limits stated in the laboratory's SOP for concentration of solutions (90-110%).
Duration of treatment / exposure:
14 d in control and low and mid dose groups; 2 d in high dose group
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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: Dose levels were selected based on data available for strcuturally related substances

Examinations

Parental animals: Observations and examinations:
MORTALITY: Yes
- Time schedule: twice daily

CLINICAL SIGNS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The weight of food consumed by each cage of males and females was recorded weekly, following allocation.



Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
organ weights: Adrenal glands, Epididymides, Kidneys, Liver, Ovaries with oviducts, Prostate gland (dorsolateral and ventral), Seminal vesicles with coagulating glands, Spleen, Thymus (where present), Thyroid, Uterus – cervix

Fixation/Preservation: Abnormalities, Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Caecum, Clitoral gland, Duodenum, Epididymides (left and right), Jejunum (including Peyer’s patch), Kidneys, Liver, Mammary gland - Females, Mammary gland -Males, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland (dorsolateral and ventral), Sciatic nerve,
Seminal vesicles with coagulating glands, Spleen, Stomach, Rectum, Testes , Thymus (where present), Thyroid, Uterus – cervix, Vagina
Postmortem examinations (offspring):
n.a.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.
Reproductive indices:
n.a.
Offspring viability indices:
n.a.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection and salivationwere observed in male and female animals dosed at 300mg/kg/day.
Piloerection, from slight to moderate, was observed in all males from Day 4 up to Days 11 or 12, and in 6 out of 10 females, mainly during the second week of the dosing period.
All males and females treated at 300mg/kg/day showed salivation during the last week of treatment. This sign was also recorded in one male (No. X1330038) and two females (Nos: X1330027 and X1330031) dosed at 100mg/kg/day but in a single occasion, on Days 2 and 3, respectively. These signs were considered treatment related but not adverse.
Surviving animals of Group 4 did not receive any treatment from Day 3 and during the recovery period clinical signs were no longer evident.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of eleven cases of deaths occurred during the study.
Eight males (nos. X1330062, X1330064, X1330066, X1330068, X1330072, X1330076, X1330078 and X1330080) and three females (nos. X1330061, X1330069 and X1330077), treated at 1000mg/kg/day (Group 4), were found dead or, in one instance (male no. X1330080), sacrificed for humane reasons on Day 3 of the study.
The treatment-related clinical signs recorded in all these animals, just prior their deaths and mainly on Day 2, were ataxia, decreased activity, kyphosis, staining of the ventral region, piloerection and salivation. The most relevant changes observed at post mortem examination in these high dose male and female ratswere yellow, brownand/or red staining of ventral or urogenital region and/or
muzzle and dark and/or red colour of brain. All the reported macroscopic observations were considered not to clearly establish the cause which led to humane sacrifice or death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No relevant changes in body weight were seen in Groups 2 and 3 animals, when compared to controls.
A statistically significant reduction in body weight gain (-54%) was noted in males treated at 300 mg/kg/day on Day 8. However, since the mean body weights were not significantly affected, this reduction is considered treatment related but not adverse considering that the differences were no longer evident on Day 15, showing a full reversibility.
Body weight gain of females treated at 300 mg/kg/day were found to be statistically increased on Day 8, compared to controls, but this was only due to an unexpected reduction in weight gain of control animal and thus considered not treatment related.
Mean body weight and body weight gain of animals treated at 100mg/kg/day were in line with control animals.
No change in body weight or body weight gain were appreciated in animals of Group 4 dosed at 1000 mg/kg/day after the interruption of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in food consumption were recorded during the treatment phase in animals of Groups 2 and 3, when compared to controls.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley rats of both sexes after repeated dosing with C12 AAPB, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring in agreement with the OECD guideline 421. The animals, 10 per sex, were assigned to four groups and administered orally, by gavage, at dose levels of 100, 300 and 1000mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10mL/kg.


Due to unexpected mortality and adverse effects at the very beginning of the treatment period, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.


Therefore, in agreement with the Sponsor, it was decided to interrupt the treatment at 1000 mg/kg/day from Day 3 of the study and to investigate only the toxicity of the test item, C12 AAPB, changing the current study design to a 2-week oral preliminary non GLP study.


 


Mortality


Eleven cases of premature death, 8 males and 3 females receiving 1000mg/kg/day (Group 4) were found dead on Day 2 of treatment or, in one instance (one male), sacrificed for humane reasons on Day 3 of treatment.


Ataxia, decreased activity, hunched posture (kyphosis), staining of the ventral region, piloerection and salivation were the treatment-related clinical signs recorded in all these animals just prior their deaths. At the post mortem examination, yellow, brown and/or red staining of ventral or urogenital region and/or muzzle and dark and/or red colour of brain were observed. All the reported macroscopic observations were considered contributory to the cause of deaths.


 


Clinical signs


Piloerection and salivation were the two most relevant treatment-related clinical signs recorded in male and female animals dosed at 300mg/kg/day. The signs were considered treatment related but not adverse.


 


Body weight and body weight gain


Changes noted in body weight gain in groups 2 and 3 were considered incidental or not adverse.


 


Food consumption


No differences in food consumption were recorded during the study in animals of Groups 2 and 3, when compared to controls.


 


Terminal body weight and organ weights


No changes were observed in terminal body weight, or absolute and relative organ weights of control and treated animals that completed the treatment or in animals of Group 4 after 14 days of the recovery period.


 


Macroscopic observations


The most relevant change observed in rats treated at 300 and 1000mg/kg/day of both sexes was thickened stomach (non glandular region), when compared to controls.


 


Conclusion


Based on the results obtained in this study, it can be concluded:


 


The dose level of 1000 mg/kg/day induced an acute toxicity when administered to Sprague Dawley rats as demonstrated by mortality and severe signs such ataxia, hunched posture and decreased activity. Treatment related findings described as thickened stomach (non glandular region), were observed in animals after 12 days of recovery period.


 


The dose level of 300 mg/kg/day induced treatment related clinical signs such as piloerection and salivation, reduction in body weight gain and thickened stomach (non glandular region) observed at the macroscopic examination. These signs were considered treatment-related, but not adverse.


 


The dose level of 300 mg/kg/day is considered the NOAEL (No Observed Adverse Effect Level) for this study.