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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2020 - 19 March 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 preliminary study was to investigate the toxicity, with particular reference to potential local gastric irritating effect, of C12 AAPB in New Zealand White (NZW) rabbits, following oral administration by gavage, for 2 consecutive weeks.
4 females were dosed with 0, 100, 300, and 600 mg/kg bw/d.
The animals were checked twice a day for mortality and daily monitored for clinical signs.
The bodyweight and food consumption were measured twice a week. Detailed macroscopic examination, organ weights and histopathology of the gastrointestinal tract, were carried out.
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:
N-(carboxymethyl)-3-(dodecanoylamino)-N,N-dimethylpropan-1-aminium hydroxide
Test material form:
solid - liquid: aqueous solution

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l.
- Age at study initiation:
- Weight at study initiation:
- Housing: individually in polycarbonate/stainless steel cages with perforated
NorylTM floor suspended over trays
- Diet (e.g. ad libitum): rabbit diet (Mucedola 2 RB 15,Mucedola S.r.l., Via G.
Galilei 4, 20019 SettimoMilanese (MI), Italy); ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19±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 preparation was made up to once a week (concentration of 20, 60 and 120mg/mL). The concentration was calculated and expressed in terms of test item corrected for purity (30.43%).
Details on mating procedure:
animals were not mated in this preliminary dose range finding study
Duration of treatment / exposure:
14 d
Frequency of treatment:
daily
Duration of test:
14 d
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.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

Examinations

Maternal examinations:
MORTALITY: Yes
- Time schedule: twice a day


CLINICAL SIGNS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
The animals were weighed on the day of allocation to treatment group, on the day that treatment commenced, twiceweekly thereafter (with the exception of the high dose females which died on Day 3) and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured on the same intervals as body weight measurements following allocation.

WATER CONSUMPTION AND COMPOUND INTAKE: No


POST-MORTEM EXAMINATIONS: Yes
- examination of the external surface and orifices; special attention was paid to the organs of the gastrointestinal tract
- Organ weights: see table below
- Tissues fixed and preserved: see table below
- Histopathological examination: see table below


Ovaries and uterine content:
n.a. (only preliminary dose range finding study in non-pregnant animals)
Fetal examinations:
n.a. (only preliminary dose range finding study in non-pregnant animals)
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 aModified 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.
Indices:
n.a. (only preliminary dose range finding study in non-pregnant animals)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe clinical signs including dyspnoea, pale appearance, red nasal discharge, coldness to touch, brown staining in the perianal region and decreased activity were recorded at 300 and 600mg/kg/day. Reduced or liquid faeces were observed in the cage.
Reduced faeces and reduced food intake were recorded at 100mg/kg/day. Dyspnoea, transparent nasal discharge, rales and emaciated appearance were also observed in one low dose female (no. E04480011) in single occasions.
Mortality:
mortality observed, treatment-related
Description (incidence):
Eight cases of premature death occurred during the study. Two high dose (nos. E0448025 and E0448031) and one mid-dose (no. E0448017) females were found dead on Day 3 and Day 5 of the study, respectively, whereas the remaining females of Group 4 (nos. E0448027 and E0448029) and Group 3 (nos. E0448019, E0448021 and E0448023) were sacrificed for humane reasons on the same days as the found dead animals. on Day 3 and Day 5 of the study, respectively.

Found dead
Animal nos. E0448025 and E0448031 (Group 4): At post mortem examination, the most relevant findings were dark and/or red areas/ colour of the stomach (pyloric and/or fundic region) and brown fluid content in the small and large intestinal tract (jejunum, ileum, caecum, colon and/or rectum) whereas the microscopic evaluation could not be performed due to the autolytic status of the organs and tissues collected for the evaluation.
Animal no. E0448017 (Group 3): At post mortem examination, the most relevant finding was dark fluid content in the small and large intestinal tract (jejunum, ileum, caecum and colon). Microscopically, the most relevant findings were erosion of the mucosal epithelium of cardial, fundic and pyloric regions of the stomach associated with mucosal haemorrhage and chronic inflammation of tunica muscularis of pyloric region.

Humane sacrifices
Animal nos. E0448027, E0448029 (Group 4) and E0448019, E0448021 and E0448023 (Group 3): Macroscopically, the most relevant findings observedwere dark and/or red areas/ colour of the stomach (pyloric and/or fundic region) and brown fluid content in the intestinal tract (duodenum, jejunum, ileum, caecum and/or colon). Microscopically, the most relevant findings were erosion of the mucosal epithelium associated with haemorrhage of cardiac, fundic and/or pyloric regions of the stomach and chronic inflammation of submucosa and
tunica muscularis of fundic and/or pyloric regions of the stomach. The findings affecting the gastric tract could be considered the factor contributory to the ill status or death of the mid- and high dose treated females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Marked reduced body weight was observed at 300mg/kg on the last day of measurement (Day 5 of study).
Reduced body weight was noted at 100mg/kg/day. Two females had a more pronounced decrease starting from Day 5 onward for animal no. E0448011 and between Day 12 and the end (Day 15) for animal no. E0448009. No changes were seen in the other low dose females.
A slight decrease (approximately 10%), although not statistically significant, was observed on terminal body weight of low dose females sacrificed at term when compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Severe reduced food consumption was noted at 300mg/kg on Day 5.
Similarly to body weight, reduced food consumption was recorded at 100 mg/kg/day, starting from Day 5 onward for animal no. E0448011 and between Day 12 and the end for animal no. E0448009. No changes were seen in the other low dose females.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The statistically significant decrease in absolute and relative uterus weight (approximately 38% for relative uterus weight) noted in low dose females sacrificed at termwhen compared to the controls was considered of doubtful interpretation, as not corroborated by histopathological evaluation. No other changes were observed in absolute and relative organ weights of control and low dose treated females that completed the treatment period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were noted in the stomach of a single female rabbit treated with the low dose (no. E0448009). The pyloric region of the stomach was characterised by the presence of multiple pinpoint red areas associated with a single depressed dark red area. The macroscopic changes correlated microscopically with erosion, haemorrhage and chronic inflammation. No other changes were noted at post mortem examination in the remaining organ/ tissues.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were seen in the stomach of female no. E0448009 (low dose group) and consisted of erosion of the mucosal epithelium associated with haemorrhage and chronic inflammation of the submucosa tunica muscularis of fundic region of the stomach. No other treatment related changes were observed in the gastrointestinal tract of the remaining three low dose females.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

Remarks on result:
not measured/tested

Maternal abnormalities

Abnormalities:
not examined

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined

Effect levels (fetuses)

Remarks on result:
not measured/tested

Fetal abnormalities

Abnormalities:
not examined

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A 2 week toxicity study with C12 AAPB was conducted in New Zealand White (NZW) female rabbits with particular aim to investigate its potential local gastric irritating effect. Female rabbits received the test item, dissolved in softened water, at dose levels on 100, 300 and 600 mg/kg/day and the dose volume was set at 5mL/kg.
Severe signs of toxicity were seen a few days after the start of treatment with premature mortality at 600 and also at 300mg/kg/day. The animals showed marked clinical signs that included decreased activity, dyspnoea, nasal discharge, rales, coldness to touch, reduced or liquid faeces. Decreased body weight and food consumption were also recorded. The necropsy revealed findings in the stomach as the factor contributory to the ill status or
death of these animals.
At low dose (100mg/kg/day), two females showed reduced food consumption and body weight during the last week of treatment. Dyspnoea, rales and reduced faeces were also recorded at clinical observations. The macroscopic observations revealed the presence of multiple pinpoint red areas associated with a single depressed dark red area in the stomach that correlated with microscopically findings of erosion, haemorrhage and chronic inflammation in one low dose female only.
No changes were seen in the organ weights.
In conclusion, based on the results obtained in this investigative study, signs of toxicity were seen at all dose levels. The treatment-related findings at histopathological examination of the stomach confirmed the potential gastric irritant effect of C12 AAPB.
Executive summary:

A 2 week toxicity study with C12 AAPB was conducted in New Zealand White (NZW) female rabbits with particular aim to investigate its potential local gastric irritating effect.

The test item, dissolved in softened water, was administered by oral gavage, according to the following experimental design:

 

Group

Treatment (mg/kg/day)+

Concentration (mg/mL)

Dose Level

Number of animals

1

0

0

Control

4 females

2

100

20

Low

4 females

3

300

60

Mid

4 females

4

600

120

High

4 females

 

+: in terms of test item corrected for purity (30.43%)

 

Control animal received softened water as vehicle during the same treatment period.

The animals were checked twice a day for mortality and daily monitored for clinical signs.

The bodyweight and food consumption were measured twice a week. Detailed macroscopic examination, organ weights and histopathology of the gastrointestinal tract, were carried out.

 

Mortality

On Day 3, two females at 600mg/kg/day, were found dead and the remaining females were sacrificed for humane reasons. On Day 5, one female at 300mg/kg/day was found dead and the remaining females were sacrificed for humane reasons.

At necropsy, the findings affecting the stomach could be considered the factor contributory to the ill status or death of these animals.

No mortality occurred at 100mg/kg/day.

 

Clinical signs

Severe clinical signs including dyspnoea, pale appearance, red nasal discharge, coldness to touch, brown staining in the perianal region and decreased activity were recorded at 300 and 600mg/kg/day. Reduced or liquid faeces were observed in the cage.

Reduced faeces and reduced food intake were recorded at 100mg/kg/day. Dyspnoea, transparent nasal discharge, rales and emaciated appearance were also observed in one low dose female.

 

Body weight

On Day 5, marked reduced body weight was observed in females dosed at 300mg/kg/day (-15% respect to Day 1). Body weight was reduced at 100mg/kg/day, especially in two females.

 

Food consumption

Severe reduced food consumption was noted at 300mg/kg on Day 5. A decrease in food consumption was recorded at 100mg/kg/day starting from Day 5 onward.

 

Terminal body weight and organ weights

A slight decrease was observed in terminal body weight of low dose females sacrificed at term when compared to controls. The statistically significant decrease in absolute and relative uterus weight was considered of doubtful interpretation, as not corroborated by histopathological evaluation.

 

Macroscopic observations

Treatment-related changes were noted in the pyloric region of the stomach of a single low dose female rabbit and was characterised by the presence of multiple pinpoint red areas associated with a single depressed dark red area. The macroscopic changes correlated at histopathological examination with erosion, haemorrhage and chronic inflammation.

 

Microscopic observations

At final sacrifice, treatment-related findings were seen in the stomach of one low dose female and consisted of erosion of the mucosal epithelium associated with haemorrhage and chronic inflammation of tunica muscularis and/or submucosal of fundic region of the stomach.

 

Conclusion

In conclusion, based on the results obtained in this investigative study, signs of toxicity were seen at all dose levels. The treatment-related findings at histopathological examination of the stomach confirmed the potential gastric irritant effect of C12 AAPB.