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Diss Factsheets

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:
Experimental start and completion dates: 14 November 2017 to 14 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ECHA have agreed that a developmental toxicity study can be conduced with 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C8 18 acyl derivs., hydroxides, inner salts. The end point will be addressed for the target substance 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts by reading across from this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD Guidelines for Testing Chemicals, No. 414, Prenatal Developmental Toxicity Study, 22 January 2001
Deviations:
yes
Remarks:
Randomisation was made in SPSS and not in Provantis, as indicated in the Study Plan. No impact on the results or study integrity.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts
EC Number:
939-455-3
Molecular formula:
Molecular formula, SMILES notation, InChl and Structural formula cannot be given as the substance is a UVCB.
IUPAC Name:
1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Solvay Novecare
- Lot/batch number of test material: UP6C10X10
- Appearance: Clear liquid
- Expiration date of the lot/batch: 11 March 2018
- Purity (sultaine content): 35.4%, Correction factor: 2.82

- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, below 70 RH%) protected from light
- Stability under storage conditions: Stable
- Stability under test conditions: Stable for the duration of the study.
- Solubility and stability of the test substance in the vehicle: Stability in the vehicle proven for at least 21 days when stored at room temperature (20±5°C).
- Reactivity of the test substance with the vehicle (if applicable): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated in distilled water at appropriate test concentrations

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Formulated in distilled water at appropriate test concentrations

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Test item pH (as received): 8.4

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar rats (Crl:WI(Han))
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats at least 12 weeks old at mating.
- Weight at study initiation: 200-260g (the variation did not exceed ± 20% of the mean weight at Gestation Day 0)
- Fasting period before study: Not stated
- Housing: Individual housing, Type III polycarbonate cages used during acclimatisation, Type II polycarbonate cages used during mating and gestation period
were used during the acclimatisation period.
- Diet (ad libitum): sniff® SM Autoclavable Complete Diet for Rats/Mice, Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water (ad libitum): Tap water (in water bottles) as for human consumption
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 24.8°C (target: 22 ± 3°C)
- Humidity (%): 30-56% (target: 30-70%)
- Air changes (per hr): 15-20/hr
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To: 14 November 2017 (first mating) to 14 December 2017 (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulated in the vehicle (distilled water) at appropriate concentrations according to the dose level and volume selected. Proven stability in the vehicle (within the concentration range used) for at least 21 days when stored at room temperature (20±5°C).

VEHICLE
- Concentration in vehicle: 13.3, 40 and 120 mg/mL (dose levels selected based on a dose range finding study)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. 8130917
- Supplier: Hungaro-Gal Kft.
- Expiry date: 4 March 2018
- Storage conditions: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration was performed at the test site using a validated LC-MS method. Representative samples were taken from the test item formulations two times during the study (during the first and last week of treatment). Samples were taken in duplicate (5 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Formulation samples were kept at room temperature until shipment. Samples (both sets) were shipped as soon as possible after collection for concentration measurement to the Principal Investigator. The validated analytical method (FPBSTUDY-172-VAL1-REPORT-01; CiToxLAB Hungary Ltd. study code: 17/133-901AN) was used for the determination of the test item content in the vehicle

All test item formulations were within the range of 94.8-104.1% of nominal concentration. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1 male : 1 female
- Length of cohabitation: Approximately 2-3 hours until at least 24 sperm positivefemales/group were attained
- Further matings after two unsuccessful attempts: Not stated
- Verification of same strain and source of both sexes: Yes, Hannover Wistar rats (CRLHan) sourced from Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually
Duration of treatment / exposure:
The control or test item dose formulations were administered to mated female rats daily by oral gavage on a 7 days/week basis, at approximately similar times, from gestation day 6 to 19 (GD 19)
Frequency of treatment:
Daily treatment from gestation day 6 to gestation day 19
Duration of test:
20 days (Gestation day 0, no treatment on gestation days 0 to 5, daily dosing from gestation day 6 to 19 (treatment period) , necropsy on day 20)
Doses / concentrationsopen allclose all
Dose / conc.:
66.7 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated females: Control (0)
26 mated females: Low dose - 66.7 mg/kg bw/day
26 mated females: Mid dose - 200 mg/kg bw/day
27 mated females: High dose - 600 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were selected based on the available information of the chemical nature and characteristics of the test item and available results from a non-pregnant dose range finding study and developmental toxicity screening test. Based on these results, 600 mg/kg bw/day was selected as high dose.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day). Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One general clinical observation was made on the first day (p.m.), on the afternoon on those days when detailed clinical observation was made in the morning. Additional general clinical observations were made for confirmatory reason on November 30, post dose, when an animal was found dead. Detailed clinical observation were made only once on necropsy days (a.m).

Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly. On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which was considered to confirm implantation).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision on gestation day 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20, or on the day of death (or when found dead).

FOOD CONSUMPTION
Food was measured with precision on gestation day 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including gestation day 0-6, 6-20 and 0-20

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20. Caesarean section and necropsy with macroscopic examination was performed on gestation day 20.
- Organs/ Tissues examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. Overies and uterus. Placentas were examined macroscopically.

Each foetus was subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined
Approximately half of each litter was subjected to visceral examination, the other half processed for skeletal examination.
For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened, and the thymus and great arteries examined.
For the foetuses subjected to skeletal examination, the abdominal region was opened,and the viscera and skin of foetuses removed, the cadaver was then fixed and stained.

All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: All per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Data were collected using the software PROVANTIS v.9 (maternal data, Caesarean section and necropsy data), or were recorded on the appropriate forms from the relevant SOPs of Citoxlab Hungary Ltd. (foetal evaluation data) then tabulated with PROVANTIS v.9 or Microsoft Office Excel 2010.
The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.
Indices:
In accordance with OECD 414 observed effects were evaluated to record all (raw) numbers used in calculating all percentages or indices
Historical control data:
Historical Control Data of Hannover Wistar Rats (CRL:WI(HAN)) was provided for:
Summary of Foetal Body Weight Data
Summary of External Foetal Examinations
Summary of Visceral Foetal Examinations
Summary of Skeletal Foetal Examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Noisy respiration was observed in 3/23 animals in the low, 12/24 animals in the mid, and 20/25 animals in the high dose group. Piloerection was present in 10/24 animals in the mid, and all animals of the high dose group. One animal (number 4505) in the high dose showed the clinical signs of noisy or slight to moderate laboured respiration, hunched back, slightly decreased activity, red discharge around the vulva and the nose. The increased presence of noisy respiration and piloerection indicates a test item related effect and is consistent with reported signs from other gavage studies with this test item. This surfactant substance, in very small amounts, can cause local effects if there is a slight reflux from the stomach, or if test item on the gavage tube contaminates the upper oesophageal area, with some substance contaminating the upper respiratory tract. All the other symptoms were attributed as secondary to the respiratory effects. The clinical signs were considered to be caused by a local effect, not a systemic toxicity effect. Animals in the Control group were symptom free.

Clear maternal toxicity was obseved at 600 mg/kg bw/day, with clinical signs associated with a local respiratory effect, signs of stomach irritation, and lower food intake and bodyweight than controls (Average body weight on GD 20 was Ca.13% below control). In the mid dose group noisy respiration and piloerection was observed in almost half of the animals, the group had a transient lower food intake. It was concluded that slight maternal toxicity was observed at the mid dose with no adverse effect on body weight. A few animals in the low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity for the group as a whole.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two high dose animals were found dead. Deaths were attributed to the strong surfactant properties of the test Item entering the upper respiratory tract as noted by the rapid onset of symptoms. It was considered likely that a small amount of test Item entered the upper respiratory tract. The deaths were not attributed to systemic toxicity of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant body weight reduction was observed at 600 mg/kg bw/day) from GD 8 to the end of the treatment. These changes resulted in a statistically significant reduction of body weight gain, corrected body weight gain and net body weight gain values during the treatment period (GD 6-20) and the entire study (GD 0-20) when compared to the control values. GD 20 mean body weight was 13% below the control mean value, confirming an adverse maternal effect of test item.

At 200 mg/kg bw/day there was a slight but statistically significantly lower body weight gain (p<0.05) for the treatment period (GD 6-20) but no relevant statistical difference for the entire study period or for the individual weighing days. The differences seen in the Mid dose were not considered to be a clearly adverse effect of treatment.

No test item related effect on body weight was observed at 66.7 mg/kg bw/day when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain or corrected body weight gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value for this group.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Description (incidence and severity):
Only pertinent behavioural changes were recorded in line with guidance requirements
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/day, 12/25 animals presented diffuse or multifocal thickening of the non-glandular mucosal region of the stomach. These effects are considered to be test item related, local adverse effects, probably an irritant effect in the stomach. No macroscopic findings were observed in the control or lower treatment groups.

In the two high dose animals found dead, non-collapsed lungs were the common finding. In one animal, dark red discoloration was observed in all lung lobes (multifocal), in the stomach (in the glandular mucosa), and in the thymus. These findings are considered to be agonal changes. In one animal, multifocal thickness of the non-glandular mucosal region of the stomach was observed, which is consistent with macroscopic findings in the evaluated high dose animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead foetus was present in the 200 mg/kg bw/day dose group. None were observed in either the control, low or high (66.7 or 600 mg/kg bw/day) dose groups.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Clear maternal toxicity was observed at 600 mg/kg bw/day with clinical signs associated with a local respiratory effect, signs of stomach irritation, and lower food intake and bodyweight than controls. At 200 mg/kg bw/day fewer clinical signs were observed in less animals with a transient lower food intake. It was concluded that there was a slight maternal toxicity at 200 mg/kg bw/day with no adverse effect on body weight. A few animals at 66.7 mg/kg bw/day had respiratory effects with no consequences and was not considered to reflect a significant maternal toxicity.

The total number of retarded foetuses was statistical significant at 600 mg/kg bw/day, and was associated with maternal toxicity. A small increased incidence of retarded ossification at 600 mg/kg bw/day was also considered to be compatible with the observed maternal toxicity.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
66.7 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly increase of unossified or incomplete ossification in the 600 mg/kg bw/day litter correlates with the reduced foetal body weight. This is compatible with maternal toxicity with reduced body weight in the high dose.
Reduction in number of live offspring:
not examined
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:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Teratogenicity
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on the lack of any developmental effects in any treatment group.
Key result
Dose descriptor:
NOAEL
Remarks:
Embryotoxicity
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on the lack of any test-item related intrauterine effect in any treatment group.
Key result
Dose descriptor:
NOAEL
Remarks:
Foetotoxicity
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on the lack of any adverse developmental effects in any treatment group.

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Table 1. Summary of pregnancy data




















































Parameters


 



Dose (mg/kg bw/day)


 



 



0



66.7



200



600



Number of mated females



24



26



26



27



Pre-terminal death or euthanasia



0



0



0



2



Number of non-pregnant females



1



3



2



0



Number of females with ≤ 5 implantation sites



0



0



1



0



Number of evaluated females on GD20 (Caesarean section)



23



23



24



25



Table 2. Summary of the intrauterine evaluation

















































































































































Parameters



Dose (mg/kg bw/day)



stats.



0



66.7



200



600



Number of evaluated dams



23



23



24



25



NS



Mean number of corpora lutea



12.04



11.78



11.67



11.24



NS



Preimplantation loss, mean



1.04



1.70



1.75



1.24



NS



Preimplantation loss (%), mean



8.79



14.09



15.25



10.60



NS



Mean number of implantations



11.0



10.09



9.92



10.0



NS



Early embryonic loss, mean



0.17



0.26



0.17



0.20



NS



Early embryonic loss (%), mean



1.48



2.25



2.25



2.15



NS



Late embryonic loss, mean



0.22



0.00



0.13



0.08



NS



Late embryonic loss (%), mean



1.80



0.00



1.50



0.73



NS



Dead foetuses, mean



0.00



0.00



0.04



0.00



NS



Dead foetuses (%), mean



0.00



0.00



0.38



0.00



NS



Post-implantation loss, mean



0.00



0.26



0.33



0.28



NS



Post-implantation loss (%), mean



3.28



2.55



4.14



2.89



NS



Total intrauterine mortality, mean



1.43



1.96



2.08



1.52



NS



Total intrauterine mortality (%), mean



11.97



16.34



18.36



13.35



NS



Viable foetuses, mean



10.61



9.83



9.58



9.72



NS



Notes: Most important parameters are shown in bold.
NS: Statistically not significant when compared to the vehicle control.


Table 3. Examination of viable foetuses









































































































Parameters



Dose (mg/kg bw/day)



Stats.



0



66.7



200



600



Number of examined litters



23



23



24



25



NS



Viable foetuses, mean



10.61



9.83



9.58



9.72



NS



Male foetuses, mean



5.13



4.91



4.71



5.24



NS



Female foetuses, mean



5.48



4.91



4.88



4.48



NS



Total number of foetuses



244



226



230



243



NS



Total number of male foetuses



118



113



113



131



NS



Total number of female foetuses



126



113



117



112



NS



Sex distribution (% of males / females)



49/51



50/50



50/50



53/47



NS



Mean foetal weight / litter (g)



3.43



3.49



3.51



3.45



NS



Number of foetuses with retarded body weight



10



17



12



33**



NS



Number of affected litters (with runts)



8



8



6



11



NS



Notes: Most important parameters are shown in bold.
NS: Statistically not significant when compared to the vehicle control.
CH2: Chi Square test; **= p<0.01


Table 4. Summary table of the external abnormalities









































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



670



Total number of examined foetuses



244



226



230



243



6889



Total number of intact (normal) foetuses



244



226



230



243



--



HC: historical control


Table 5. Summary table of the visceral abnormalities

























































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



670



Total number of examined foetuses



121



114



115



121



3450



Total number of intact (normal) foetuses



116



108



109



121*



--



Total number of foetuses / litters


with malformation



1/1



0/0



2/2



0/0



--



Total number of foetuses / litters


with variation



4/4



6/6



4/3



0/0*



--



Notes: Numbers represent the number of abnormalities / number of affected litters.
HC: historical control
*= p<0.05; Chi Square test


Table 6. Details of the visceral abnormalities




























































































































































































































































































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



670



Total number of examined foetuses



121



114



115



121



3450



Visceral malformations



Kidney, misshapen (Small)



litter incidence



n



1



0



0



0



1



%



4.3



0.0



0.0



0.0



0.149



Foetal incidence



n



1



0



0



0



1



%



0.826



0.000



0.000



0.000



0.029



Abnormal Lung Lobes



litter incidence



n



0



0



1



0



1



%



0.0



0.0



4.2



0.0



0.149



Foetal incidence



n



0



0



1



0



1



%



0.000



0.000



0.870



0.000



0.029



Testis, malpositioned



litter incidence



n



0



0



1



0



--



%



0.0



0.0



4.2



0.0



--



Foetal incidence



n



0



0



1



0



--



%



0.000



0.000



0.870



0.000



--



Visceral variations



Brachiocephalic trunk, short



litter incidence



n



2



1



0



0



45



%



8.7



4.3



0.0



0.0



6.7



Foetal incidence



n



2



1



0



0



53



%



1.653



0.877



0.000



0.000



1.536



Renal papilla, absent



litter incidence



n



0



0



1



0



--



%



0.0



0.0



4.2



0.0



--



Foetal incidence



n



0



0



1



0



--



%



0.000



0.000



0.870



0.000



--



Renal pelvis, dilated



litter incidence



n



0



0



1



0



--



%



0.0



0.0



4.2



0.0



--



Foetal incidence



n



0



0



1



0



--



%



0.000



0.000



0.870



0.000



--



Carotid artery, malpositioned



litter incidence



n



0



0



1



0



1



%



0.0



0.0



4.2



0.0



0.149



Foetal incidence



n



0



0



1



0



1



%



0.000



0.000



0.870



0.000



0.029



Notes: Numbers represent the number (n) or ratio (%) of abnormalities.
HC: historical control (data provided where considered useful)
No statistically significant differences were noted compared to the control group.



















































































































































































































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



670



Total number of examined foetuses



121



114



115



121



3450



Visceral variations



Kidney, malpositioned



litter incidence



n



0



1



0



0



1



%



0.0



4.3



0.0



0.0



0.149



Foetal incidence



n



0



1



0



0



1



%



0.000



0.877



0.000



0.000



0.029



Adrenal gland, malpositioned



litter incidence



n



0



1



0



0



1



%



0.0



4.3



0.0



0.0



0.149



Foetal incidence



n



0



1



0



0



1



%



0.000



0.877



0.000



0.000



0.029



Ureter convoluted



litter incidence



n



0



0



1



0



7



%



0.000



0.000



4.167



0.000



1.045



Foetal incidence



n



0



0



1



0



7



%



0.000



0.000



0.870



0.000



0.203



Thymic cord



litter incidence



n



0



3



1



0



69



%



0.000



13.043



4.167



0.000



10.299



Foetal incidence



n



0



3



3



0



82



%



0.000



2.632



1.739



0.000



2.377



Renal papilla, small



litter incidence



n



2



1



0



0



20



%



8.7



4.3



0.0



0.0



2.985



Foetal incidence



n



2



1



0



0



22



%



1.653



0.877



0.000



0.000



0.638



Notes: Numbers represent the number (n) or ratio (%) of abnormalities.
HC: historical control (data provided where considered useful)
No statistically significant differences were noted compared to the control group.


Table 7. Summary table of the skeletal abnormalities

























































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



669



Total number of examined foetuses



123



112



115



122



3435



Total number of intact (normal) foetuses



111



93



98



90 CH**



--



Total number of foetuses / litters


with malformation



2/2



0/0



1/1



1/1



--



Total number of foetuses / litters


with variation



10/7



19CH*/11



16/9



31CH/15



--



Notes: Numbers represent the number of abnormalities / number of affected litters.
HC: historical control
CH: Chi2 test; * = p < 0.05 ** = p < 0.01


Table 8. Details of the skeletal abnormalities













































































































































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



669



Total number of examined foetuses



123



112



115



122



3435



Skeletal malformations



Malformed Vertebrae (Split,


Fused)



litter incidence



n



0



0



1



0



3



%



0.0



0.0



4.2



0.0



0.448



Foetal incidence



n



0



0



1



0



3



%



0.000



0.000



0.870



0.000



0.087



Rib, Fused



litter incidence



n



0



0



0



1



--



%



0.000



0.000



0.000



4.000



--



Foetal incidence



n



0



0



0



1



--



%



0.000



0.000



0.000



0.820



--



Transverse process fused;


Pelvic girdle, malpositioned#



litter incidence



n



2



0



0



0



6



%



8.696



0.000



0.000



0.000



0.897



Foetal incidence



n



2



0



0



0



6



%



1.626



0.000



0.000



0.000



0.175



Notes: Numbers represent the number (n) or ratio (%) of abnormalities.
HC: historical control (data provided where considered useful)
No statistically significant differences were noted compared to the control group.
#: Historical control database contains Transverse processes, fused


Table 8. Details of the skeletal abnormalities (continued)


































































































































































































































































































































































































Parameters



Dose (mg/kg bw/day)



HC data



0



66.7



200



600



Total number of examined litters



23



23



24



25



669



Total number of examined foetuses



123



112



115



122



3435



Skeletal malformations



Skull: 3 or More Bones,


Incomplete Ossification



litter incidence



n



1



3



1



4



123



%



4.3



13.0



4.2



16.0



18.358



Foetal incidence



n



1



5



1



5



153



%



0.813



4.464



0.870



4.098



4.454



Skull: Hyoid, body, unossified



litter incidence



n



0



1



0



1



--



%



0.000



4.348



0.000



4.000



--



Foetal incidence



n



0



1



0



3



--



%



0.000



0.893



0.000



2.459



--



Sternum: Ossified Sternebra (4


or less) #



litter incidence



n



5



5



5



9



269



%



21.7



21.7



20.8



36.0



40.2



Foetal incidence



n



5



7



10



18CH**



385



%



4.065



6.250



8.696



14.554



11.208



Ribs: Wavy



litter incidence



n



2



6



2



7



198



%



8.7



26.1



8.3



28.0



29.596



Foetal incidence



n



3



10CH*



3



11CH*



311



%



2.439



8.929



2.609



9.016



5.054



Ribs: Rib or Cartilage


Interrupted



litter incidence



n



1



0



0



2



4



%



4.348



0.000



0.000



8.000



0.598



Foetal incidence



n



1



0



0



2



6



%



0.813



0.000



0.000



1.639



0.175



Vertebrae: 2 or More Dumbbell


or Asymmetric Ossification



litter incidence



n



0



1



1



1



85



%



0.000



4.348



4.167



4.000



12.706



Foetal incidence



n



0



1



2



1



92



%



0.000



0.893



1.739



0.820



2.697



Vertebrae: Sacral Unossified



litter incidence



n



0



0



1



0



--



%



0.000



0.000



4.167



0.000



--



Foetal incidence



n



0



0



1



0



--



%



0.000



0.000



0.870



0.000



--



Bipartite Ossification



litter incidence



n



0



0



2



0



27



%



0.000



0.000



8.333



0.000



4.036



Foetal incidence



n



0



0



2



0



27



%



0.000



0.000



1.739



0.000



0.786



Pubis unossified



litter incidence



n



0



0



1



1



6



%



0.000



0.000



4.167



4.000



0.897



Foetal incidence



n



0



0



1



1



6



%



0.000



0.000



0.870



0.820



0.175



Limbs (C/T): Tarsal ≤3



litter incidence



n



1



1



1



1



34



%



4.348



4.348



4.167



4.000



5.082



Foetal incidence



n



1



1



2



1



49



%



0.813



0.893



1.739



0.820



1.426



Notes: Numbers represent the number (n) or ratio (%) of abnormalities.
HC: historical control (data provided where considered useful)
#: Ossified sternebra (3 or less) is recorded in the historical control database
CH: Chi2 test; * = p < 0.05; ** = p < 0.01


Table 9. Summary of Bodyweight Data















































































Female



 



Day(s) Relative to Start Date



0



3



6



8



10



12



14



16



18



20



0
mg/kg bw/day



Mean
SD



221.6 R
10.8



230.3 R
15.7



242.6 R
12.6



248.4
14.2



254.6
14.5



265.7
14.7



273.6
15.8



286.0
17.4



308.8
20.7



334.6
25.1



66.7 mg/kg bw/day



Mean
SD



221.3
8.0



232.0
8.6



241.8
9.2



245.9
11.5



252.1
12.6



259.8
14.6



269.4
14.6



281.5
14.3



303.3
15.5



328.5
18.5



200 mg/kg bw/day



Mean
SD



222.8
9.0



232.7
9.6



244.2
12.2



246.4
12.9



251.3
14.1



257.8
14.7



266.1
15.7



278.8
15.3



298.1
19.3



321.3
21.7



600 mg/kg bw/day



Mean
SD



222.1
12.8



231.1
14.4



240.6
14.1



233.8uu
16.4



235.7dd
18.1



242.8dd
17.8



250.1dd
18.5



260.9dd
17.9



272.1dd
24.5



290.8dd
26.2



Statistical Test: Citoxlab DT Transformation: Automatic
Comments and Markers


Measurement         Group      Sex           Day          Marker    Comment


Bodyweight.            1CG         Female    0              R              Automatic Transformation: Rank


Bodyweight.            1CG         Female   3              R              Automatic Transformation: Rank


Bodyweight.            1CG         Female    6              R              Automatic Transformation: Rank


Bodyweight.            1CG         Female    8              R,kk         Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01


Bodyweight.            1CG         Female    10            I,aa          Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01


Bodyweight.            1CG        Female    12            I,aa          Automatic Transformation: Identity (No   Transformation), (All Groups) Test: Analysis of Variance p < 0.01


 Bodyweight.           1CG        Female    14            I,aa          Automatic Transformation: Identity (No  Transformation), (All Groups) Test: Analysis of Variance p < 0.01


Bodyweight.            4HDG     Female    8              uu            Test: Dunn 2 Sided p < 0.01


Bodyweight.            4HDG      Female    10            dd            Test: Dunnett 2 Sided p < 0.01


Bodyweight.            4HDG      Female    12            dd            Test: Dunnett 2 Sided p < 0.01


Bodyweight.             4HDG      Female    14            dd            Test: Dunnett 2 Sided p < 0.01


Bodyweight.             1CG        Female    16            I,aa          Automatic Transformation: Identity (No  Transformation), (All Groups) Test: Analysis of Variance p < 0.01


Bodyweight.             1CG        Female    18            I,aa          Automatic Transformation: Identity (No  Transformation), (All Groups) Test: Analysis of Variance p < 0.01


Bodyweight.             1CG        Female   20            I,aa          Automatic Transformation: Identity (No  Transformation), (All Groups) Test: Analysis of Variance p < 0.01


Bodyweight.            4HDG      Female    16            dd            Test: Dunnett 2 Sided p < 0.01


Bodyweight.             4HDG      Female    18            dd            Test: Dunnett 2 Sided p < 0.01


Bodyweight.             4HDG      Female    20            dd            Test: Dunnett 2 Sided p < 0.01


Table 10. Selected body weight parameters









































































Parameters



Dose (mg/kg bw/day)



Stats.



0



66.7



200



600



Number of evaluated dams



23



23



24



25



-



Body weight on GD20 (g)



334.6



328.5



321.3



290.8**



D



Body weight gain GD6-20 (g)



92.0



86.7



77.1*



50.2**



D



Body weight gain GD0-20 (g)



113.0



107.1



98.5



68.7**



U



Corrected body weight on GD20 (g)



271.2



269.0



262.4



237.0**



U



Corrected body weight gain GD0-20 (g)



49.6



47.6



39.6



14.9**



U



Net body weight gain GD6-20 (g)



28.6



27.1



18.2*



-3.6**



U



Notes: Body weight data were rounded to one decimal place. Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.
*= p<0.05; **= p<0.01; D: Dunnett two sided test.; U: Dunn two sided test


 

Applicant's summary and conclusion

Conclusions:
C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days 6 to 19 at 600 mg/kg bw/day induced maternal toxicity, with clinical signs associated with a local respiratory effect, reduced food intake, reduced body weight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. All maternal changes at 600 mg/kg bw/day were compatible with being related to local irritancy of the test item, with no evidence of any direct systemic toxicity. The 200 mg/kg bw/day had lesser signs with no adverse effect on body weight, there was evidence of slight maternal toxicity. A few animals at 66.7 mg/kg bw/day had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

At 600 mg/kg bw/day, the incidence of runts was higher, and the incidence of retarded ossification were statistically higher compared to the control group; these observations were attributed to maternal toxicity. Theese effects were not evident at lower dose groups. There were no malformations or developmental effects attributed to test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived:
NOAELmaternal toxicity: 66.7 mg/kg bw/day, based on significant maternal effects at 600 mg/kg bw/day and clinical signs of local effects at 200 mg/kg bw/day. The maternal effects were all attributed to local effects, there was no evidence of direct systemic toxicity in any group.
NOAELembryotoxicity: 600 mg/kg bw/day, based on the lack of any test-item related intrauterine effect in any treatment group.
NOAELfoetotoxicity: 600 mg/kg bw/day, based on the lack of any adverse developmental effects in any treatment group.
NOAELteratogenecity: 600 mg/kg bw/day, based on the lack of any developmental effects in any treatment group.
Executive summary:

A developmental toxicity study was performed to assess the effects of the test item C8-18 alkylamidopropyl hydroxysultaine on the embryonic and foetal development (inclusive of organogenesis period) of Hannover Wistar rats in their first pregnancy. The study was perfomed in accordence with OECD 414. The dams (one control and three test item treated groups) were treated daily by oral (gavage) at a dose volume of 5 mL/kg bw from gestation day 6 up to and including gestation day 19. Control dams were treated with the vehicle (distilled water). Caesarean sections, necropsy of dams and examination of uterine contents were performed on gestation day 20. Doses were selected based on available information of the test item chemical nature and characteristics and available results froma a dose range finding study and developmental toxicity screening test. Doses of 66.7, 200 and 600 mg/kg bw/day were selected based overt toxicity at 800 mg/kg bw/day in previous testing. Test item formulations were analysed for concentration twice during the treatment period using LC-MS. Simultaneously, vehicle control formulations were analysed. Parameters monitored included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams was 23 in both the control and 66.7 mg/kg bw/day groups, 24 at 200 mg/kg bw/day and 25 at 600 mg/kg bw/day) dose groups, respectively. No histopathology evaluation was performed.


 


Based on the above the following no-observed-adverse-effect (NOAEL) levels were derived:


NOAELmaternal toxicity: 66.7 mg/kg bw/day, based on significant maternal effects at 600 mg/kg bw/day and clinical signs of local effects at 200 mg/kg bw/day. The maternal effects were all attributed to local effects, there was no evidence of direct systemic toxicity in any group.


NOAELembryotoxicity: 600 mg/kg bw/day, based on the lack of any test-item related intrauterine effect in any treatment group.


NOAELfoetotoxicity: 600 mg/kg bw/day, based on the lack of any adverse developmental effects in any treatment group.


NOAELteratogenecity: 600 mg/kg bw/day, based on the lack of any developmental effects in any treatment group.