Registration Dossier

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:
From 14 November 2017 to 07 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
UP6C10X10
- Expiration date of the lot/batch:
11 March 2018
- Manufacture date: 11 March 2016
- Purity (sultaine content): 35.4%

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 test conditions:

- Solubility and stability of the test substance in the solvent/vehicle:
During the method validation, 21-day stability of the Test Item in the selected vehicle was obtained. The Test Item proved to be stable on 20±5°C in the concentration range of 1.6-220
mg/L (when adjusted for purity).

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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: at least 12 weeks old at mating.
- Weight at study initiation: 200-260 g at Gestation Day 0
- Fasting period before study: no
- Housing: Standard laboratory conditions; individual housing
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany), ad libitum
- Water (e.g. ad libitum): The animals were provided tap water (in water bottles), ad libitum
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 14 November 2017 To: 14 December 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (distilled water) at the following concentrations: 13.3, 40 and 120 mg/mL for the Low, Mid, and High dose groupe, respectively. Formulations were prepared prior to administration to the animals at the appropriate frequency (not more than 10 days before use). A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
As the test item is a surfactant with foaming/irritation potential, in order to avoid as much as possible aspiration into the respiratory tract susceptible to cause unspecific toxicity, attention was paid, to very carefully clean the outside of the gavage tube each time the syringe was filled, and to ensure that the cannula used was the correct length to ensure the test item is dispensed in the stomach and not into the oesophagus.

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 (Liquid Chromatography – Mass Spectrometry) method. Representative samples were taken from the test item formulations two times during the study (during the first week and just before the 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.

Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male : 1 female
- Length of cohabitation: approximately 2-3 hours
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:
from gestation day 6 (GD 6) to gestation day 19 (GD 19)
Frequency of treatment:
Daily
Duration of test:
21 days
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:
number of mated females/group:
Control: n= 24
Low dose: n = 26
Mid dose: n = 26
High dose: n = 27
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected by the Study Director and the Sponsor based on available information of the chemical nature and characteristics of the test item and available results of a dose range finding study (DRF) [9], and a developmental toxicity screening test [10]. In the DRF study no signs of systemic toxicity were observed up to and including the 600 mg/kg bw/day dose level. However, in the developmental toxicity screening test, severe maternal toxicity (reduced body weight and food consumption) was observed at 800 mg/kg bw/day, and a similar, less expressed effect was seen in the 600 mg/kg bw/day dose group. Animal deaths occurred in the preliminary teratology study, they are considered to be due to treatment, and not related to the systemic toxicity of the test item; there was no observable effect on the pregnancy of the animals. In a previous DRF study (information supplied by the Sponsor) there was mortality at 1000 mg/kg bw/day so it was clear that the High dose should not be too close to this level.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Only one general clinical observation was made on the first day (in the afternoon), 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 one high dose animal was found dead. Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20, or on the day of death (for found dead animals).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

OTHER:
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 is considered to confirm implantation).
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:
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.
In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter- group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Indices:
Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D. - Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss, as well as foetal death
- Preimplantation loss: %, group mean
-Postimplantation loss: %, group mean

Foetal Data:
- Sex distribution: %, group mean
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities*/litter: %, group mean
- Visceral abnormalities*/litter: %, group mean
- Skeletal abnormalities*/litter: %, group mean
Historical control data:
not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Evaluated animals:
Animals in the Control group were symptom free.
Noisy respiration was observed in 3 out of 23 animals in the Low, 12 out of 24 animals in the Mid, and 20 out of 25 animals in the High dose group. Piloerection was present in 10 out of 24 animals in the Mid, and in 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 similar to 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.

Non-pregnant animals:
Noisy respiration was observed in 1 out of 3 Low dose, and 1 out of 2 Mid dose animals. Piloerection was observed in 1 out of 2 Mid dose animals. These clinical signs were considered to be related to the test item as described above.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two High dose animals were found dead in the study. One Animal showed hunched back, piloerection from the day before death, and piloerection, hunched back, moderately noisy, gasping respiration on the morning of the death. The other animal showed abnormal skin colour (white) on the limbs and pinna, as well as extremely decreased activity, prostration, and tonic convulsion after treatment. Considering the strong surfactant properties of the Test Item, and the rapid progress of the symptoms, it is 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 in the High dose group (600 mg/kg bw/day) from GD 8 up 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. The GD 20 mean body weight was 13% below the control mean value, confirming an adverse maternal effect of test item.
In the Mid dose group 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 in the Low dose group (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. (see details in Table 1)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption in the High dose group (600 mg/kg bw/day) was statistically lower during the whole dosing and treatment period (GD 6-20 and GD 0- 20; 30% and 22.4% below controls, respectively). In the Mid dose group, food consumption was statistically significantly lower between GD 12 and GD 14, and when calculated for the whole treatment period (GD 6-20) but was a relatively small (< 10%) difference). No differences in the food consumption values were found in the Low (66.7 mg/kg bw/day) dose group when compared to the control group. These lower food intake values in the High and Mid dose groups were considered to be test item related effects although the difference in the Mid dose was not enough to be considered a clearly adverse effect of the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed for any of the test item treated groups in the gravid uterine weight parameter when compared to the control value. The gravid uterine weights in the dose groups were in line with the body weights for these groups (see details in table 1).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Evaluated animals:
No macroscopic findings were observed in the Control, Low and Mid dose groups. Diffuse or multifocal thickening of the non-glandular mucosal region of the stomach was observed in 12 out of 25 High dose (600 mg/kg bw/day) animals. These findings are considered to be test item related, local adverse effects, probably an irritant effect in the stomach.

Non-pregnant animals:
No macroscopic findings were observed for non-pregnant animals in any dose groups.

Found dead animals:
Two High dose animals were found dead during the study. Non-collapsed lungs were the common finding in these animals. 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 in correlation with the macroscopic findings in the evaluated High dose animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.
The early and the late embryonic loss values of the test item treated groups were comparable with control. One dead foetus was present in one litter of the Mid dose group, and no dead foetus was found in any of the other (Control, Low, and High) dose groups. There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups (see details in table 2).
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 one litter of the Mid dose group, and no dead foetus was found in any of the other (Control, Low, and High) dose groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Hundred and three females (24 for the Control, 26 for the Low and Mid, 27 for the High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 23 in the Control group, 23 in the Low dose group (66.7 mg/kg bw/day), 24 in the Mid dose group (200 mg/kg bw/day) and 25 in the High dose group (600 mg/kg bw/day). Summary of pregnancy data is seen in Table 3.
Other effects:
no effects observed
Description (incidence and severity):
Corpora lutea: The mean number of corpora lutea was comparable with the control in all test item treated groups.
Evaluation of placentas: No abnormalities were observed on the placentas of any animals in the Control, Low (66.7 mg/kg bw/day), Mid (200 mg/kg bw/day) or High (600 mg/kg bw/day) dose groups.

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:
mortality
body weight and weight gain
food consumption and compound intake
other: The maternal effects were all attributed to local effects due to the surfactants properties of the test substance.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean foetal weight per litter in the test item did not differ significantly from the control mean value (see details in Table 4).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of viable foetuses was comparable with the control mean in all test item treated groups (see details in Table 4).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups (see details in Table 4).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no external variations or malformations in this study (see details in Table 5).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The skeletal findings in the Low and Mid dose groups correspond with the current historical control (HC) or the concurrent study control data, or were considered to be incidental findings without dose response. The increased skeletal variations in the High dose group are considered to be a secondary effect of the low food intake and reduced body weight of the dams (see details in Tables 6 and 7).
Malformations such as Malformed Vertebrae (Split, Fused) in the Mid (1/115 foetus) dose group; Fused Rib in the High (1/122 foetus) dose group and Lumbar-Sacral Transverse process, fused, Pelvic girdle, malpositioned in the Control (2/123 foetuses in 2 litters) dose group were observed.

These findings correspond with the current historical control or occur only in study Control group data and have isolated occurrence that was considered incidental, ascribed to individual variability and not related to the test item.

In the case of most skeletal variations the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

The incidence of statistically significantly higher occurrence of unossified or incomplete ossification (sternum, ribs vertebrae, pubis) in the High dose group litter is in correlation with the reduced foetal body weight. This is compatible with maternal toxicity with reduced body weight in the High dose. The only other statistically different finding in the High dose was Wavy Ribs, which had an incidence fully in line with Historical incidence and hence is not considered to be an effect of test item.

When maternal toxicity affects food intake, causing a body weight reduction in the dams, the otherwise normal development of the foetuses can be delayed – some foetuses may grow more slowly; hence in this study the significantly higher number of retarded foetuses in the High dose group, see 9.6.3, often with delayed ossification. It must be noted that this type of secondary toxicity effect does not result in increased number of malformation of the foetuses [12-14].

In conclusion, all of the foetal findings for the Low and Mid dose groups, in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment. In the High dose group, small increases in the incidence of retarded ossification were attributed as secondary to the maternal body weight and not a direct foetal effect of the test item. Based on these results the test item did not affect adversely the intrauterine development.
Visceral malformations:
no effects observed
Description (incidence and severity):
All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence (in the case of convoluted ureter, which was present only in the Mid dose group, with a greater occurrence than in the historical control), therefore considered as incidental findings (see details in Tables 8 and 9).
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of retarded foetuses (runts) were significantly higher in the High dose group. The number of affected litters was similar in all test item treated groups as in the Control group, although the statistical difference correlates with an increased incidence of retarded ossification in this group, attributed to low maternal body weight (see details in Table 4).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects reported
Remarks on result:
other: highest dose tested
Key result
Dose descriptor:
NOAEC
Remarks:
foetotoxicity
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects reported
Remarks on result:
other:
Remarks:
highest dose tested
Key result
Dose descriptor:
NOAEC
Remarks:
teratogenecity
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects reported
Remarks on result:
other:
Remarks:
highest dose tested

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of  maternal body weight parameters

Parameters

Dose (mg/kg bw/day)

 

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

Gravid Uterus (g)

63.4

59.5

58.9

53.8

NS

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

Table 2: Summary of the intrauterine evaluation

 

Parameters

Dose (mg/kg bw/day)

 

0

66.7

200

600

 

Number of evaluated dams

23

23

24

25

 

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

11.00

10.09

9.92

10.00

NS

Early embryonic loss, mean

0.17

0.26

0.17

0.20

NS

Early embryonic loss (%), mean

1.48

2.55

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

Postimplantation loss, mean

0.39

0.26

0.33

0.28

NS

Postimplantation 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: 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 4: Examination of viable foetuses

 

 

Parameters

Dose (mg/kg bw/day)

 

0

66.7

200

600

 

Number of examined litters

23

23

24

25

 

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**

CH2

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 5: Summary table of the external abnormalities

 

 

Parameter

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

--

Note: HC: historical control

Table 6: Summary table of the skeletal abnormalities

 

 

Parameter

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

90CH**

--

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: Chi2test; * = p < 0.05 ** = p < 0.01

 

Table 7: Details of the skeletal abnormalities

 

 

Parameter

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

Skeletalmalformations

 

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 7: Details of the skeletal abnormalities (continued)

 

Parameter

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

Skeletalvariations

 

Skull: 3 or More Bones, Incomplete Ossification

Litter incidence

n

1

3

1

4

123

%

4.3

13.0

4.2

16.0

18.385

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

14.754

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

9.054

 

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

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: Chi2test; * = p < 0.05; ** = p < 0.01


Table 7: Details of the skeletal abnormalities (continued)

 

Parameter

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

Skeletalvariations

 

 

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)

 

Table 8: Summary table of the visceral abnormalities

 

Parameter

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 9: Details of the visceral abnormalities

 

Parameter

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

Litterincidence

n

2

1

0

0

45

%

8.7

4.3

0.0

0.0

6.7

Foetalincidence

n

2

1

0

0

53

%

1.653

0.877

0.000

0.000

1.536

 

 

Renal papilla, absent

Litterincidence

n

0

0

1

0

--

%

0.0

0.0

4.2

0.0

--

Foetalincidence

n

0

0

1

0

--

%

0.000

0.000

0.870

0.000

--

 

 

Renal pelvis, dilated

Litterincidence

n

0

0

1

0

--

%

0.0

0.0

4.2

0.0

--

Foetalincidence

n

0

0

1

0

--

%

0.000

0.000

0.870

0.000

--

 

 

Carotid artery, malpositioned

Litterincidence

n

0

0

1

0

1

%

0.0

0.0

4.2

0.0

0.149

Foetalincidence

n

0

0

1

0

1

%

0.000

0.000

0.870

0.000

0.029

Table 9: Details of the visceral abnormalities (Continued)

 

Parameter

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

Litterincidence

n

0

1

0

0

1

%

0.0

4.3

0.0

0.0

0.149

Foetalincidence

n

0

1

0

0

1

%

0.000

0.877

0.000

0.000

0.029

 

 

Adrenal gland, malpositioned

Litterincidence

n

0

1

0

0

1

%

0.0

4.3

0.0

0.0

0.149

Foetalincidence

n

0

1

0

0

1

%

0.000

0.877

0.000

0.000

0.029

 

 

Ureter convoluted

Litterincidence

n

0

0

1

0

7

%

0.000

0.000

4.167

0.000

1.045

Foetalincidence

n

0

0

1

0

7

%

0.000

0.000

0.870

0.000

0.203

 

 

Thymic cord

Litterincidence

n

0

3

1

0

69

%

0.000

13.043

4.167

0.000

10.299

Foetalincidence

n

0

3

2

0

82

%

0.000

2.632

1.739

0.000

2.377

 

 

Renal papilla, small

Litterincidence

n

2

1

0

0

20

%

8.7

4.3

0.0

0.0

2.985

Foetalincidence

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.

Applicant's summary and conclusion

Conclusions:
In conclusion, C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 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 the maternal changes in the High dose group were compatible with being related to the local irritancy of the test item, there was no evidence of direct systemic toxicity.

The Mid dose of 200 mg/kg bw/day had lesser signs with no adverse effect on body weight, but there was evidence of a slight maternal toxicity. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

In the High dose group, 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. There were no such effects in the Mid or Low 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; bBased on the lack of any developmental effects in any treatment group.
Executive summary:

This developmental toxicity study was performed according to OECD Test Guideline 414 to assess the effects of the test item C8-18 alkylamidopropyl hydroxysultaine on the embryonic and foetal development (including the organogenesis period) of HannoverWistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (distilled water) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The doses were selected based on available information of the chemical nature and characteristics of the test item and available results of a dose range finding study,and a developmental toxicity screening test. Based on this information, the doses of 66.7, 200 and 600 mg/kg bw/day were deemed suitable for the purpose of the study.

Test item formulations were analysed for concentration two times during the treatment period using a validated LC-MS method. Simultaneously, vehicle control formulations were analysed for concentration.

Parameters monitored during the study included mortality and clinical observations, bodyweight,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 the Control, 23 in the Low (66.7 mg/kg bw/day), 24 in the Mid (200 mg/kg bw/day) and 25 in the High  (600 mg/kg bw/day) dose groups, respectively.

 

Results:

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.

Two animals were found dead in the High dose groups, with respiratory symptoms suggesting that their death was caused by the strong surfactant activity (a local effect, not attributed to systemic toxicity) of the Test Item.

Apart from incidental findings, noisy respiration and piloerection were present in almost half of the animals in the Mid dose group (in 12 and 10 animals from the 24 evaluated dams,respectively) and dominantly present (in 20 and 25 animals from the 25 evaluated dams, respectively) in the High dose group. A few Low dose animals were similarly affected. These adverse effects were considered related to a local irritant type effect of the test item. There were no signs of systemic toxicity.

Statistically significant lower body weight was observed in the High dose group from GD 8 up to the end of the treatment, with significantly reduced body weight gain, corrected body weight gain and net body weight gain values. These changes were considered to be test item related adverse effects, probably secondary to the local respiratory and stomach effects of the test item as observed in the clinical signs and at necropsy.

There were no test item related adverse effect on maternal body weight / body weight gain and maternal corrected body weight / net body weight gain in the Low and Mid dose groups (66.7 and 200 mg/kg bw/day, respectively).

A relatively large, statistically significant reduction of the food consumption was observed in the High dose animals after the start of the treatment, this reduction contributed to the lower body weight in this group. A minor difference in the Mid dose group (<10%) may have been related to treatment but had no significant consequences.

At necropsy of the dams, no remarkable external observations related to test item treatment were recorded for any pregnant animals during necropsy. Diffuse or multifocal thickening of the non-glandular mucosal region of the stomach was observed in the High dose animals, indicating a test item related adverse local effect. No abnormalities were observed on the placentas in any examined groups.

The maternal findings showed a clear maternal toxicity at the High dose, 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 approximately 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 is concluded that there was a slight maternal toxicity at the Mid dose,but there was 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 awhole.

There were no toxicologically significant differences,or test item related-changes in the examined parameters of the intrauterine development up to and including 600 mg/kg bw/day.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

The total number of retarded foetuses was statistical significantly higher in the High dose animals, associated with maternal toxicity.

There were no test item related effects on external or visceral development of foetuses inthestudy.AsmallincreasedincidenceofretardedossificationintheHighdosegroup was considered to be compatible with the maternal toxicity in this group.There were no other differences observed between the groups at foetal examination.

Foetal malformations observed in the study were all considered to be incidental, they showed no dose dependency, thus not regarded as a test item related effect.

In conclusion, C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600mg/kg bw/day induced maternal toxicity, with clinical signs associated with a local respiratory effect, reduced food intake, reduced bodyweight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. All the maternal changes in the High dose group were compatible with being related to the local irritancy of the test item, there was no evidence of direct systemic toxicity.

 

The Mid dose of 200mg/kg bw/day had lesser signs with no adverse effect on body weight, but there was evidence of a slight maternal toxicity. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

 

In the High dose group, 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. There were no such effects in the Mid or Low 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.