Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 8 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 414 with any major deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

acclimation period followed for 4 or 5 days instead of 5 days (due to receipt of animals on D1 or D2 post-coitum)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

acclimation period followed for 4 or 5 days instead of 5 days (due to receipt of animals on D1 or D2 post-coitum)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

EC Number:

939-579-8

Molecular formula:

Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na

IUPAC Name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Details on test material:

- Name of test material (as cited in study report): CHIMEXANE HB
- Physical state: Yellowish thick gel
- Analytical purity: 53.8 %
- Correction factor: 1.86
- Lot/batch No.: 0137998
- Date of receipt: 21 April 2008
- Expiration date of the lot/batch: April 2009
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: 225-322 g (mean: 267 g)
- Housing: Animals were housed individually in wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France) distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: About 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: dietary admixture, in A04 C powder maintenance diet

Details on exposure:

DIET PREPARATION
- A premix at 20000 ppm of test item in the diet was prepared and then each concentration was prepared by diluting the premix with the diet. On each day of check of concentration, the premix prepared at 20000 ppm was kept for possible analysis until knowledge of the results.
- Storage temperature of food: Dietary admixtures were prepared on a weekly basis and were stored in closed bags at room temperature and protected from light prior to use.

STABILITY
- Satisfactory homogeneity and stability of dosage forms prepared at 500 or 20000 ppm was demonstrated during the study (Study No. 34831 AHS) over an 8-day storage period in open feeders at room temperature and over a 15-day period in closed bags at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration of the test item in samples of each control and test item dietary admixtures prepared for use on the first day of treatment of the first female and on the last day of treatment of the last female was determined.
- All analyses were performed by the validated analytical method (34830 VAA) of the provided by the Sponsor.
- Acceptance criterion: Measured concentration = nominal concentration ± 20 %.

Results: Test concentrations in the administered dietary admixtures analyzed the first day of treatment of the first female and on the last day of treatment of the last female remained within an acceptable range of - 12 % to + 14 % of variation compared to the nominal values.

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant; the females were mated at the breeder's facilities (Janvier, Le Genest-Saint-Isle, France).
- Proof of pregnancy: Detection of vaginal plug referred to as Day 0 post-coitum (p.c.).

Duration of treatment / exposure:

15 days (Days 6-20 p.c.)

Frequency of treatment:

Once daily; animals were allowed the dietary admixture ad libitum

Duration of test:

22 days (Days 0-21 p.c.)

Doses / concentrations

Remarks:

Doses / Concentrations:
675, 2025 and 6075 ppm of active content (equivalent to target dose-levels of 50, 150 and 450 mg active content/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of 4-week toxicity study (Study No. 31265 TSR) in which the test item was administered by dietary admixture at the target dose-levels of 150, 450 and 1000 mg/kg bw/day.
- Rationale for animal assignment: Before the beginning of the treatment period, the animals were allocated to the groups, according to a stratified procedure based on body weight recorded on Day 2 p.c., to ensure comparatively similar mean body weights among groups.

Examinations

Maternal examinations:

CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Once a day before the start of treatment and then twice a day during treatment period, including weekends and public holidays.
- Clinical signs: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Achieved intake of the test item was calculated on a weekly basis for each treated group as follows:
D = C x FC/BW; where, D = achieved dosage (mg/kg bw/day), C = nominal concentration of active test item (ppm: mg test item/10^6mg food), FC = mean food consumption (g/animal/day), BW = mean body weight (g)

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: On Day 21 p.c., females were sacrificed by inhalation of carbon dioxide followed by cervical dislocation and were subjected to a macroscopic post-mortem examination.

OTHER:
Preservation of tissues:
- Liver of females (control and 150 mg/kg bw/day) and placentas of females (50 and 450 mg/kg bw/day) were sampled and kept preserved in 10 % buffered formalin.

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 and distribution of implantations: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of dead and live fetuses: Yes
- Gross evaluation of placentas: Yes

Fetal examinations:

EXAMINATION OF FETUSES:
- Live fetuses were sacrificed by a subcutaneous injection of thiopental sodium.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus [excluding one autolyzed fetus from dam treated at 450 mg/kg bw/day] was determined at the time of hysterectomy by visual assessment of the anogenital distance and was confirmed by examination of the sexual organs at the time of detailed dissection of the soft tissues or at evisceration.
- External examinations: Yes [all per litter excluding one autolyzed fetus from dam treated at 450 mg/kg bw/day]; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed in Harison’s fluid for examination of the structures of the head.
- Skeletal examinations: Yes [all the remaining litters]; eviscerated and fixed with ethyl alcohol, stained with alizarin red S and alcian blue and examined for skeletal abnormalities including the observation of all the bone structures of the head, spine, rib cage, pelvis and limbs.

Statistics:

- Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
- Percentage values were compared by the Fisher exact probability test.

Indices:

- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live fetuses) / Number of implantation sites] X 100
- Fetal or litter incidence: (Total number of fetuses or litters with a particular finding / Total number of fetuses or litters examined)X 100
- Mean proportion of affected fetuses: (Sum of proportion of fetuses affected in each litter / Total number of litters examined) X 100

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy status:
- All mated animals were pregnant, except four, one and three females exposed to 50, 150 and 450 mg active content/kg bw/day, respectively (i.e. 59, 169 and 467 mg achieved active content/kg bw/day).

Mortality and clinical signs:
- No mortality and no clinical signs were observed.

Body weight and body weight change:
- Lower mean body weight gains were observed at 450 mg active content/kg bw/day (i.e. 467 mg achieved active content/kg bw/day) with statistical significance for the periods GD 6 to 9 (8 g vs. 23 g, p<0.001) and GD 15 to 18 (29 g vs. 43 g, p<0.001), when compared to the mean control values and this was considered to be treatment-related.
- Mean body weights were statistically significantly lower than controls from GD 9; the mean body weight towards the end of the dosing period (GD 18) and the terminal body weight (GD 21) were approximately -10 % lower than controls.
- Carcass weight and the net weight change from GD 6 were considered to have been adversely affected by the test item treatment. The mean gravid uterus weight was also slightly lower but this correlates to the difference in the mean number of fetuses per female. This body weight change was considered to be test item treatment-related.
- At 50 and 150 mg active content/kg bw/day (i.e. 59 and 169 mg achieved active content/kg bw/day), the mean body weight gain and mean body weight were considered to have been unaffected by the test item treatment. The gravid uterus weight, the carcass weight and the net weight change from GD 6 were also unaffected by the test item treatment.

Food consumption:
- At 450 mg active content/kg bw/day (i.e. 467 mg achieved active content/kg bw/day) lower mean food consumption was noted throughout the study, with a more marked effect at the start of the dosing period (interval days GD 6 to 9: - 25 %, p<0.001), when compared to controls and this difference was considered to be test item treatment-related.
- At 50 and 150 mg active content/kg bw/day (i.e. 59 and 169 mg achieved active content/kg bw/day), mean food consumption was unaffected by the test item treatment.

Maternal necropsy findings:
- There were no necropsy findings which were attributable to the test item treatment.
- At 50 mg active content/kg bw/day (i.e. 59 mg achieved active content/kg bw/day), one female had one enlarged placenta. At 150 mg active content/kg bw/day, one female presented two fused placentas, with a single implantation site. At 450 mg active content/kg bw/day, one female showed six enlarged placenta and one female had two fused placentas.
- These observations were considered to be at background level and were not ascribed to the test item treatment.

Litter data:
- There were no treatment-related effects on the mean numbers of corpora lutea, implantations or fetuses or on the pre- and post-implantation losses.
- At 450 mg active content/kg bw/day (i.e. 467 mg achieved active content/kg bw/day), the mean number of fetuses per animal was minimally lower than controls. This was considered not to be treatment-related since the numbers of corpora lutea and implantation sites were also minimally lower than those of controls.
- At 150 mg active content/kg bw/day (i.e. 467 mg achieved active content/kg bw/day), the mean percentage of post-implantation loss was slightly higher than controls (8.2 % versus 4.5 %, not statistically significant); this was due to a slightly higher number of late resorptions (6 versus 1 in controls, p<0.05) mainly from one litter which had four late resorptions, and was considered not to be toxicologically significant.
- Sex ratio and mean fetal body weight were similar in the control and treated groups.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External observations:
- Total of 264, 261, 228 and 228 (total of 229 high-dose fetuses but one of them was autolyzed) fetuses from the control, 50, 150 and 450 mg target active content/kg bw/day dose-groups (i.e. 0, 59, 169 and 467 mg achieved active content/kg bw/day) were examined and no test item treatment-related external malformations or variations in any fetus was observed.

Soft tissue observations:
- Total of 125, 124, 109 and 108 fetuses distributed between 20 litters (except at 150 mg active content/kg bw/day in which 19 litters were evaluated) from the control, 50, 150 and 450 mg active content/kg bw/day dose-groups were examined and no test item treatment-related soft tissue malformations were noted.
- At 450 mg active content/kg bw/day, one female had three fetuses with absent innominate artery. Due to the low litter and fetal incidence of this observation, and in the absence of any abnormality of the other related tissues/vessels, a relationship to the test item treatment was excluded.

Skeletal and cartilage observations:
- Total of 139, 136, 119 and 120 fetuses distributed between 20 litters from the control, 50, 150 and 450 mg active content/kg bw/day dose-groups were examined and none of the skeletal malformations or variations noted were considered to be test item treatment-related.
- At 450 mg active content/kg bw/day, two fetuses from one litter presented many skeletal variations (mainly incomplete ossifications of parietal, 1st to 4th and 6th sternebrae, unossified centrum of cervical or caudal vertebrae, unossified metacarpal, metatarsal and/or proximal phalanx) or skeletal malformations (unossified hemisternebra and incomplete ossification of the hemisternebra in one fetus), but the corresponding cartilage was present.

Effect levels (fetuses)

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 7.8.2/1: Mean achieved dosages (mg/kg bw/day) between gestation Days 6 and 21

Concentration (ppm)	675	2025	6075
Target dose-levels (mg/kg bw/day)	50	150	450
GD 6 to 21	59	169	467
(% from target dose-level)	(+18)	(+13)	(+4)

 

GD: gestation day

The mean achieved dosages increased in a nearly dose-proportional manner and mean values were very close to the targeted dose-levels of 50, 150 or 450 mg active content/kg bw/day.

Table 7.8.2/2: Mean body weight and body weight change (g)

Dose-level (mg/kg bw/day)	0	50	150	450
Mean body weight change
GD 6-9	23	20	26	8 #
GD 15-18	43	43	39	29 #
GD 6-21	154	159	157	116 #
Mean body weight
GD 6	268	269	262	269
GD 18	385	386	373	346 #
GD 21	422	428	419	385**
Gravid uterus weight	100.6	101.8	92.5	87.4
Carcass weight	321.5	326.2	326.3	297.3*
Net weight change from GD 6	53.5	57.0	64.1	28.4 #

 

GD: gestation day; *: p<0.05; **: p<0.01; #: p<0.001

Table 7.8.2/3: Changes in mean food consumption (g/animal/day)

Dose-level (mg/kg bw/day)	0	50	150	450
GD 6-9 (% from controls)	24	25 (+4)	25 (+4)	18 # (-25)
GD 15-18 (% from controls)	31	32 (+3)	30 (-3)	26 # (-16)
GD 18-21 (% from controls)	27	31** (+15)	29 (+7)	25 (-7)

 

GD: gestation day; **: p<0.01; #: p<0.001

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the No Observed Effect Level (NOEL) of CHIMEXANE HB was considered to be 2025 ppm (equivalent to 150 mg active content/kg bw/day) for maternal toxicity and 6075 ppm (equivalent to 450 mg active content/kg bw/day) for developmental toxicity in Sprague-Dawley rats.

Executive summary:

In a GLP-compliant prenatal developmental toxicity study performed according to OECD Guideline 414, CHIMEXANE HB was administered by dietary admixture to groups of mated female Sprague-Dawley, Rj Han: SD rats (24/dose) at the dose levels of 0, 675, 2025 and 6075 ppm active content (equivalent to target dose-levels of 0, 50, 150 and 450 mg/kg bw/day) from Days 6 to 20 post-coitum. Clinical signs and mortality were checked daily. Maternal body weight and food consumption were recorded approximately every 3 days. On Day 21 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, uterine scars, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

All mated animals were pregnant, except four, one and three females exposed to 50, 150 and 450 mg active content/kg bw/day, respectively. No mortality and no clinical signs were observed. At 450 mg active content/kg bw/day (i.e. 467 mg achieved active content/kg bw/day), low mean body weights and food consumption were noted throughout the study and transiently low mean body weight gain was noted at the start and end of the treatment period. The gravid uterus weight, carcass weight and the net weight change from Gestation Day 6 were lower than controls. None of the pregnancy parameters (including mean number of live fetuses per litter, sex ratio, and fetal body weight) were affected by the test item treatment. No treatment-related necropsy findings were noted. External and soft tissue examinations of the fetuses did not reveal any test item-related effect. The examination of the skeleton and cartilage of the fetuses did not reveal any test item treatment-related malformation or variation. No adverse effects related to the treatment were observed at 50 and 150 mg active content/kg bw/day (i.e. 59 and 169 mg achieved active content/kg bw/day).

Under the test conditions, the No Observed Effect Level (NOEL) of CHIMEXANE HB was considered to be 2025 ppm (equivalent to 150 mg active content/kg bw/day) for maternal toxicity and 6075 ppm (equivalent to 450 mg active content/kg bw/day) for developmental toxicity in Sprague-Dawley rats.