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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 2011 - 6 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
the animals were acclimated to the study conditions for a period of 4 or 5 days before the beginning of the treatment period instead of a period of at least 5 days, 
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 11-AMINOUNDECANOIC ACID
- Physical state: white powder
- Lot/batch No.: 11.08.481
- Analytical purity: 98.57 (first receipt) and 90.9 % (second receipt)
- Expiry date: 1 september 2012 (first receipt) and 10 August 2013 (second receipt)
- Storage conditions: at room temperature, away from humidity and under nitrogen atmosphere.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France (Châtillon-sur-Chalaronne, France)
- Age at study initiation: approximately 18 - 20 weeks old on the day of treatment
- Mean body weight at study initiation: 3421 g (range: 2795 g to 3805 g)
- Fasting period before study: no
- Housing: noryl cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: 14 November 2011 to 17 February 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 10.0, 33.3 and 100.0 mg/mL. No correction factor was applied.
Homogeneity and stability of the dosage forms at 1 and 110 mg/mL were demonstrated for up to 10 days at +4°C and protected from light.
The test item dosage forms were prepared for use up to 10 days, stored at +4°C prior to use and delivered in brown flasks into crushed ice.

VEHICLE:
The vehicle was 0.5% carboxymethylcellulose aqueous solution (3 mL/kg) in drinking water prepared using:
- drinking water, treated by reverse osmosis using ELIX 5 plus apparatus (Millipore SA, Saint Quentin-en-Yvelines, France),
- carboxymethylcellulose, batch No. 100M0219V, supplied by Sigma (Saint-Quentin-Fallavier, France).

- Concentration in vehicle: 10.0, 33.3 and 100.0 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: The test item concentrations in the administered dosage forms analyzed in weeks 1 and 4 remained within an acceptable Range of variations (-8.5% to -0.4%) when compared to the nominal values (± 15%).
Dosage forms at 1 and 110 mg/mL were found to be homogeneous and stable after 10 days at +4°C and protected from light (CiToxLAB France Study No. 37498 AHS).
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: visual assessment (time-mated female rabbits obtained from the breeder).
Duration of treatment / exposure:
day 6 to day 28 post-coitum
Frequency of treatment:
Daily
Duration of test:
29 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
22-23 females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous Maximum Tolerated Dose toxicity study by oral route (gavage) in non-pregnant rabbits and of a preliminary study of prenatal developmental toxicity by oral route (gavage) in rabbits (CIT/Study No. 37442 TSL).

In the Maximum Tolerated Dose toxicity study by oral route (gavage) in non-pregnant rabbits, animals (three female rabbits per group) received 300 or 1000 mg/kg/day for 5 days and after a 3-day treatment-free period, 300 or 600 mg/kg/day for 5 days, respectively. In group 1 (300 → 300 mg/kg/day), there were no clinical signs, no biologically significant effects on body weight or food consumption which might be attributed to the treatment with the test item. In group 2 (1000 → 600 mg/kg/day), 1/3 females had absence of feces and was emaciated on study termination (up to -915 g body weight loss (-26.4%) vs. body weight on study) and had no food consumption. Therefore, 1000 mg/kg/day was considered to be an excessive high dose-level. 600 mg/kg/day was selected as a high-dose for the preliminary study of prenatal developmental toxicity by oral route (gavage) in rabbits.

In the preliminary study of prenatal developmental toxicity by oral route (gavage) in rabbits, animals (six mated female rabbits per group) received 150, 300 or 600 mg/kg/day from day 6 to day 28 p.c.. There were no treatment-related clinical signs. In the high-dose group, 1/6 females with an history of prolonged body weight decrease and reduced food consumption, aborted on day 26 p.c. Slight to moderate dose related reduction in mean body weight were observed at 300 mg/kg/day (up to -7.3% vs. controls on day 24 p.c.) and at 600 mg/kg/day (up to -9.0% vs. controls on day 19 p.c.).
Reductions in mean food consumption were observed in all treated groups. These reductions were moderate at 150 and 300 mg/kg/day (up to -33.6% on days 12-15 p.c. and -25% on days 15-19 p.c. vs. controls, respectively) and severe at 600 mg/kg/day (-58.3% on days 6-9 p.c. vs. controls, p < 0.001 and -43.5% on days 9-12 p.c. vs. controls, p < 0.01). On hysterectomies and on external fetal examination, there were no treatment related findings.

Taking into account the abortion and the severe decrease in mean food consumption in the high dose-group, 600 mg/kg/day was considered to be an excessive high dose-level for a further definitive study.

Therefore, 300 mg/kg/day was selected as the high dose-level. The low-dose and mid dose-levels were selected using a ratio representing a 3-fold interval (i.e. 30 and 100 mg/kg/day).

- Rationale for animal assignment: stratified procedure base.

Study design in the main study:
Three groups of 22 mated female KBL New Zealand White rabbits were administered the test item, 11-aminundecanoic acid (batch 11.08.481), once daily from day 6 to day 28 p.c., by gavage at dosages of 30, 100 or 300 mg/kg/day (groups 2 to 4). An additional group of 22 mated females received the vehicle CMC, 0.5% under the same experimental conditions. A dosage volume of 3 ml/kg was used. Following poor clinical conditions during the acclimation period and following inconclusive results, it was decided at the request of the sponsor to repeat the experiment at the high dose-level (300 mg/kg/day) in order to ascertain whether or not the test item treatemnt was associated with fetal observations in recorded in group 4 (300 mg/kg/day) and wether or not the maternal condition prior to dosing had modify the potential susceptibility of the animals to the test item. Two additional groups of 23 and 22 mated female KB rabbits were administered under the same experimental conditions as above, etither the vehicle (group 5) or the test item at 300 mg/kg/day (group 6).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: on days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c., and prior to premature sacrifice.

FOOD CONSUMPTION:
- Time schedule: on the following intervals: days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on day 29 post-coitum.
- Examined: principal thoracic and abdominal organs.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including::
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Number of uterine scars, evaluation of placenta.
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
- Other : number dead and live, body weight, sex
Statistics:
Mean values were compared by a one-way analysis of variance and a Dunnett's test.
Percentage values were compared by a Fisher exact probability test.
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality and Abortion

There were no mortality or abortion in the control and 100 mg/kg/day groups.

30 mg/kg/day
Dam No. X30044 was sacrificed on day 29 p.c. after aborting 15 fetuses (five placenta in the bedding). This female had an emaciated appearance from day 28 p.c.. At necropsy, there were no findings.
This finding was observed on day 29 p.c. as a results of an early delivery (total gestation period in rabbit is about 30 days). Therefore a treatment-related effect was considered unlikely.

300 mg/kg/day
Dam No. 30075 was sacrificed on day 13 p.c. after evidence of abortion (blood in the bedding). This female lostosed weight since day 2 p.c., had an absence of feces from day 8 p.c. and, an emaciated appearance on day 9 p.c. and from day 12 p.c.. This dam had no food consumption from the start of the study. At necropsy, there were no findings that could be related to treatment. Taking into account the sanitary status of this female during the pre treatment period, a relationship to the treatment was considered unlikely.
Dam No. 30083 was sacrificed on day 24 p.c. after evidence of abortion (blood in the bedding). This female had an emaciated appearance from day 23 p.c. and a cutaneous lesion on neck from day 20 p.c.. At necropsy, there were no findings that could be related to treatment. However, taking into account both the sanitary status of this female the days preceding abortion (significant decrease in body weight change during the period of days 6 to 15 p.c. or day 6 to 19 p.c., a relationship to the treatment cannot be ruled out.

When evaluating in detail individual data on body weight, body weight change or food consumption, there were the following observations:

- Dosing period:

Mean body weight changes on the period of days 6 to 9 p.c. in the 100 and 300 mg/kg/day groups were 26 and -2 g vs. 47 g in the control group, respectively. Mean food consumption in the 300 mg/kg/day group on the period of days 6 to 9 p.c. was 115 g vs. 148 g in the control group. These slight but not significant decreases in body weight and food consumption at 300 mg/kg/day associated with the abortion of one female are most probably indicative of maternal toxicity.

- Pre-dosing period:
On an individual basis, both in the control and test item-treated groups, there were dams with repeated episodes of weight loss and almost no food consumption (<10g/day) before treatment.
Episodes of body weight (BW) loss and almost no food consumption (FC<10 g/day) during days 2 to 6 before treatment before treatment as detailed below :


Females No. X30011:
BW: -305 g (-9.8%)
FC: 0 to 5 g/day (days 2 to 6 p.c.)

X30018:
BW: -50 g (-1.5%)
FC: 0 g/day (days 2 to 4 p.c.)
X30029:
BW: -325 g (-9.1%)
FC: 0 to 5 g/day (days 2 to 6 p.c.)

X30043:
BW: -450 g (-12.2%)
FC: 0 to 5 g/day (days 2 to 5 p.c.)

X30065:
BW: -325 g (-8.8%)
FC: 0 to 5 g/day (days 4 to 6 p.c.)

X30073:
BW: -65 g (-2.0%)
FC: 2 g/day (days 2 to 4 p.c.)

X30075:
BW: -410 g (-11.1%)
FC: 0 g/day (days 4 to 6 p.c.)

X30076:
BW: -225 g (-5.9%)
FC: 0 g/day (days 4 to 6 p.c.)

X30079:
BW: -245 g (-6.6%)
FC: 0 g/day (days 5 to 6 p.c.)





Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
When compared with the control group, there were no evidence of adverse effects of the treatment with the test item on reproductive parameters,
numbers of corpora lutea or implantations.

Fetal weight and sex :
There was no evidence of an adverse effect of the treatment with the test item on mean fetal body weights or fetal sex ratios.

External variation examination :
There were no effects on the incidence of external variations per fetuses or per litter.

External malformation examination:

Group 1 (vehicle control): one fetus (X30011-09) had a short tail (this finding was not observed in the Historical Control Data).

Group 3 (100 mg/kg/day): one fetus (X30057-03) had a spina bifida (occulta). This finding was not observed in control groups from other studies (Historical Control Data) and also observed in one fetus from the high-dose group but with no skeletal observation correlates. Spina bifida was also a common malformation reported in this animal species and strain (Charles River Historical Control Data, 2008-2010). Therefore a treatment-related effect was considered unlikely.

Group 4 (300 mg/kg/day): five fetuses from three different litters (X30076-01, X30081-07, X30087-01, X30087-03 and X30087-08) had a series of malformations (limb/extremities and/or central nervous system) for which a test item treatment-related effect cannot be excluded. All malformations were observed at litter and fetal incidences higher than the upper limit of the Historical Control Data.

Group 5 (vehicle control): fetuses had no external malformations.

Group 6 (300 mg/kg/day): fetuses had no external malformations.

Litter and fetal incidences of external malformations in group 4 were higher than the upper limit of the Historical Control Data while not external malformations were recorded at the same dose-level in group 6. Therefore, a relationship to the treatment was not excluded.


Soft tissues malformation
There were no soft tissue malformations in fetuses from group (vehicle control group), and, from group 2 (30 mg/kg/day).

Group 3 (100 mg/kg/day): one fetus (X30065-03) had a retroesophageal subclavian artery. This malformation was previously recorded in one fetus in control group 5 and in control groups from other studies at higher litter and fetal incidences (Historical Control Data). Therefore a relationship to treatment with the test item was considered unlikely.

Group 4 (300 mg/kg/day): one fetus (X30088-02) had an absent subclavian artery. This malformation was previously recorded in the Historical Control Data. Two fetuses from the same litter (X30087-08 and X30087-03) had a series of malformations (cardiovascular defects, misshapen cerebrum, open diaphragm, absent, gall bladder and/or adrenals). Some of these malformations were also recorded in the Historical Control Data (cardiovascular effects, absent gallbladder) or are regularly reported in public literature (Morita et al., 1987 and Stadler et al., 1983). However, for other malformations recorded in these fetuses (misshapen cerebrum, open diaphragm, absent adrenals) a test item treatment-related effect can not be excluded.

Group 6 (300 mg/kg/day): two fetuses from the same litter (X30116-02 and X30116-05) had malpositioned kidneys with short ureters. Malpositioned kidney is a malformation previously recorded in Historical Control Data.


Skeletal examination:
There were no singificant changes at cartilage examination and the incidence. The types of fetal variations findings recorded in groups 4 and 6 (300 mg/kg/day) are generally considered to represent slightly delayed ossification rather than dysmorphogenic changes.
There were no effect that could be related to the treatment at 30 and 100 mg/kg/day as the incidence of changes were either not dose-related or within the historical control data.

Group 2 (30 mg/kg/day): there were no skeletal malformations recorded at incidence higher than the upper limit of the Historical Control Data.

Group 3 (100 mg/kg/day): absent cervical hemivertebra(e) (fetus X30052-03), absent thoracic hemivertebra(e) (fetuses X30046-07, X30065-04, X30065-05) and absent rib(s) (fetus X30046 07) were not observed in the control group and not recorded in control groups from other studies (Historical Control Data). These findings were not observed in the higher dose group and only recorded in a few fetuses. Therefore, these findings were not considered to be treatment-related.


Group 4 (300 mg/kg/day): findings not recorded in the control group or in control groups from other studies (Historical Control Data) concerned two fetuses from two different litters: X30076 02 with absent thoracic hemivertebra(e) and X30087-03 with multiple severe malformations (cervical vertebra(e) with open arch, misshapen scapula, absent sternum and a series of abnormalities of limb and extremities). Based on litter incidences, a test item treatment-related effect cannot be excluded.

Group 6 (300 mg/kg/day): findings not recorded in the control groups (misaligned and fused lumbar vertebra(e) or extra sternebra) concerned the same fetuses. These malformations were recorded at incidences lower than the upper limit of the Historical Control Data, therefore a test item treatment-related effect was considered unlikely.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAELs obtained in the oral developmental toxicity study in rabbits are:
- maternal toxicity: 100 mg/kg/day
- development: 100 mg/kg/day
Executive summary:

The objective of this prenatal developmental toxicity study performed according to guideline OECD 414 was to evaluate the potential toxic effects of the test item, 11-AMINOUNDECANOIC ACID,on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (day 6 to day 28 post‑coitum inclusive).

Three groups of 22 mated female KBL NewWhite rabbits were administered the test item, 11-AMINOUNDECANOIC ACID(batch No. 11.08.481), once daily from day 6 to day 28p.c., by gavage, at dosages of 30, 100 or 300 mg/kg/day (groups 2 to 4). An additional group of 22 mated females received the vehicle, 0.5% (w/v) carboxymethylcellulose, under the same experimental conditions and acted as the control group (group 1).A dose volume of 3 mL/kg/day was used.

In order to ascertain whether or not the test-item treatment was associated with fetal observations recorded in the high-dose group 4,it was decided at the request of the sponsor to complete the study by repeating administration at the high dose-level. Two additional groups of 23 and 22 mated female KBL New Zealand White rabbits were administered either the vehicle (group 5) or the test item, 11-AMINOUNDECANOIC ACID (batch No. 11.08.481) at 300 mg/kg/day (group 6) under the same experimental conditions as above.

Results

 

On an individual basis, both in control group 1 and test item-treated groups 2 to 4, there were dams with repeated episodes of weight loss and almost no food consumption (<10 g/day) before treatment. 

The inclusion of the two additional groups (group 5 and group 6) revealed/confirmed that the animals entering the study in groups 1 to 4 were not at the expected health conditions (body weight losses, reduced or absence of food consumption during the predosing period).

 

At termination on day 29p.c., there were 20, 21, 22, 17, 19 and 21 dams with live fetuses in groups 1 to 6, respectively.

 In the 30 mg/kg/day group 2, no findings were observed that were considered to be related to the treatment with the test item.

 In the 100 mg/kg/day group 3,the findings recorded in this dose group were observed at a low incidence and only in a few fetuses. Most findings were also recorded in the Historical Control Data at comparable incidences. Therefore it was concluded that the effects observed in this group were not related to treatment with the test item.

In the 300 mg/kg/day group 4, there were:

.         Maternal toxicity indicated by:

o      one aborted female on day 13p.c.and one aborted female on day 24p.c.,

o      not statistically significant decreases (especially recorded during the period of days 6 to 9 p.c.) on mean body weight, mean body weight change or on mean food consumption.

.         Fetal toxicity indicated by:

o      slightly higher incidences of external, soft tissue and skeletal malformations.

 

In the 300 mg/kg/day group 6, there were:

.         Maternal toxicity indicated by:

o      statistically significant reductions in mean body weight and mean body weight change,

o      statistically significant decreases in mean food consumption.

.         No fetal toxicity.

Conclusion

 

The test item, 11-AMINOUNDECANOIC ACID (batch No. 11.08.481), was administered by gavage, once daily, from days 6 to 28 p.c., inclusive, to mated female KBL New Zealand White rabbits at dosages of 30, 100 or 300 mg/kg/day.

Slight to moderate maternal toxicity was recorded in females given 300 mg/kg/day (group 4 and 6) while no maternal toxicity was recorded at 30 and 100 mg/kg/day.

The oral administration of 11-AMINOUNDECANOIC ACID to the dams at all dose-levels had no influence on gestational parameters. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio and the pre-and the post-implantation losses were all unaffected by the treatment.

Fetal examinations revealed no influence of the test compound on sex ratio and fetal body weight. The fetal findings associated with slight maternal toxicity observed in group 4 (300 mg/kg/day) were not recorded in group 6 (300 mg/kg/day) in the presence of maternal toxicity, therefore a treatment related effect could not be ascertained.

 

On the basis of the results obtained in this study:

.         The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day, based on decreased mean body weight gain and food consumption at 300 mg/kg/day,

.         The NOAEL for embryo-fetal development was considered to be 100 mg/kg/day based on dubious findings recorded at 300 mg/kg/day in a context of maternal toxicity.