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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 July 2014 - 05 September 2014
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:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptanoic acid
EC Number:
203-838-7
EC Name:
Heptanoic acid
Cas Number:
111-14-8
Molecular formula:
C7H14O2
IUPAC Name:
heptanoic acid
Details on test material:
- Name of test material: Heptanoic acid
- Physical state: colorless liquid
- Lot/batch No.: 1404017
- Analytical purity: 99.46%
- Expiry date: 29 January 2017
- Storage condition: at room temperature.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France).
- Age/Weight at insemination: the females were approximately 17-19 weeks old and had a mean body weight of 3807 g
- Housing: the animals were individually housed in noryl cages (Tecniplast, 65.3 cm x 65.3 cm x 45 cm).
- Diet: Type 110C (SAFE, Augy, France) free access
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 14 days before insemination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 10 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8 h/16 h.

IN-LIFE DATES: 28 July 2014 to 05 September 2014.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. It was mixed with the required quantity of vehicle. Care was taken to not use plastic but glass materials with the test item or test item formulations.
No correction factor was applied.
The test item dose formulations were prepared and stored according to the following available stability data:
- concentration of 335 mg/mL: stability (9 days at room temperature and protected from light after 15 minutes of magnetic stirring) considered to cover the highest concentration used in this study (333 mg/mL),
- concentrations of 30 and 100 mg/mL: stability (11 days at room temperature and protected from light after 20 hours of magnetic stirring, 1 hour without stirring and then 1 hour of magnetic stirring again) considered to cover the lowest concentrations used in this study (33.3 and 100 mg/mL).

The control formulation was prepared and stored under the same conditions as the high-dose formulation.
Dose formulations were delivered to the study room at room temperature and protected from light, after at least 20 hours of magnetic stirring at room temperature and protected from light. During delivery of formulations to the animal room, stirring was not stopped for more than 1 hour.

VEHICLE
- Justification for use and choice of vehicle: homogenous formulation with this vehi cle
- Concentration in vehicle: 0, 33.3, 100 and 333 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: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: see paragraph PREPARATION OF DOSING SOLUTIONS
Details on mating procedure:
Insemination on site. Day of insemination = Day 0 p.c.
Duration of treatment / exposure:
Day 6 to day 28 post-coitum (p.c.).
Frequency of treatment:
Daily
Duration of test:
23 days.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 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 preliminary developmental toxicity study in New Zealand White rabbits conducted at 100, 300 and 1000 mg/kg/day in 0.5% (w/v) carboxymethylcellulose aqueous solution. Except erythema or dryness around the mouth from generally Day 16 p.c. at the high-dose especially, there were no obvious effects of the test item treatment.

- Rationale for animal assignment: stratification procedure.

Examinations

Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time each day, for the recording of clinical signs (including any evidence of abortion).

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded:
* twice a week before insemination,
* on Days 0, 3, 6, 9, 12, 15, 19, 24 and 29 p.c.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded:
* once a week before insemination,
* for the following intervals: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on day 29 p.c.
- 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
- Number of fetuses (live and dead).
Fetal examinations:
- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: all live fetuses per litter
- Skeletal examinations: Yes: all live fetuses per litter
- Other: body weight, sex.
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
Historical control data:
SEE DOC ATTACHED BELOW

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths at 0, 100 or 300 mg/kg/day.
At 1000 mg/kg/day, two pregnant females were found dead. One female was found dead on Day 17 p.c. and the other female was found dead on Day 13 p.c. They had loud and abdominal breathing for 2 or 6 days before death. One of these females lost weight and nearly did not eat from the first day of treatment and, at necropsy, had brownish nodules in the right lung. These clinical signs and deaths were considered to be related to the test item treatment. At 300 and 1000 mg/kg/day, animals were noted to be difficult to restrain and dose.

Clinical signs (see table 1)
The most relevant test item treatment-related clinical sign was dryness around the mouth at 300 and 1000 mg/kg/day, generally starting around mid-gestation.
Loud breathing was also recorded in surviving females on the day after or from the last day of treatment. It was considered to be test item treatment-related.
There were three females at 300 mg/kg/day which lost blood from vagina around Day 23 p.c. or from Day 27 p.c. There was no dose-relationship and there were no effects on hysterectomy data of these females. This clinical sign was thus considered to be of no toxicological significance.

Body weight (see table 2)
At 100 mg/kg/day, there were no toxicological significant effects.
At 300 mg/kg/day, the mean body weight changes observed at the end of the dosing period from day 24 p.c. was considered to be test item treatment-related.
At 1000 mg/kg/day, the mean body weight losses observed at the beginning and the end of the dosing period were considered to be test item treatment-related.
However, there was no toxicological impact on mean body weights.

Food consumption (see table 3)
At 100 mg/kg/day, there was no effect of the test item treatment.
At 300 mg/kg/day, the lower mean food consumption recorded from day 24 p.c. was considered to be related to the test item treatment.
At 1000 mg/kg/day, the statistically significant lower mean food consumptions recorded during the dosing period were considered to be related to the test item treatment.

MATERNAL TERMINAL EXAMINATIONS
Net body weight change
There were no effects on mean carcass weight, mean gravid uterus weight, or mean net body weight change.

Hysterectomy data
There were no effects on mean hysterectomy data.

In the control group and at 100 mg/kg/day, there were one female in each group with implant scars only. At 300 and 1000 mg/kg/day, there was one female in each group with aplasia of one uterine horn.
These findings were not attributed to the test item treatment because they were recorded for isolated animals and/or also in the control group, and uterine horn aplasia is a congenital abnormality.

Macroscopic post-mortem examination (see table 4)
The higher incidence of findings in the stomach noted in the test item-treated groups compared with controls was considered to be related to the corrosive properties of the test item.
The other macroscopic post-mortem findings were not ascribed to the test item treatment in view of their low incidence and were thus considered as incidental findings.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: stomach

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Sex of fetuses
There were no effects on mean percentage of male fetuses.

Fetal weights
There were no effects on mean fetal body weight.
The slightly lower mean fetal body weight noted at 300 mg/kg/day was due to the slightly higher mean number of live fetuses compared with controls. Moreover, there were no dose-relationship and no statistical significance. This change was not considered to be test item treatment-related.

External, soft tissue and skeletal examinations
External examination
There were no external variations or malformations ascribed to the test item treatment (very low incidence and/or included in historical control data).

Soft tissue examination (see table 5)
Gallbladder translucent contents were observed in a few litters in each test item-treated group, with statistical significance at 100 mg/kg/day and mainly at 1000 mg/kg/day. This finding was not described in Historical Control Data or in the control group and was considered to be test item treatment-related but of minor toxicological significance (variation, low incidence). Moreover, there were no macroscopic findings on liver of these fetuses indicative of a potential obstruction of bile ducts, as discoloration for example.
A few litters in each test item-treated groups had fetuses with finding in the liver vs. none in the control group. As there was no statistical significance and as the main finding (coloured focus) was included in the historical control data while the two other findings were observed in single fetuses, there were not considered to be of toxicological significance.
There were no test item treatment-related soft tissue malformations (very low incidence and/or seen in Historical Control Data).
There were particularly one fetus at 300 mg/kg/day and one at 1000 mg/kg/day with at least two malformations in the heart and/or its blood vessels. A relationship with the test item treatment was considered to be unlikely as most of the findings were seen in Historical Control Data, since there was no dose-relationship and as they were of very low incidence.

Skeletal and cartilage examinations
There were no test item treatment-related effects on cartilages.
The slightly higher incidence of litters with fetuses having unossified/incompletely ossified 1st metacarpal(s) at 300 mg/kg/day compared with controls was not considered to be toxicologically significant in view of the absence of statistical significance and the fact that these findings were included in the Historical Control Data.
There were no test item treatment-related skeletal malformations.

Fetal malformation discussion
There were 14 fetuses from 13 litters with malformations (external, soft tissues and/or skeletal) in the study.
Most of the malformations noted at 300 and 1000 mg/kg/day were also found in the control group and/or in recent Historical Control Data.
The other malformations (malpositioned adrenal, ectrodactyly, absent forepaw phalanx, absent aortic arch and dilated left pulmonary artery) were observed in single fetuses, linked to other malformations described below (absent aortic arch, dilated pulmonary artery, absent forepaw phalanx) and/or also observed in the past in our laboratory in New Zealand White rabbit litters (ectrodactyly, seen in old Historical Control Data).
There were no fetal malformations ascribed to the test item treatment.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
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

Clinical signs:

Table 1:

Dose-level (mg/kg/day)

0

100

300

1000

Dryness around the mouth

 

 

7/24

22/23

Loud breathing

 

 

1/24

4/23

Reddish vaginal discharge and Blood in the bedding

 

 

3/24

 

Body weight and body weight changes:

Table 2:

Dose-level (mg/kg/day)

0

100

300

1000

Body weight

 

 

 

 

Day 6p.c.

3883

3895
(0)

3930
(+1)

3962
(+2)

Day 29p.c.

4192

4139
(-1)

4167
(-1)

4217
(+1)

Body weight change

 

 

 

 

Days 6 - 29p.c.

+309

+244

+238

+233

Days 6 - 9p.c.

+18

+7

+13

-45**

Days 9 - 12p.c.

+37

+38

+47

+61

Days 24 - 29p.c.

+33

+14

-29*

-14

Statistically significance: *: p<0.05; **: p<0.01; in brackets and italic: differences from controls (%).

Food consumption

Table 3:

Dose-level (mg/kg/day)

0

100

300

1000

Days 6 - 9p.c.

177

172
(-3)

179
(+1)

144*
(-19)

Days 15 - 19p.c.

188

164
(-13)

181
(-4)

156*
(-17)

Days 24 - 29p.c.

105

93
(-11)

77#
(-27)

73#
(-30)

Statistically significance: *: p<0.05, #: p<0.001; in brackets and italic: differences from controls (%).

Table 4:

Dose-level (mg/kg/day)

0

100

300

1000

Stomach mucosa:

Colored deposit (brownish)

Colored (reddish)

Colored area(s) (often reddish and depressed)

Colored focus (i)

Thickened

 

6

 

 

 

13

1

 

 

 

12

2

2

 

1a

2

10

1

2

Total number of animals with at least one finding in stomach:

6/24

14/23

14/24

15/23

a: dead animal.

Macroscopic examination:

Table 5:

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Variations

 

 

 

 

 

Translucent contents in the gallbladder

 

13.6 (2.5)

8.3 (1.6)

19.0 (2.5)

-

Liver: whitish colored focus

 

9.1 (1.0)

4.2 (0.4)

9.5 (1.0)

[0-12.5]

Liver: colored nodule

 

 

4.2 (0.4)

 

[0-6.3]

Liver: irregular color

 

4.5 (0.5)

 

4.8 (0.5)

-

Malformations

 

 

 

 

 

Cardiomegaly

 

 

4.2 (0.4)a

 

[0.0-6.3]

Absent left kidney/ureter

 

 

4.2 (0.4)

 

[0.0-3.1]

Malpositioned adrenal

 

 

4.2 (0.4)

 

-

Dilated brachiocephalic trunk

 

 

 

4.8 (0.5)b

[0.0-3.1]

Absent aortic arch

4.3 (0.5)

 

 

4.8 (0.5)b

-

Dilated left pulmonary artery

 

 

 

4.8 (0.5)b

-

Dilated aortic arch

4.3 (0.5)

 

4.2 (0.4)a

 

[0.0-10.5]

Statistically different from controls: *: p<0.05; a: one same fetus; b: one same fetus; HCD: Historical Control Data 2008-2013 [litter incidences], except liver: colored nodule: 2006-2010; -: not in HCD.

Applicant's summary and conclusion

Conclusions:
The test item's NOAELs in rats in an oral developmental toxicity study were:
- maternal toxicity: 300 mg/kg/day
- embryo/fetal development: 1000 mg/kg/day
Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item 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 (p.c.) inclusive) at dose levels of 100, 300 or 1000 mg/kg/day..

On Day 29 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal/cartilage abnormalities.

The following changes were observed:

There were no unscheduled deaths except at 1000 mg/kg/day where two pregnant females were found dead around mid-gestation; this was considered to be related to the test item treatment.

The most relevant test item treatment-related clinical sign was dryness around the mouth at 300 and 1000 mg/kg/day. Loud breathing was also recorded in a few females treated at 300 and 1000 mg/kg/day, included the dead females.

At 300 and 1000 mg/kg/day, there were slightly mean body weight losses at the beginning (1000 mg/kg/day) and at the end (both doses) of the dosing period. Mean food consumption was reduced at treatment initiation and towards mid-gestation (p<0.05) at 1000 mg/kg/day but mainly at the end of the treatment at 300 and 1000 mg/kg/day (p<0.001).

At necropsy, the high incidence of findings in the stomach (i.e. reddish mucosa, reddish area on the mucosa, thickened mucosa, reddish foci on the mucosa) in all test item-treated groups was considered to be due to the corrosive properties of the test item.

There were no test item treatment-related external, soft tissue or skeletal malformations or variations.

 

Conclusion

The test item was administered by gavage daily from Day 6 to Day 28 p.c .to inseminated female New Zealand White rabbits at 100, 300 and 1000 mg/kg/day.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day, based mainly on the 2 dead dams at 1000 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day, based on the absence of adverse effects in the fetuses.