Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

There is a key reproductive/developmental screening study performed on heptanoic acid (Hoberman, Argus Research laboratories, 1990). Although this study does not meet completely OECD guideline standards for reproductive or developmental toxicity, it provide an extensive data set that indicate a low potential for reproductive or developmental toxicity.

Four groups of virgin Crl CD rats were administered oral dose levels of 0, 200, 1000 or 2000 mg/kg bw/day of heptanoic acid by gavage once daily, 7 days prior to cohabitation, through cohabitation (maximum of 7 days), gestation, delivery, and a 4 -day post-parturition period. Clinical signs, body weight and feed consumption were recorded throughout the study. Mating performance, fertility, duration of gestation/parturition, maternal behavior, litter sizes, dystocia, number of implantation sites, and gross lesions at necropsy were also examined. Offspring were examined for viability, sex, external morphology, and body weight at birth and on day 4 postpartum.

The article caused the death of one and 3 rats in the 1000 and 2000 mg/kg bw/day dose groups, respectively. These rats generally had excess salivation, rales, urine-stained abdominal fur, ungroomed coat, red nasal discharge (chromorrhinorrhea), and/or decreased motor activity occur. Bright red mottled lungs fur noted and revealed by necropsy in one of the high dosage group rats that was found dead was considered effect of the test article. No other gross lesions were caused by the test article. Clinical signs at 200 mg/kg bw/day in dams (8/10 animals) during premating and gestation included a significant increase in rales. This effect was not reported during the lactation period. In the 1000 and 2000 mg/kg bw/day dose group, significant increases in the incidence of rales were reported in 10/10 and 6/7 animals respectively. At 1000 and 2000 mg/kg dose level excess salivation was reported in all animals during premating, and gestation. Excess salivation continued during lactation only in the high-dose group. The 1000 and 2000 mg/kg/day dosages of the test article caused remarkable weight loss after the first dosage. The 2000 mg/kg bw/day group showed reduced body weight gains during premating, and significantly decreased average maternal body weights on days 10 and 16 of gestation. Body weight changes on days 1 to 7 of the study averaged +8.8, +10.8, +7.8 and +4.0 g in the control, low, middle and high dosage groups, respectively. Absolute and relative maternal feed consumption values were reduced for the 1000 and 2000 mg/kg/day dosage groups, as compared with the control group values during the premating period. On days 0 to 21 of gestation, maternal body weight changes averaged +132.8, +134.6, +140.5 and +126.0 g in the control, low, middle and high dosage groups, respectively. Maternal body weight changes in the control, low, middle and high dosage groups averaged +0.1, -1.6, +12.0 and 14.5 g, respectively, and maternal body weights averaged 310.0, 307.6, 324.4 and 275.0 g, on day 4 postpartum. During gestation and lactation period, absolute and relative maternal feed consumption values tended to be reduced for the high dosage group. The high-dose was also associated with reduced mating and fertility that were related to mortality. The duration of cohabitation and fertility and gestation indices at 200, 1000, or 2000 mg/kg bw/day were not different from comparable indices in the control group. No biologically relevant or statistically significant differences in the number of implantations, duration of gestation, the percentage of dams delivering one or more live pups, and the pup viability index were observed. No malformations or gross lesions were observed in pups at any dose levels.The authors concluded that the dose level of 200 mg/kg bw/day of heptanoic acid had no significant adverse effects on the reproductive performance of female Sprague-Dawley rats or the growth or developmental of their offspring.

 

 


Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 september 1989 To 26 october 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 2c: Comparable to guideline study with acceptable restrictions.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The test item was not admistered to male rats (deviation to the guideline).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Crl:CD (SD) BR
- Age at study initiation: (P) Females: around 12 wks; Males: around 23 weeks.
- Weight at study initiation: (P) Males: no data g; Females: 236-277 g.
- Fasting period before study: no
- Housing: Individually in wire-bottomed stainless-steel cages suspended above absorbent paper liners, except during the cohabitation and postparturition periods.
- Diet : ad libitum (Rodent Chow #5002, Ralston Purina)
- Water : ad libitum (local water passed through a reverse osmosis membrane)
- Acclimation period: approximately two weeks prior to the first day of intubation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 35-65
- Air changes (per hr): a minimum of 10
- Photoperiod : 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 19 September 1989 To: 26 October 1989
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: B129 was dissolved in corn oil. The solutions were prepared weekly at the test facility. All prepared solutions were refrigerated and protected from light in amber glass bottles.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 40, 200 and 400 mg/ml
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): MAR 16 90B and AUG 11 90B
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 7 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each concentration were reserved on the first day the dosage solutions were prepared and also from the last batches prepared. Samples were shipped to the sponsor for analysis. No data on results was presented in the study report.
Duration of treatment / exposure:
Females: premating period (7 days) + mating period (maximum of 7 days) + gestation (22 days) + lactation (4 days)
Males: untreated (Male rats were used only as breeders)
Frequency of treatment:
Once daily
Details on study schedule:
Appropriate dosages of the test article were given to the female rats for seven days prior to and then through cohabitation (maximum seven days), presumed gestation, delivery and a four-day lactation/postparturition period (dams than delivered litters).
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 females per dose.
Males were untreated.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the dosage period


BODY WEIGHT: Yes
- Time schedule for examinations: daily during the dosage period


Oestrous cyclicity (parental animals):
not evaluated
Sperm parameters (parental animals):
not evaluated
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups,
- stillbirths,
- live births,
- postnatal mortality,
- presence of gross anomalies,
- body weight gain between days 1 and 4,
- physical or behavioural abnormalities.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: not examined
- Maternal animals: All surviving animals on day 4 of lactation, or on day 25 post-coitum for females which had not delivered.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations


HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries from all dams and any observed gross lesions were preserved in neutral buffered 10 % formalin for possible future evaluation.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination)


Statistics:
Maternal and pup incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Baseline maternal body weight data, body weight changes during the premating, gestation and lactation periods, feed consumption data and litter averages for pup body weights and percentage of male pups were analized using Bartlett's test of Homogeneity of Variances and the analysis of variance, when appropriate.
Natural delivery parameters involving discrete data were evaluated using the Kruskal-Wallis Test procedures previously described.
Reproductive indices:
Fertility index = (number of pregnant female rats/ rats mated)
Gestation index = (number of females with live litters/ pregnant rats)
Offspring viability indices:
not calculated
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The test article caused the deaths of one 1000 mg/kg/day dosage group rat on day 17 of gestation and three 2000 mg/kg/day dosage group rats. Two high dosage group rats died during the premating dosage period, and one high dosage group rat died on day 3 of the gestation period. Rales occurred for significant numbers of 200, 1000 and 2000 mg/kg/day dosage group rats duringthe premating and gestation periods, and for significant numbers of 1000 and 2000 mg/kg/day dosage group rats during lactation. Excess salivation occurred for significant numbers of 1000 and 2000 mg/kg/day dosage group rats during the premating and gestation periods, and for significant numbers of 2000 mg/kg/day dosage group rats during lactation. Significant numbers of 2000 mg/kg/day dosage group rats had labored breathing, ungroomed coat and decreased motor activity during gestation. Other clinical observations that were considered effects of the 1000 and/or 2000 mg/kg/day dosages of the test article, but did not occur at statistically significant incidences, were urine-stained abdominal fur, chromorrhinorrhea, oral exudate, ungroomed coat, absence of motor activity and ataxia.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The 1000 and 2000 mg/kg/day dosages of heptanoic acid caused weight loss after the first dosage; the 2000 mg/kg/day dosage tented to cause reduced body weight gains during the premating period. During gestation, the 2000 mg/kg/dayndosage of thetest article tended to reduce maternal body weights on day 10 and 16 of gestation. The 2000 mg/kg/day dosage of the test article also tended to reduce maternal body weight gain during lactation, and maternal body weight was significantly reduced on day 4 postpartum, as compared with the control group value.
Absolute and relative feed consumption values tended to be reduced for the 2000 mg/kg/day dosage group during the premating, gestation and lactation periods.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The 2000 mg/kg/day dosage of the test article tended to reduce mating and fertility, observations that were interralated with deaths. The duration of coabitation and the fertility and gestation indices were not significantly affected by the test article.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross lesions attributable to the test article occurred for one 2000 mg/kg/day dosage group rat that was found dead on day 15 of the study, prior to mating. This rat had slight urine-staining of the abdominal fur and bright red, mottled lungs. No other gross lesions were caused by the test article.

Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
< 200 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: At this dose, test substance induced rales among a significant number of rats at 200 mg/kg/day. The 1000 and 2000 mg/kg/day dosages caused deaths and weight losses;
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
No biologically relevant or statistically significant differences among the groups in the pup viability indices were noted.

BODY WEIGHT (OFFSPRING)
Pups body weights tended to be reduced for the high dosage group on day 4 of lactation.

GROSS PATHOLOGY (OFFSPRING)
Administration of the test article to the dams at dosages as high as 2000 mg/kg/day did not cause malformations or gross lesions in the pups.
Dose descriptor:
NOAEL
Remarks:
(offspring)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No adverse effect at 1000 mg/kg/day
Reproductive effects observed:
not specified
Conclusions:
Under the conditions of this test, heptanoic acid induced maternal toxicity. Deaths occurred at 1000 and 2000 mg/kg/day and significant numbers of rat with rales were induced at each tested dose. Heptanoic acid had no effect on mating, fertility, gestation, delivery and did not cause malformations or gross lesions in the pups.
Executive summary:

In a reproductive and developmental toxicity test (Alan, 1990), heptanoic acid was administered orally via gavage once daily to Sprague-Dawley virgin female rats (ten rats per group) at dosages of 0 (vehicle, corn oil), 200, 1000 and 2000 mg/kg/day. All dosages were given at a dosage volume of 5 ml/kg and adjusted daily for body weight changes. The rats were observed for clinical signs, deaths and /or deliveries of litter. Body weights were recorded daily during the dosage period and feed consumption values were recorded periodically. All dams were necropsied and examined for gross lesions. The litters were examined for numbers, viability, body weights, sex ratios and external morphology of the pups. Delivered pups were additionally examined for viability, clinical observations and body weights during a four-day postparturition period. The pups were necropsied when found dead.

One female and three females dead at 1000 and 2000 mg/kg/day respectively. One high dosage group rat had urine-stained abdominal fur and bright red, mottled lungs. During the premating and gestation periods, rales occurred for significant numbers of rats given dosages of 200 to 2000 mg/kg/day of the test article and for significant numbers of 1000 and 2000 mg/kg/day dosage group rats during lactation. Significant numbers of 1000 and 2000 mg/kg/day dosage group rats excess salivation occur during the premating and gestation periods. During lactation excess salivation occurred for significant numbers of 2000 mg/kg/day dosage group rats. During gestation, significant numbers of 2000 mg/kg/day dose group rats had labored breathing, ungroomed coat and decreased motor activity. The 1000 and 2000 mg/kg/day dosages of the test article caused weight loss after the first dosage; the 2000 mg/kg/day dosage tended to cause reduced body weight gains during the premating period. During gestation, the 2000 mg/kg/day dosage tended to reduce maternal body weights on days 10 and 16 of gestation. The 2000 mg/kg/day dosage of the test article also tended to reduce maternal body weight gain during lactation, and maternal body weight was significantly reduced on day 4 postpartum, as compared with the control group value. Absolute and relative feed consumption values tended to be reduced for the 2000 mg/kg/day dosage group during the premating, gestation and lactation periods. The high dosage group litters tended to have reduced pup body weights on day 4 postpartum. No biologically relevant or statistically significant differences occurred among the groups in the average number of implantations, the duration of gestation, the percentage of dams delivering one or more live pups and the pup viability index. Administration of heptanoic acid to dams at dosages as high as 2000 mg/kg/day did not cause malformations or gross lesions in the pups.

The maternal no-observable-adverse-effect-level (NOAEL) for heptanoic acid was less than 200 mg/kg/day. The 200 mg/kg/day dosage of heptanoic acid caused significant numbers of rats to have rales. The 1000 and 2000 mg/kg/day dosages caused deaths and weight losses; maternal body weight gain and feed consumption values tended to be reduced for the 2000 mg/kg/day dosage group throughout the study, with maternal body weights significantly reduced on days 10 and 16 of gestation and day 4 of lactation. The NOAEL for heptanoic acid in the offspring was 1000 mg/kg/day. The 2000 mg/kg/day dosage tended to reduce pup body weight on day 4 postpartum.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information
 Two GLP guideline studies (OECD 414) are available in rats (Serota, 1982) and rabbits (Bentz, 2015).
    
    
    
    

 

In an embryo/fetal toxicity and teratogenesis study (Serota, 1992) , groups of 22 pregnant Sprague-Dawley rats were given 0 (corn oil vehicle), 1000 mg/kg bw/day of heptanoic acid on days 6 -15 of pregnancy. Measurement of maternal body weights and food consumption and gross pathology revealed no evidence of maternal toxicity. Measurement of mean ovarian weight, uterine weight, litter size, pregnancy rates, corpora lutea, implantation sites, implantation efficiency, fetal viability, fetal size and sex, gross pathology, and visceral and skeletal examinations of fetuses revealed that there was no evidence of embryo toxicity, fetal toxicity, or teratogenesis related to administration of heptanoic acid.

 

In an embryofetal toxicity and teratogenesis study (Bentz, 2015), the test item, Heptanoic acid (batch No. 1404017), 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.

 

Link to relevant study records

Referenceopen allclose all

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.
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
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.
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.
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.
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
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.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: stomach
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.
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
Abnormalities:
no effects observed
Developmental effects observed:
no

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.

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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Study period:
From: 7 October 1982 To: 7 November 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 2e: Study well documented, meets generally accepted scientific principles, acceptable restrictions.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to determine dose levels that would be tolerated in pregnant females for a subsequent teratology screening study. Test substance was administered by oral intubation at three different dose levels from days 6-15 of gestation. Criteria evaluated for compound effect were mortality, clinical signs, body weights, food consumption, water consumption, gross pathology, and ovarian and uterine data.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: 12 weeks old
- Weight at study initiation: 177-285 g
- Fasting period before study: no
- Housing: no data
- Diet: ad libitum (Purina Rodent Laboratory Chow)
- Water: ad libitum (tap water)
- Acclimation period: three weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 25-56
- Air changes: no data
- Photoperiod: no data


IN-LIFE DATES: From: 7 October 1982 To: 7 November 1982
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: appropriate amounts of each compound were weighed for each dose level and added to the required volume of vehicle. Each solution was then stirred for five minutes.



Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: a maximum of sixteen days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Days 6 through 15 of gestation
Frequency of treatment:
Daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, and 15 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 16
- Organs examined: lung, kidneys and stomach


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
- Other:
Fetal examinations:
None
Statistics:
Individual body weights, body weight changes, food consumption values, and water consumption values of the control group were compared statistically to data of the treated groups of the same sex by Bartlett's test for homogeneity of variance. This was followed by a one-way classification analysis. If ANOVA of homogeneous data was significant, Scheffe's multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA heterogeneous data was significant, Games and Howell's multiple pairwise comparison procedure was used to compare the group mean values. Survival through Day 16 was analyzed by life table technique. Ovarian and uterine data were analyzed on a per litter basis using a nonparametric comparison of group means. All analyses were evaluated at the 5.0 % one-tailed probability level.
Indices:
Implantation efficiency
Percent resorptions
Percent dead fetuses
Percent live fetuses
Historical control data:
No
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No apparent treatment-related effects were observed. Effects in group dosed with heptanoic acid were comparable to control.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this test, heptanoic acid was well tolerated by female. Indeed, no toxicity was observed.
Executive summary:

In this range finding study (Hazleton, 1982), heptanoic acid was administered by oral intubation at three different dose levels (six inseminated females/dose level) from Days 6 -15 of gestation. One group of six inseminated females received a corn oil vehicle and served as the control group. Criteria evaluated for compound effect were mortality, clinical signs, body weights, food consumption, water consumption, gross pathology, and ovarian and uterine data. No apparent treatment-related effects were observed. Effects in group dosed with heptanoic acid were comparable to control. Based on these results, the dose level of 1000 mg/kg/bw was chosen for the main study.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 may 1982 To 24 June 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 2b: Guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only one dose was used but the dose was chosen with the range finding study
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: Crl:COBS
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Kingston, New York
- Age at study initiation: sexually mature animals
- Weight at study initiation: 234.6 g (+/- 20.66 ) (female control)/ 233.7 g (+/- 21.25)
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum (Purina Rodent Laboratory Chow)
- Water: ad libitum (tap water)
- Acclimation period: approximately nine weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-25
- Humidity (%): 57 (+/- 4.8)
- Air changes (per hr): no data
- Photoperiod : 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: 11 May 1982 To: 24 June 1982
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The amount of compound required was weighed, on an Arbor balance, into pre-calibrated graduated beakers, filled to volume with corn oil, and stirred on a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): 80235
- Purity: no data
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until gestation or for a 3-week period

Duration of treatment / exposure:
From Days 6 through 15 of gestation
Frequency of treatment:
Daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: preliminary study results (Maternal tolerance study in rats from Hazleton, 1982)
- Rationale for animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 6, 9, 12, 15, and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Brain, pituitary, thymus, lung, liver, spleen, kidney, stomach, intestines
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 early resorptions: Yes
- Number of late resorptions: Yes
- Number of resorptions: Yes

- Other: number and distribution of dead and live fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one-third per litter
- Skeletal examinations: Yes: two-thirds per litter
- Head examinations: Yes: all per litter
Statistics:
Survival was analyzed by the National Cancer Institute Package.
Mean values were analyzed by Box's test for homogeneity of variances. This test was followed by a one-way classification analysis of variance if the variances proved to be homogeneous. If the variance proved to be heterogeneous, a rank transformation of data was performed, which was followed by Box's test and ANOVA. If ANOVA of untransformed or transformed data was significant, Dunett's T-test was used for control vs. treatment group mean comparisons. Pregnancy rates were analyzed by a test of multiple proportions using one degree of freedom Chi-square test with Yates' continuity correction.
Percentage values were compared to those of the control group by Fisher's "Exact" test for proportions. All pairwise comparisons were evaluated at 5.0 % probability (one-tailed) level.
Indices:
Overall survival rate (percent) = (number of female surviving to Day 20/ number of females placed in study) x 100
Pregnancy Rate (percent) = (Number of females pregnant/ number of females placed in breeding) x 100
Mean implantation Efficiency (percent) = Group mean of ([implantation per litter/corpora lutea per litter] x 100)
Mean Incidence of resorptions (percent) = Group mean of ([resorptions per litter/implantations per litter] x 100)
Mean Incidence of fetal mortality (percent) = group mean of ([dead fetuses per litter/implantations per litter] x 100)
Mean incidence of fetal viability (percent) = Group mean of ([live fetuses per litter/implantations per litter] x 100
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
The survival rates were comparable to control. No deleterious effects were reported.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No visceral and/or skeletal anomalies were noted.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1:Summary of ovarian, uterine, and litter data

control C-181
Mean number of Corpora lutea 15.9 14.7
Mean number of Implantations 13.5 12.8
Mean number of resorptions 1.2 0.6
Mean number of fetuses (dead) 0.0 0.0
Mean number of fetuses (live) 12.4 12.2
Mean implantation efficiency (%) 85.7 88.0
Mean incidence of resorption (%) 9.2 4.3
Mean incidence of fetal mortality (%) 0.0 0.0
Mean incidence of fetal viability (%) 90.8 95.7

Table 2:Summary of gross pathology findings in fetuses

Control C-181
Number of fetuses examined 191 175
Number of fetuses appearing normal 169 166
Pathology observed - Ureter dilated
- ureter hydroureter
- ureter undulated
- umbilical hernia
- situs inversus
- small
- Ureter dilated
- ureter hydroureter
- ureter undulated
Conclusions:
Under the conditions of this test, heptanoic acid did not induce maternal or embryo/fetal toxicity and teratogenic effects.
Executive summary:

In this study (Serota, 1982), the embryo/fetal toxicity and teratogenic effects of Heptanoic acid was evaluated when administered by oral gavage to pregnant rats from Days 6 through 15 of gestation. The test material was administered to a group of 22 female rats at a single dose level of 1000 mg/kg bw. The control group received only corn oil. Maternal, ovarian, uterine, litter, and fetal data were evaluated for evidence of compound effects. No maternal toxicity was observed. No fetotoxic effects (resorptions, fetal viability, fetal weights and fetal lengths) were observed. In conclusion, no incidence of teratogenic effects was noted in offspring. The maternal no-observable-adverse-effect-level (NOAEL) for heptanoic acid was 1000 mg/kg/day. The NOAEL for heptanoic acid in the offspring was 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2014 - 12 June 2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
other: Preliminary study to the OECD 414 guideline study
Deviations:
no
GLP compliance:
no
Remarks:
The GLP study is not required for a preliminary study
Limit test:
no
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 at study initiation: approximately 17-19 weeks old on the day of treatment
- Mean body weight at study initiation: 3690 g (range: 3410 g to 4025 g)
- Fasting period before study: no
- Housing: individually housed in noryl cages (Tecniplast, 65.3 cm x 65.3 cm x 45 cm)
- Diet: pelleted diet “type 110C”, batch Nos. 13353 and 14021 (SAFE, Augy, France)
- 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 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): 8h dark/16h light.

IN-LIFE DATES: 30 April 2014 to 12 June 2014.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous solution at 0.5% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. The test item 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 (Teflon®-coated magnetic stir bar had to be used to stir the doses 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) considered to cover the highest concentration used in this study (333 mg/mL),
- concentration of 30 mg/mL: stability (5 hours at room temperature and protected from light) 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.

VEHICLE
- Concentration in vehicle: 33.3, 100 and 333 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg/day.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable.
Details on mating procedure:
Insemination at the test site facility.
The day of insemination was designated Day 0 p.c.
Duration of treatment / exposure:
Day 6 to Day 28 p.c. inclusive.
Frequency of treatment:
Daily
Duration of test:
23 days
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:
5 inseminated 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 maximum tolerated dose study in non-pregnant New Zealand White rabbits (CiToxLAB France/Study No. 40896 TSL). In this study, there were no effects on clinical condition, mean body weight changes, mean food consumption and no macroscopic post-mortem findings after 7 days at 1000 mg/kg/day in 0.5% (w/v) carboxymethylcellulose aqueous solution.

- Rationale for animal assignment: computerized stratification procedure based on the last body weight recorded before insemination.
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: once a day before the treatment period and at least twice a day during treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: from arrival, the animals were 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 of day, for the recording of clinical signs.

BODY WEIGHT (GAIN):
- Time schedule: twice a week before insemination and 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 animal was recorded twice during the week before insemination and for the following intervals: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.. Any obvious spillage of food was documented.

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: No
- Skeletal examinations: No
- Head examinations: No
- Other: number dead and live and body weight.
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
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
At termination on Day 29 p.c., there were 3/5, 4/5, 4/5 and 3/5 dams with live fetuses in the control, 100, 300 and 1000 mg/kg/day groups, respectively.
There were no premature deaths and the main clinical sign was dryness around the mouth at 1000 mg/kg/day (preceded by erythema in one female), which was considered as minor toxicological significance.
At 1000 mg/kg/day, the transient statistically significantly lower mean food consumption noted during the first 3 days of treatment ( 32% vs. controls, p<0.05) was considered to be related to the test item treatment. Higher mean body weight gain from days 19 to 24 p.c. (+152 g/female/day vs. +65 in controls, p<0.01) was considered to be minimally toxicologically significant and there were no significant test item-related effects on mean body weights and mean body weight gain over the treatment period (Days 6-29 p.c.). No relevant changes in mean body weight or food consumption were noted at 100 or 300 mg/kg/day.
There were no effects on mean body weight, mean carcass weight, mean net body weight change, mean gravid uterus weight and mean hysterectomy data at any dose-level. There were no toxicologically relevant macroscopic changes at necropsy.
Dose descriptor:
other: Maximum Tolerated Dose
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: at external fetal examination

Details on embryotoxic / teratogenic effects:
There were no effects on mean fetal body weight as well as no external variations or malformations at any dose-level.
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
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The test item was administered by gavage, once daily, from Days 6 to 28 p.c., inclusive, to inseminated New Zealand White female rabbits at dosages of 100, 300 and 1000 mg/kg/day.
On the basis of the results obtained in this study, the dose-level of 1000 mg/kg/day was not considered to have exceeded the maximum tolerated dose in pregnant females and was considered relevant to be used for a main prenatal development study in rabbits.
Executive summary:

The objective of this non-GLP preliminary 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 100, 300 or 1000 mg/kg/day, in order to select dose-levels for a further main study.

.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 and examined for external abnormalities.

 

At 1000 mg/kg/day, the transient statistically significantly lower mean food consumption noted during the first 3 days of treatment (-32% vs. controls, p<0.05) was considered to be related to the test item treatment. Higher mean body weight gain from days 19 to 24 p.c. (+152 g/female/day vs. +65 in controls, p<0.01) was considered to be minimally toxicologically significant and therewere no significant test item-related effects on mean body weights and mean body weight gain over the treatment period (Days 6-29 p.c.). No relevant changes in mean body weight or food consumption were noted at 100 or 300 mg/kg/day.

There were no effects on mean body weight,mean carcass weight, mean net body weight change, mean gravid uterus weight and mean hysterectomy data at any dose-level. There were no toxicologically relevant macroscopic changes at dams necropsy and no effects on mean fetal body weight as well as no external variations or malformations at any dose-level.

 

On the basis of the results obtained in this study, the dose-level of 1000 mg/kg/day was not considered to have exceeded the maximum tolerated dose in pregnant females and was considered relevant to be used for a main prenatal development study in rabbits.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an embryo/fetal toxicity and teratogenesis study (Serota, 1992) , groups of 22 pregnant Sprague-Dawley rats were given 0 (corn oil vehicle), 1000 mg/kg bw/day of heptanoic acid on days 6 -15 of pregnancy. Measurement of maternal body weights and food consumption and gross pathology revealed no evidence of maternal toxicity. Measurement of mean ovarian weight, uterine weight, litter size, pregnancy rates, corpora lutea, implantation sites, implantation efficiency, fetal viability, fetal size and sex, gross pathology, and visceral and skeletal examinations of fetuses revealed that there was no evidence of embryo toxicity, fetal toxicity, or teratogenesis related to administration of heptanoic acid.

In an embryofetal toxicity and teratogenesis study (Bentz, 2015), the test item, Heptanoic acid (batch No. 1404017), 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.

 

Justification for classification or non-classification

Based on the lack of histopathology of reproductive organs including the testes and ovaries in repeat dose study, the lack of effect on female fertility, reproduction organs and prenatal paramaters at dose-level of 1000 mg/kg/day, no classification for fertility is warranted according to CLP regulation.

Based on the lack of effects on embryonic and fetal development in the previous cited study at dose-levels of 1000 mg/kg/day, no classification for developmental toxicity is required for Heptanoic acid according to CLP regulation..

Additional information