Registration Dossier

Administrative data

Description of key information

There are two repeated toxicity studies performed according to GLP:


1-Repeated Dose 28-day oral rat toxicity study (similar to OECD 407- Terrill, 1990)
Rat (female/male): NOAEL = 1750 mg/kg bw/day and LOAEL = 3500 mg/kg bw/day (similar to OECD 407)


2-Repeated dose 90-days oral rat toxicity study (similar to OECD 40- Bentz, 2015)
Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2014 - 14 August 2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
addition of seminology and estrus cycle examinations
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: approximately 5 weeks old on the first day of treatment
- Mean body weight at study initiation: The males had a mean body weight of 135 g (range: 116 g to 152 g) and the females had a mean body weight of 123 g (range: 105 g to 143 g)
- Housing: the animals were housed by two, by sex and group, in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 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.

IN-LIFE DATES: 14 May 2014 to 14 August 2014.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. It was mixed with the required quantity of vehicle. Care was taken to not use plastic or metal but glass materials with the test item or test item formulations.
No correction factor was applied.
Dose formulations were prepared and stored according to the available stability data. They were delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: suitable solution obtained with this vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 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: not assessed, dose formulation is a solution
Stability: stable after 11 days of storage at room temperature and protected from light from 10 to 200 mg/mL.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of previous studies.

In a 4-week rat toxicity study, Heptanoic acid was administered daily by the oral route to male and female Sprague Dawley rats at dose-levels of 0, 875, 1750 and 3500 mg/kg/day for 27 days. The test item was administered in corn oil. In this study, the animals given 3500 mg/kg/day exhibited wheezing, lower body weight change and total food consumption (males only), gross lesions of the stomach and microscopic lesions in the non-glandular region of the stomach. The few signs exhibited by the animals receiving 1750 mg/kg/day, or less, suggested that these animals were rather unaffected by treatment (wheezing in 4/10 females at 1750 mg/kg/day). Based on decreased body weights and food consumption, gross lesions of the stomach, and microscopic lesions of the non glandular region of the stomach at 3500 mg/kg/day, the Lowest Observable Adverse Effect Level (LOAEL) and the No Observable Adverse Effect Level (NOAEL) for Heptanoic acid were concluded to be 3500 and 1750 mg/kg/day, respectively.

In a reproductive and developmental screening toxicity test, 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 for 7 days before mating, through mating, gestation and until Day 4 post-partum. The maternal 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 with rales. The 1000 mg/kg/day dosage caused 1/10 death during gestation, rales and a slight body weight loss between Days 1 and 2 of treatment. The 2000 mg/kg/day dosage caused three deaths during the study, mostly rales, and weight losses between Days 1 and 2 of treatment; maternal body weight gain and food 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 the progeny was 1000 mg/kg/day based on reduced pup body weight on Day 4 post-partum at 2000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORTALITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

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

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, then on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week, over a 7 day period, during the study.

OPHTHALMOLOGIC EXAMINATION:
- Time schedule: on all animals before the beginning of the treatment period and on control and high-dose animals on one occasion at the end of the treatment period.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: once, in week 11 (before the daily treatment).

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

HISTOPATHOLOGY:
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table for animals of the control and high-dose groups (groups 1 and 4), on forestomach for animals of the low- and intermediate-dose groups (groups 2 and 3) and on all macroscopic lesions.

In addition testicular staging was performed for control and high-dose males (groups 1 and 4).
Other examinations:
Estrus cycle
- Time schedule: for 21 consecutive days before the end of the treatment period.

Seminology
- Time schedule: before sacrifice at the end of the treatment period under anesthesia on each male.
Epididymal sperm motility
Epididymal sperm morphology
Epididymal sperm count
Testicular sperm count
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
At 1000 mg/kg/day, one male died during treatment in week 12 and one female was found cannibalised in week 4. No toxicologically relevant clinical signs were observed in both animals before death. At necropsy the male had red and firm lungs and at microscopic examination, both animals had slight congestion/hemorrhage and moderate (female) or marked (male) edema in the lungs. Pathology examination did not allow determining the cause of deaths.
These deaths were considered to be likely unrelated to the test item administration (low incidence of deaths, different time of death, and different kind of deaths).

CLINICAL SIGNS:
There were no Heptanoic acid-related clinical signs in animals treated at 100 or 300 mg/kg/day during the study.

At 1000 mg/kg/day, ptyalism was observed on several occasions in 6/10 females and in 8/10 males and was considered to be of minimal toxicological significance.
Loud breathing was noted in one female and in one male treated at 1000 mg/kg, from Day 3 to Day 14 or 17. This clinical sign was considered to be of no toxicological significance since it was transient and with a low incidence.

A mass on mammary right side was observed in one male treated at 100 mg/kg/day, from week 6 until the end of the study (adenocarcinoma seen at microscopic examination). This was considered incidental since it was observed in one animal without relationship to dose-levels.
Other clinical signs (alopecia, thinning of hair, increase in size of urogenital region or on lip, abnormal growth of teeth, nodosities on right forelimb, soiled boy parts, chromorhynorrhea, scabs) were observed in a few animals among the test item-treated groups, and were deemed incidental since they can be commonly seen in animals from this species and in studies of this duration, were transient and/or showed no relationship to dose-levels in incidence or severity.

BODY WEIGHT:
There were no toxicologically relevant Heptanoic acid-related changes in mean body weight and mean body weight gain during the study.
There were occasional statistical differences in mean body weight gains at 100, 300 and 1000 mg/kg/day; they were considered to be fortuitous as there was no dose-relationship and they were transient and isolated.

FOOD CONSUMPTION:
At 1000 mg/kg/day and compared to controls, a statistically significant lower or higher mean food consumption was observed in week 9 (female; -23% from controls) or in weeks 12 and 13 (males; +10 and +16%). This was considered as of limited toxicological importancenot toxicologically relevant since it was transient, slight and/or isolated (transient and/or less than ± 20% of differences from controls)..
There were no toxicologically relevant Heptanoic acid-related effects on mean food consumption at the lower dose-levels.

OPHTHALMOLOGIC EXAMINATION:
There were no Heptanoic acid-related effects on ophthalmological parameters.

ESTRUS CYCLE:
There were no statistical significant Heptanoic acid-related changes on mean estrous cycle length and mean number of cycles. However a trend to an increase in mean estrous cycle length was observed in females from the dose-level of 300 mg/kg/day. This was considered to be Heptanoic acid-related but of minor no toxicological significance in absence of statistical significance and of differences in estrous cycles at microscopic examination of the genital tract.

NEUROBEHAVIOURAL EXAMINATION:
There were no toxicologically relevant Heptanoic acid-related effects at the Functional Observation Battery including motor activity (horizontal movements and rearing).

Differences from controls in reactivity to handling, exophtalmos, urination, pupil size, pupillary reflex or forelimbs grip strength were observed in isolated animals (generally in males) at all dose-levels. In view of the slight severity and incidence and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the Heptanoic acid treatment. Other changes including alopecia, scabs and increase in size of urogenital region were observed in some animals in all groups, correlated with clinical signs observed and were deemed incidental.

At 300 mg/kg/day, lower number of horizontal movements and rearing were noted in 1/10 males compared to controls; there were no correlating clinical signs during the study. This finding was observed with a low incidence, without dose relationship and was therefore not considered Heptanoic acid-related.

HAEMATOLOGY:
There were no toxicologically relevant Heptanoic acid-related effects in mean hematology parameters. The statistical differences observed sporadically on isolated parameters were not considered biologically relevant.
At 100 mg/kg/day, there was one male with changes in white (mainly neutrophils) and red blood cell counts, hemoglobin concentration, hematocrit (PCV) and reticulocyte percentage. These changes were considered incidental since they were of low amplitude and not dose-related.

CLINICAL CHEMISTRY:
There were no toxicologically relevant Heptanoic acid-related effects in mean biochemistry parameters. The statistical differences observed sporadically on isolated parameters were not considered biologically relevant.

URINALYSIS:
There were no toxicologically relevant Heptanoic acid-related effects on mean urinalysis parameters.
A dose-related higher presence of magnesium ammonium phosphate crystals was noted in females from 300 mg/kg/day (mean group grade of 1.4 and 1.7 vs. 0.8 in controls) but as there were no other relevant findings in laboratory investigations and renal pathology they were considered with no toxicological importance. There was no dose-relationship in this parameter in males despite the presence of a few males with more numerous magnesium ammonium phosphate crystals than in controls.

SEMINOLOGY:
There were no toxicologically relevant Heptanoic acid-related effects on epididymal sperm motility, morphology and count or on testicular sperm head count and daily sperm production rate.

When compared with controls, mean testicular sperm count and daily sperm production rate were lower at 1000 mg/kg/day (-19% and -18%, respectively). However, these parameters can be highly variable in Sprague-Dawley rats (Marty et al., 2009) and there were no effects on epididymides and
testes at microscopic examination. Therefore, the variations noted in mean testicular sperm counts were not considered to be toxicologically significant.

Azoospermia was observed in 1/10 males treated at 300 mg/kg/day. This was considered to be incidental in view of the isolated occurrence and since no relationship to dose-levels was noted.

ORGAN WEIGHTS:
No test item-related organ weight differences were observed.
The few organ weight changes were considered to be of minor magnitude, not dose-related and/or the contribution of slightly higher body weight in males at 100 or 1000 mg/kg/day. In the absence of associated microscopic findings, these differences were considered not to be related to test item administration.
This included the higher absolute and relative spleen weights in males at 1000 mg/kg/day and the lower mean absolute and relative-to-body thymus weights in females treated at 100 mg/kg/day.

GROSS PATHOLOGY:
Unscheduled deaths
One male treated at 1000 mg/kg/day died during treatment on Day 78 and had red and firm lungs at necropsy. The cause of death of this animal could not be established.

One female treated at 1000 mg/kg/day and found dead on Day 26 was cannibalized. The cause of death of this animal could not be established.

Terminal sacrifice
There were no test item-related gross findings.

The few gross findings were seen with similar incidence in both control and test item-treated animals, were seen only at minor incidence in the low or intermediate dose-group and/or correlated with common background findings. Thus they were considered not to be related to test item administration.

HISTOPATHOLOGY:
Unscheduled deaths
One male treated at 1000 mg/kg/day died during treatment on Day 78 and had red and firm lungs at necropsy, correlated with slight congestion/hemorrhage and marked edema in this organ. In the absence of any specific gross or microscopic findings, the cause of death of this animal could not be established.

One female treated at 1000 mg/kg/day and found dead on Day 26 was cannibalized. Except non specific slight congestion/hemorrhage and moderate edema in the lungs, no other noteworthy gross or microscopic findings were noted. Consequently, the cause of death of this animal could not be established.

Although the death of these high-dose group animals could not be explained, a relationship to test item was considered to be unlikely in view of the absence of noteworthy microscopic lesions, of the low incidence of these deaths and of the absence of toxicologically relevant findings in these animals.

Terminal sacrifice
Minimal to slight test item-related hyperplasia of squamous cells (or acanthosis) with hyperkeratosis were observed in the forestomach from males and females treated at 300 or 1000 mg/kg/day. In the absence of associated degenerative or inflammatory reaction, these findings were considered as non-adverse and secondary to the chemical properties of the test item; therefore they were considered to be related to the treatment with the test item.

The other microscopic findings were considered to be unrelated to test item since they were seen with similar incidence in control animals, at low severity and incidence were not dose-related and/or corresponded to background findings seen occasionally in the untreated rats of these strain and age. This included the slight hypertrophy of tubular renal cells in one female treated at 1000 mg/kg/day.

There were no differences regarding the estrous cycle between test item-treated and control females at microscopic examination of the genital tract.

A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. No test item-related findings were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The test item, Heptanoic acid, was administered daily to Sprague-Dawley rats, by oral route, at dose levels of 100, 300 or 1000 mg/kg/day for 13 weeks.
There were no adverse findings in the study.
The test item-related hyperplasia of squamous cells with hyperkeratosis observed in the forestomach from males and females treated at 300 or 1000 mg/kg/day were considered as non-adverse because of their low magnitude and because they were not associated with any degenerative or inflammatory reaction.
Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item at dose-levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). In addition, one group of ten males and ten females received the control item alone (corn oil) and acted as a control group (group 1). The control and test items were administered by oral route during a 13-week period at a constant dosage-volume of 5 mL/kg/day.

The concentration of the dose formulation was checked in study weeks 1, 5, 9 and 13.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed once a week. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and in control- and high-dose groups at the end of the treatment period. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis investigations were performed on all animals. Estrous cycle was monitored on all females during the last 3 weeks of the treatment period.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Sperm investigations were performed at sacrifice of the males. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1, 5, 9 and 13 were within the acceptable range of variation when compared to the nominal values (±10%).

 

There were no heptanoic acid-related deaths during the study and no clinical signs at 100 and 300 mg/kg/day. At 1000 mg/kg/day, treatment-related clinical signs were limited to ptyalism considered to be ofminimal toxicological significance. No findings were observed at ophthalmology examination. There were no toxicologically significant effects on mean body weight, mean body weight change and at hematology, blood biochemistry and urinalysis investigations.

At 1000 mg/kg/day and compared to controls, a statistically significant lower or higher mean food consumption was observed in week 9 (female; -23% from controls) or in weeks 12 and 13 (males; +10 and +16%) considered of limited toxicological importance.

There were no relevant effects at the Functional Observation Battery including motor activity and inmean sperm parameters (epididymal motility, morphology and count, testicular sperm count). There were no toxicologically relevant effects on mean estrous cycle.

 

There were no test item-related organ weight differences or gross findings. At microscopic examination, non-adverse findings (hyperkeratosis and hyperplasia of squamous cells) in the forestomach were found in males and females treated at 300 or 1000 mg/kg/day. The hyperplasia of squamous cells and the hyperkeratosis were considered as non-adverse because of their low magnitude and because they were not associated with any degenerative or inflammatory reaction.


Conclusion

The test item was administered daily to Sprague-Dawley rats, by oral route, at dose-levels of 100, 300 or 1000 mg/kg/day for 13 weeks.

There were no adverse findings in the study.

The test item-related hyperplasia of squamous cells with hyperkeratosis observed in the forestomach from males and females treated at 300 or 1000 mg/kg/day were considered as non-adverse because of their low magnitude and because they were not associated with any degenerative or inflammatory reaction.

Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

28-days oral study in rats (Terrill 1990) according to OECD 407:

Group (10/sex/group) of male and female Sprague-Dawley rats were given dose levels of 0, 850, 1750, or 3500 mg/kg bw of heptanoic acid by gavage in corn oil (10 ml/kg) daily for 27 days. In this study, clinical signs were monitored twice weekly and body weights and food consumption were measured weekly. At necropsy, blood was drawn and clinical chemistry, hematological determinations, and organ weights were measured. A variety of tissues were prepared and preserved in 10% formalin. All tissues from the control and high-dose groups and tissue from the heart, liver, kidneys, and gross lesions from the low- and mid-dose group were embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically.

Six animals (3 low-dose females, two high-dose females and one high-dose male) died during the study. The cause of death for five of the six unscheduled deaths was considered related to gavage administration. In one low-dose females marked pyelonephritis and inflammation throughout the lower urinary tract with an associated urinary calculus were present which was considered as a typical spontaneous lesion for this age and strain of test animal. Treatment-related findings included physical signs such as salivation, languid behavior, polypnea, dyspnea, tremors, wheezing, thin appearance, ataxia and hunch posture; these findings were observed, predominately in the high-dose animals, approximately one hour post-dosing. At the weekly observations which were performed prior to dosing only respiratory distress (wheezing) was exhibited by the high-dose males and mid- and high-dose females. The mean body weight change and mean total food consumption in high-dose males was significantly decreased compared to control males. Treatment-related pathology findings included hyperkeratosis that was present in the non-glandular region of the stomach of almost all of the high-dose male and female animals sacrificed at termination. Hyperkeratosis was generally diffuse and was graded minimal to moderate. This change correlates with the rough and thickened mucosa noted in the non-glandular region of the stomach at necropsy. The presence of this change suggests a mild local irritating effect associated with the gavage administration of heptanoic acid. There was however no evidence of degeneration/necrosis or inflammation. Microscopic examination of the non-glandular region of the stomach in the low- and mid-dose groups showed no evidence of hyperkeratosis.

In conclusion the high-dose animals (3500 mg/kg/day of heptanoic acid) exhibited wheezing; decrease in body weight change (11.5 %) and total food consumption (9.2 %) (males only); gross lesions of the stomach and microscopic lesions in the non-glandular region of the stomach (hyperkeratosis). The few signs exhibited by the animals receiving 1750 mg/kg/day, or less, suggested that these animals were unaffected by treatment.

Based on decreased body weights and food consumption, gross lesions of the stomach, and microscopic lesions of the non-glandular region of the stomach at 3500 mg/kg/day, the lowest observable adverse effect level (LOAEL) and the no observable adverse effect level (NOAEL) for heptanoic acid were concluded to be 3500 and 1750 mg/kg/day, respectively. Except for tissue already discussed, there was no evidence of histopathology of other tissue including the testes and ovaries.

90- days oral study in rats according to OCDE 408

 

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item at dose-levels of 100, 300 or 1000 mg/kg/day (groups 2 to 4). In addition, one group of ten males and ten females received the control item alone (corn oil) and acted as a control group (group 1). The control and test items were administered by oral route during a 13-week period at a constant dosage-volume of 5 mL/kg/day.

The concentration of the dose formulation was checked in study weeks 1, 5, 9 and 13.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed once a week. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and in control- and high-dose groups at the end of the treatment period. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis investigations were performed on all animals. Estrous cycle was monitored on all females during the last 3 weeks of the treatment period.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopicpost-mortemexamination. Sperm investigations were performed at sacrifice of the males. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1, 5, 9 and 13 were within the acceptable range of variation when compared to the nominal values (±10%).

 

There were no heptanoic acid-related deaths during the study and no clinical signs at 100 and 300 mg/kg/day. At 1000 mg/kg/day, treatment-related clinical signs were limited to ptyalism considered to be ofminimal toxicological significance. No findings were observed at ophthalmology examination. There were no toxicologically significant effects on mean body weight, mean body weight change and at hematology, blood biochemistry and urinalysis investigations.

At 1000 mg/kg/day and compared to controls, a statistically significant lower or higher mean food consumption was observed in week 9 (female; -23% from controls) or in weeks 12 and 13 (males; +10 and +16%) considered of limited toxicological importance.

There were no relevant effects at the Functional Observation Battery including motor activity and inmean sperm parameters (epididymal motility, morphology and count, testicular sperm count). There were no toxicologically relevant effects on mean estrous cycle.

 

There were no test item-related organ weight differences or gross findings. At microscopic examination, non-adverse findings (hyperkeratosis and hyperplasia of squamous cells) in the forestomach were found in males and females treated at 300 or 1000 mg/kg/day. The hyperplasia of squamous cells and the hyperkeratosis were considered as non-adverse because of their low magnitude and because they were not associated with any degenerative or inflammatory reaction.


Conclusion

The test item was administered daily to Sprague-Dawley rats, by oral route, at dose-levels of 100, 300 or 1000 mg/kg/day for 13 weeks.

There were no adverse findings in the study.

The test item-related hyperplasia of squamous cells with hyperkeratosis observed in the forestomach from males and females treated at 300 or 1000 mg/kg/day were considered as non-adverse because of their low magnitude and because they were not associated with any degenerative or inflammatory reaction.

Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.


Justification for classification or non-classification

No classification is warranted according to CLP criteria.