Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Mar 2022 to 06 Jul 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guideline 422. Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Justification of Route and Dose Levels
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid in rats (Test Facility Study No. 20312495, see Section 5), and in an attempt to produce graded responses to the test item. In this dose range finding study, no effects were observed on female survival, clinical signs, body weight (gain), food consumption, macroscopy and organ weights (kidney and liver) up to 1000 mg/kg/day. Therefore, this dose level was selected to be the high dose level in this Main study.
The high-dose level should produce some toxic effects, but not death nor obvious suffering.
The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

EC Number:

270-470-1

EC Name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Cas Number:

68441-66-7

Molecular formula:

not available due to complexity of the substance

IUPAC Name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Test material form:

liquid

Details on test material:

Identification: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Batch (Lot) Number: 2019194336
Expiry date: 03 June 2022 / 03 December 2023
Physical Description: Clear light yellow liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature

Additional information
Test Facility test item number: 212207/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name):
Trade names: HATCOL® 1106, HATCOL® 2926, HATCOL® 3326
CAS number: 68441-66-7
Stability at higher temperatures: Yes, maximum temperature: 175°C, maximum; duration: 10 minutes
Volatile: No
Specific gravity / density: 1.01
Solubility in water: <0.1 mg/L
Stability in water: Not indicated

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Justification of Test System and Number of Animals
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat.
Strain: Crl: WI(Han).
Condition: Outbred, SPF-Quality.
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Males 40.
Number of Females 48 (nulliparous and non-pregnant).
Number of Pups Expected: Approximately 480 pups (40 litters x 12 pups).
Target Age at the Initiation of the Pretest Period: Females: approximately 10-12 weeks.
Age at the Initiation of Dosing: Males 10 – 12 weeks old; Females 11 – 13 weeks old
Body Weight Range at the Initiation of Dosing: 271 – 316 g males; 183 – 242 g females

Animal Identification
Method: Each animal will be identified using a subcutaneously implanted electronic identification chip that is implanted prior to start of the pretest period (females) or treatment period (males).
Prior to start of the pretest period, reserve females will be numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment will receive identification by chip.
Pups will be identified on postnatal day (PND) 1. They will be randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurs the identification, the pups will be identified by tattoo on the feet.
Further identification marks may be applicable (e.g. tail mark with indelible ink), to be documented in the study file.

Environmental Acclimation
The animals will be allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of dosing (males).

Selection, Assignment, Replacement, and Disposition of Animals
A total of 40 females will be selected at randomization before initiation of the pretest phase.
Each selected female classified as not having regular estrous cycles during the pretest phase will be replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles will continue in the study. The supernumerary females will then be removed from the study, and their estrous cycle results will be kept in the raw data but will not be reported.
Animals will be randomly assigned to groups at arrival. Males and females will be randomized separately. Animals in poor health will not be assigned to groups.
At least upon receipt of the animals, a health inspection will be performed and any assigned animals considered unsuitable for use in the study will be replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
After initiation of dosing, study animals may be replaced during the replacement period with alternate animals in the event of accidental injury, non-test item-related health issues, or similar circumstances. The alternate animals may be used as replacements on the study within 1 to 3 days.
The disposition of all animals will be documented in the study records.
On PND 4, eight pups from each litter of equal sex distribution (if possible) will be selected to reduce variability among the litters. The non-selected pups will be culled on PND 4.

HUSBANDRY
Housing
Caging: On arrival and during the pretest (females only) and pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females will be cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males will be housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females will be individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females will be housed in Makrolon plastic cages (MIII type, height 18 cm). Pups will be housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, F0-animals will be housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Cages containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the
study records. Cages will be arranged on the racks according to a Latin-square model.
Animals will be separated during designated procedures/activities.

Cage Identification: Color-coded cage card indicating at least Test Facility Study No., group, animal identification number.

Animal Enrichment
Animals will be socially housed for psychological/environmental enrichment and will be provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.

Environmental Conditions
The target conditions for animal room environment will be as follows:
Temperature: 18 to 24 °C.
Humidity: 40 to 70%.
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures)
Ventilation: At least 10 air changes per hour.
The actual daily mean temperature during the study period was 19 to 21C with an actual daily mean relative humidity of 35 to 67%. The values that were outside the targeted mean humidity range occurred on two days with a minimum of 35% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study

Food
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
Type: Pellets (alternate diet may be provided on individual animal basis as warranted as approved by the Study Director).
Frequency: Ad libitum, except during designated procedures. During motor activity measurements, animals will not have access to food for a maximum of 2 hours.
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the Supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles. During motor activity measurements, animals will not have access to water for a maximum of 2 hours.
Analysis: Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Preparation of Formulations
Dose formulations will be divided into aliquots where required to allow to be dispensed on each dosing occasion.
The dosing formulations will be removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations will be kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle will be continuously stirred until and during dosing.
Adjustments will be made for specific gravity of the vehicle and test item. No correction will be made for the purity/composition of the test item.

Details on mating procedure:

Frequency: Daily, after a minimum of 14 days of treatment. The mating period will consist of a maximum of 14 consecutive days.
Procedure: Animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating will be confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day will be designated Day 0 post-coitum. Once mating has occurred, the males and females will be separated.
A maximum of 14 days will be allowed for mating, after which females who have not shown evidence of mating will be separated from their males. In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (nonselected male if possible) will be used for re-mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below will be performed using a validated analytical procedure (Test Facility Study No. 20312499). Full details of the study design and results can be found in Section 8 - Analytical methods-CRL Den Bsoch-20312499.

Concentration and Homogeneity Analysis
Storage Conditions: Temperature set to maintain 18-22°C.
Acceptance Criteria: For concentration, mean sample concentration results within or equal to ± 10% for solutions and ± 15% for suspensions of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.

Stability Analysis
Stability analyses will be performed, prior to the first dosing, in conjunction with the method development and validation study (Test Facility Study No. 20312499) to demonstrate if the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data will be retained in the study records of No. 20312499.

Duration of treatment / exposure:

Males: 29 days
Females: 42 – 64 days

Frequency of treatment:

Daily

Details on study schedule:

Dose Route: Oral gavage
Frequency: Once daily
Duration: Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Females will not be dosed during littering.
Pups will not be treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Method: The first day of dosing will be designated as Day 1 (exception: Alternate animals used for replacement after Day 1 will assume the day of the animal being replaced). The dose volume for each animal will be based on the most recent body weight measurement. The dose formulations will be stirred continuously during dose administration and doses will be given using a plastic feeding tube.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals per dose group (10 males/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

No further details specified in the study report.

Positive control:

Not required for a study of this design.

Examinations

Parental animals: Observations and examinations:

Mortality: All animals. Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.
Frequency: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency will be at least once daily.

Clinical Observations: All animals. Animals will be observed for specific clinical signs. The time of onset, grade and duration of any observed signs will be recorded. Signs will be graded for severity and the maximum grade will be predefined at 1, 2, 3 or 4. Grades will be coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs only its presence (i.e. maximum grade 1) will be scored.
Frequency: At least once daily, up to the day prior necropsy.

Arena Observations: All animals. Animals will be observed for clinical signs outside the home cage in a standard arena after dosing. The time of onset, grade and duration of any observed signs will be recorded.
Frequency: Once before the first administration of the test item and weekly during the Treatment Period.

Individual Body Weights: All animals. In order to monitor the health status animals may be weighed more often.
Frequency: On Day 1 of treatment (prior to dosing) and weekly thereafter. Mated females: On Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Food Consumption: All animals. Quantitively measured per cage.
Frequency: Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water Consumption: All animals. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.
Frequency: Regular basis through the study.

Functional Tests – F0-Generation
Frequency: Once during the Treatment Period. The 5 selected males per group
will be tested once during Week 4 of treatment and the 5 selected females per group will be tested once during the last week of lactation (i.e. PND 6-13). These tests will be performed after clinical
observations (including arena observation, if applicable).
Procedure: The following tests will be performed:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements , using a grip strength meter (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1 hour under normal laboratory light conditions) will be tested using the Kinder Scientific Motor Monitor System LLC, Poway, USA. Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or
movements of the head.

General Reproduction Data – F0-Generation
Frequency: Daily from the mating period onwards.
Procedure: Male number paired with, mating date, confirmation of pregnancy, and delivery day will be recorded. Palpation may be used to aid in confirmation of pregnancy.
The females will be allowed to litter. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started).
The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that are littering will be left undisturbed.
Cage debris of pregnant females will be examined for evidence of premature delivery. Signs of difficult or prolonged parturition will be recorded, if applicable.
Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, will be recorded, if applicable.

Clinical Pathology

Hematology
Hematology Parameters
White blood cells (WBC), Reticulocytes (absolute), Neutrophils (absolute), Red blood cell distribution (RDW), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Red blood cells (RBC), Mean corpuscular hemoglobin concentration (MCHC), Platelets
A blood smear will be prepared from each hematology sample. Blood smears are labelled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results.

Coagulation
Coagulation Parameters
Prothrombin Time (PT), Activated partial thromboplastin time (APTT)

Clinical Chemistry
Clinical Chemistry Parameters
Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total bilirubin, Chloride, Bile acids, Calcium, Urea, Inorganic phosphate (Inorg. Phos)

Thyroid Hormone
Thyroid Hormone Parameters
Thyroxine (T4), Thyroid-stimulating hormone (TSH; only if required)

After clotting and centrifugation, serum will be used as listed below.
F0-males: 150 μL serum for measurement of total T4 and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH) (added by study plan amendment if applicable).
F0-females: The serum will be used for possible future measurement of total T4 and TSH (added by study plan amendment if applicable).
Serum samples retained for possible future analysis will be maintained by the Test Facility in the freezer (≤ -75°C). Under these storage conditions, samples will be stable for 6 months.
Any remaining sample will be discarded prior to finalization of the report.

Oestrous cyclicity (parental animals):

Frequency: Daily vaginal lavage will be performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage will continue for those females with no evidence of copulation until termination of the mating period.
End of Treatment - on the day of necropsy, a vaginal lavage will also be taken to determine the stage of estrus. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously.
Procedure: Estrous cycles will be evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):

Not assessed as part of this study.

Litter observations:

Mortality: All pups. Pups will be observed within their cage unless necessary for identification or confirmation of possible findings.
Frequency: The number of live and dead pups will be determined on PND 1 and daily thereafter.

Clinical Observations: All pups. Only days on which clinical signs are present between the first and last litter check will be given in the respective report tables.
Frequency: At least once daily, up to the day prior to necropsy.

Individual Body Weights: All pups. Live pups will be individually weighted.
Frequency: On PND 1, 4, 7 and 13.

Sex: All pups. Sex will be externally determined.
Frequency: On PND 1 and 4.

Anogenital Distance: All pups. Anogenital distance (AGD) will be measured for all live pups. The AGD will be normalized to the cube root of body weight.
Frequency: On PND 1.

Areola/Nipple Retention: All males pups in each litter. Examination for the number of areola/nipples.
Frequency: On PND 13.

Culling: All litters. To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) will be selected. Blood samples will be collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, will not be done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable.

Thyroid Hormone
Thyroid Hormone Parameters: Thyroxine (T4); Thyroid-stimulating hormone (TSH; only if required)
After clotting and centrifugation, serum will be used as listed below.
PND 4 pups: The pooled serum will be used for possible future measurement of total T4 (added by study plan amendment if applicable).
PND 14-16 pups: 150 μL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH (added by study plan amendment if applicable).

Postmortem examinations (parental animals):

Unscheduled Deaths/Euthanasia
If an animal dies on study, a necropsy will be conducted and specified tissues will be saved, but not weighed. If necessary, the animal will be refrigerated to minimize autolysis.
Animals may be euthanized for humane reasons as per Test Facility SOPs. These animals will be deeply anesthetized using isoflurane and subsequently exsanguinated. They will undergo necropsy, and specified tissues will be retained, but not weighed.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia will have a terminal body weight recorded and will be deeply anesthetized using isoflurane and subsequently exsanguinated.

Scheduled necropsies are summarized below:
Males (which sire or fail to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which deliver: PND 14-16.
Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27.
Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
All males surviving to scheduled necropsy will be fasted overnight with a maximum of 24 hours before necropsy. Water will be available. F0-females will not be fasted overnight.

Necropsy
All animals will be subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites will be recorded for all paired females.

Organ weights
The organs will be weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Organ weights will not be recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs will be weighed together. Organ weights as a percent of body weight (using the terminal body weight) will be calculated.

Tissue Collection and Preservation
Representative samples of the tissues will be collected from all animals and preserved in 10% neutral buffered formalin or modified Davidson's solution as detailed in Test Facility SOPs. Additional tissue samples may be collected to elucidate abnormal findings.

HISTOLOGY AND MICROSCOPIC EVALUATION
Histology
Tissues will be embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.

Microscopic Evaluation
Tissues will be evaluated histopathologically. Target tissues identified by the study pathologist
during microscopic evaluation will be communicated to the Study Director; tissues will be
evaluated and reported.
Special stains may be used at the discretion of the pathologist to further characterize lesions and changes identified during routine evaluation of individual animals. Any special stains will be documented in the individual animal data.

Postmortem examinations (offspring):

TERMINAL PROCEDURES – F1-GENERATION
16.1. Method of Euthanasia – F1-Generation
Pups, younger than 7 days will be euthanized by decapitation.
All remaining pups (PND 7-16), except the pups selected for blood collection, will be euthanized by an intraperitoneal injection of sodium pentobarbital.
The pups selected for blood collection on PND 14-16 will be anesthetized using isoflurane and subsequently exsanguinated.

Unscheduled Deaths – F1-Generation
Recognizable fetuses of females that die spontaneously or are euthanized in extremis will be examined externally and sexed (both externally and internally, if possible). Live fetuses will be euthanized by decapitation.
Pups that die or are euthanized before scheduled termination will also be examined externally and sexed (both externally and internally, if possible). Pups found dead during the weekend can be fixed in identified containers containing 70% ethanol if not being subjected to necropsy on the same day. The stomach of pups not surviving to the scheduled necropsy date will be examined for the presence of milk, if possible. If possible, defects or cause of death will be evaluated.

Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups will be euthanized by decapitation. Sex will be determined both externally and internally. Descriptions of all external abnormalities will be recorded. From two surplus pups per litter, blood will be collected, if possible.
All remaining pups will be euthanized on PND 14-16. Sex will be determined both externally and internally. Descriptions of all external abnormalities will be recorded. Particular attention will be paid to the external reproductive genitals to examine signs of altered development. No full histopathological examination will be performed, however, if any abnormalities will be observed during determination of sex, abnormalities may be collected and fixed in 10% buffered formalin at discretion of the Study Director. In addition, the thyroid will be collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection), and will be preserved in 10% buffered formalin.

Statistics:

Parental Variables
Body Weight Gains: Calculated against the body weight on Day 1 (pre-mating and lactation periods) or Day 0 (post-coitum period).
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Statistics for Data Collected/Processed in ToxData
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions will be analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics will be performed according to the matrix below when possible, but will exclude semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons will be made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Reproductive indices:

Mating index (%): (Number of females mated / Number of females paired) x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating.

Fertility index (%): (Number of pregnant females / Number of females mated) x 100

Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100

Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Post-implantation survival index will be expressed as 100% when the number of offspring exceeds the number of implantation sites recorded.

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Offspring viability indices:

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number if live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100

Viability index (%): (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100

Lactation index (%): Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test material-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Note to clinical signs tables: For males, “repro period” represents the mating phase. For females, “repro period” represents the mating, post-coitum and lactation phase.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gains of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight of treated animals was comparable to the control level over the treatment period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test material-related changes were noted in hematology parameters.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test material.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related changes were noted in clinical chemistry parameters up to 300 mg/kg/day.
A lower cholesterol concentration was noted (0.71x of control, not statistically significant) for females treated at 1000 mg/kg/day. Individual values of 3/5 females were below the historical control range.
Any other changes in clinical chemistry parameters, regardless of reaching statistical significance, were considered to be unrelated to treatment with the test material as these occurred in the absence of a dose-related trend.

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of T4 in males were considered unaffected by treatment with the test material.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test material.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
Motor activity was considered not to be affected by treatment with the test material. All groups showed a similar motor activity habituation profile with in general a decreasing trend in activity over the duration of the Test Period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related alterations in organ weights.
The absolute weight of ovaries in females at 1000 mg/kg/day was statistically significant lower compared to the control group. This was considered incidental based on the lack of a dose response, the absence of a microscopic correlate and the fact that it was not significant when expressed relative to body weight.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test material. All females had regular cycles of 4 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was 1/10 couple (Male No. 12 and Female No. 52) of the 100 mg/kg/day group where the female was not pregnant. In addition, there was 1/10 couple (Male No. 28 and Female No. 68) of the 300 mg/kg/day group that did not mate.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material.

Mating Index
Mating index was considered not to be affected by treatment with the test material. Except for Female No. 68 (300 mg/kg/day), all females showed evidence of mating. In absence of a dose-response this was considered unrelated to treatment.
The mating indices were 100, 100, 90 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Precoital Time
Precoital time was considered not to be affected by treatment with the test material.
All females showed evidence of mating within 4 days, except for Female Nos. 63, 66 (300 mg/kg/day) and 75 (1000 mg/kg/day) for which mating was observed after 13 or 14 days of pairing. These longer precoital times are occasionally encountered in this type of study and in the absence of any dose-related occurrence this was considered unrelated to treatment with the test item.

Number of Implantation Sites
Number of implantation sites was considered not to be affected by treatment with the test material.
The slightly lower mean number of implantation sites noted at 100 mg/kg/day (11.7 vs. 13.8 in controls) was caused by the relatively low number of implantation sites of Female Nos. 59 and 60, who had 6 and 4 implantation sites, respectively. Based on the absence of a dose response, this was considered unrelated to treatment with the test material.

Fertility Index
Fertility index was considered not to be affected by treatment with the test material.
All mated females were pregnant, with the exception of Female No. 52 (100 mg/kg/day). As this occurred in the absence of a dose-response this was considered not related to treatment with the test material.
The fertility indices were 100, 90, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Developmental Data
Gestation Index and Duration
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test material. The gestation indices were 100% for all groups.

Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-Implantation Survival Index
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test material.
Post-implantation survival index was 87, 90, 96 and 92% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Litter Size
Litter size was considered not affected by treatment with the test material.
Live litter sizes were 11.8, 10.6, 11.9 and 11.4 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
A lower mean number of living pups was recorded at 100 mg/kg/day. This was related to the lower number of implantation sites in this dose group. In absence of a dose-response, this was considered unrelated to treatment with the test material.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups surviving until scheduled necropsy that were considered to be related to treatment with the test material.
The nature and incidence of the observed clinical signs remained within the range considered normal for pups of this age or the finding was limited to a control pup.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Live Birth Index
The live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test material.
The live birth indices were 98, 100, 99 and 97% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Two pups originating from the control group (one each in Litter Nos. 47 and 49), one pup at 300 mg/kg/day (Litter No. 61) and four pups originating from two litters at 1000 mg/kg/day (Nos. 75 and 80) were found dead at first litter check. For the dead pup(s) of Litter Nos. 47, 75 and 80, no milk was present in the stomach. In addition, beginning autolysis was observed for the dead pup of Litter No. 47. The death of these pups was considered to be unrelated to treatment with the test material, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test material.
Viability indices were 100, 100, 98 and 99% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Two pups originating from one litter at 300 mg/kg/day (Litter No. 67) and one pup at 1000 mg/kg/day (Litter No. 78) were missing on PND 3 or 4 and were most likely cannibalized. These missing pups were considered to be unrelated to treatment with the test material, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test material.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test material.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and corrected for body weight) in male and female pups was considered not to be affected by treatment with the test material.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test material had no effect on areola/nipple retention.
For none of the examined male pups nipples were observed on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups surviving until scheduled necropsy that were considered to be related to treatment with the test material.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age or were limited to a control group and were therefore considered not to be related to treatment with the test material.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

REPRODUCTION DATA SUMMARY

	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
Females paired Females mated Pregnant females Females with living pups on Day 1	10 10 10 10	10 10 9 9	10 9 9 9	10 10 10 10
Mating index (%) (Females mates / Females paired) * 100   Fertility index (%) (Pregnant females / Females mated) * 100   Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	100     100     100	100     90     100	90     100     100	100     100     100

 

PRECOITAL TIME

F0-GENERATION – POST COITUM

DAY OF THE PAIRING PERIOD	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
NUMBER OF FEMALES MATED 1 2 3 4 13 14	4 2 3 1 - -	6 - 4 - - -	1 2 3 1 1 1	3 - 5 1 1 -
MEDIAN PORECOITAL TIME MEAN PRECOITAL TIME N	2 2.1 10	1 1.8 10	3 5.0 9	3 3.5 10

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

IMPLANTATION SITES SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
AT NECROPSY Implantations	MEAN ST. DEV N	13.8 2.6 10	11.7 4.2 9	12.6 1.0 9	12.8 1.5 10

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

DEVLEOPMENTAL DATA

F0-GENERATION – LACTATION

	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
LITTERS             TOTAL	10	9	9	10
DURATION OF GESTATION             MEAN (+)             ST. DEV.             N	21.4 0.5 10	21.8 0.7 9	21.3 0.5 9	21.6 0.5 10
DEAD PUPS AT FIRST LITTER CHECK             LITTERS AFFECTED (#)             TOTAL             MEAN (+)             ST. DEV.             N	2 2 0.2 0.4 10	0 0 0.0 0.0 9	1 1 0.1 0.3 9	2 4 0.4 1.0 10
LIVING PUPS AT FIRST LITTER CHECK             % OF MALES / FEMALES (#)             TOTAL             MEAN (+)             ST. DEV.             N	47 / 53 118 11.8 4.2 10	47 / 53 95 10.6 4.0 9	49 / 51 107 11.9 1.4 9	41 / 59 114 11.4 1.9 10
POSTNATAL LOSS             % OF LIVING PUPS             LITTERS AFFECTED (#)             TOTAL (#)             MEAN (+)             ST. DEV.             N	0.0 0 0 0.0 0.0 10	0.0 0 0 0.0 0.0 9	1.9 1 2 0.2 0.7 9	0.9 1 1 0.1 0.3 10
CULLED PUPS             TOTAL	43	31	33	33
LIVING PUPS DAY 4 P.P.             TOTAL             MEAN (+)             ST. DEV.             N	75 7.5 1.1 10	64 7.1 2.0 9	72 8.0 0.0 9	80 8.0 0.0 10
BREEDING LOSS DAYS 5 – 13 P.P.             % OF LIVING PUPS AT DAY 4 P.P.             LITTERS AFFECTED (#)             TOTAL (#)             MEAN (+)             ST. DEV.             N	0.0 0 0 0.0 0.0 10	0.0 0 0 0.0 0.0 9	0.0 0 0 0.0 0.0 9	0.0 0 0 0.0 0.0 10
LIVING PUPS DAY 13 P.P.             % OF MALES / FEMALES (#)             TOTAL             MEAN (+)             ST. DEV.             N	51 / 49 75 7.5 1.1 10	47 / 53 64 7.1 2.0 9	53 / 47 72 8.0 0.0 9	46 / 54 80 8.0 0.0 10

Female No. 74, pup 9 (1000 mg/kg/day); the pup was inadvertently culled on Day 5 instead of Day 4 and was therefore included in “culled pups” and  not included in “living pups day 4 p.p.” and “breading loss day 5-13 p.p.”

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher’s Exact test significant at 5% (#) or 1% (##) level

 

DEVLOPMENTAL DATA

	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
Total number of offspring born Total number of uterine implantation sites Number of live offspring on Day 1 after littering Number of live offspring on Day 4 (before culling) Number of live offspring on Day 4 (after culling) Number of live offspring on Day 13 after littering	120 138 118 118 75 75	95 105 95 95 64 64	108 113 107 105 72 72	118 128 114 113 80 80
Post-implantation survival index (%) (Total number of offspring born/Total number of uterine implantation sites) * 100   Live birth index (%) (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100   Viability index (%) (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering) * 100   Lactation index (%) (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100	87     98       100       100	90     100       100       100	96     99       98       100	92     97       99       100

Female No. 74, pup 9 (1000 mg/kg/day); the pup was inadvertently culled on Day 5 instead of on Day 4 and was therefore excluded from the calculation of the lactation index

 

BODY WEIGHTS OF PUPS (GRAM)

F0-GENERATION – LACTATION

DAY	SEX		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
1	M	MEAN ST. DEV. N	6.3 0.5 10	7.1 0.8 9	6.2 0.5 9	6.8 0.7 10
	F	MEAN ST. DEV. N	6.1 0.5 10	6.5 0.4 9	5.8 0.6 9	6.4 0.8 10
	M+F	MEAN ST. DEV. N	6.2 0.5 10	6.7 0.5 9	6.0 0.5 9	6.6 0.7 10
4	M	MEAN ST. DV. N	9.6 1.1 10	10.7 1.2 9	9.3 1.1 9	10.1 1.2 10
	F	MEAN ST. DEV. N	9.3 1.1 10	9.9 0.8 9	8.9 1.0 9	9.7 1.2 10
	M+F	MEAN ST. DEV. N	9.4 1.1 10	10.2 0.9 9	9.1 1.0 9	9.9 1.2 10
7	M	MEAN ST. DEV. N	15.9 1.7 10	17.3 1.2 9	15.3 1.6 9	16.7 1.7 10
	F	MEAN ST. DEV. N	15.5 1.7 10	16.3 1.0 9	15.0 1.1 9	16.3 1.6 10
	M+F	MEAN ST. DEV. N	15.7 1.7 10	16.7 1.0 9	15.2 1.3 9	16.5 1.6 10
13	M	MEAN ST. DEV. N	30.7 1.8 10	32.2 2.0 9	29.6 2.8 9	31.5 2.7 10
	F	MEAN ST. DEV. N	29.9 1.9 10	31.6 1.9 9	29.0 2.4 9	30.6 2.2 10
	M+F	MEAN ST. DEV. N	30.3 1.9 10	31.9 1.7 9	29.3 2.6 9	31.0 2.3 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

ANOGENITAL DISTANCE AND NIPPLE RETENTION PER GROUP

F0-GENERATION – LACTATION

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
Anogenital dist M mm	MEAN ST. DEV. N	2.76 0.16 10	2.85 0.44 9	2.56 0.31 9	2.79 0.29 10
Anogenital dist F mm	MEAN ST. DEV. N	1.18 0.20 10	1.16 0.16 9	1.06 0.17 9	1.06 0.12 10
Number of nipples	MEAN MEDIAN (+) N	0.00 0.00 10	0.00 0.00 9	0.00 0.00 9	0.00 0.00 10

# / ## Fisher’s Exact test significant at %% (#) or 1% (##)

+ / ++ Steel-test significant at %% (+) or 1% (++)

 

NORMALIZED ANOGENITAL DISTANCE SUMMARY

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PND 1
Norm anog dist M mm	MEAN ST. DEV. N	1.49 0.07 10	1.48 0.20 9	1.39 0.15 9	1.47 0.12 10
Norm anog dist F mm	MEAN ST. DEV. N	0.65 0.10 10	0.62 0.08 9	0.59 0.11 9	0.57 0.05 10

+ / ++ Steel-test significant at 5% (+) or 1% (++) level

 

Summary of Thyroid Hormone Values: F1 Generation PND 14-16

Day: 14 Relative to Birth Date

Sex: Male		Reporting Special C
		T4 (ng/mL) [G]
0 mg/kg/day Group 1	Mean SD N	60.81 11.62 10
100 mg/kg/day Group 2	Mean SD N tCtrl	64.72 9.50 9 1.06
300 mg/kg/day Group 3	Mean SD N tCtrl	60.30 11.53 9 0.99
1000 mg/kg/day Group 4	Mean SD N tCtrl	60.97 11.83 10 1.00

[G] – Anova & Dunnett

 

Summary of Thyroid Hormone Values: F1 Generation PND 14-16

Day: 14 Relative to Birth Date

Sex: Female		Reporting Special C
		T4 (ng/mL) [G]
0 mg/kg/day Group 1	Mean SD N	62.18 8.83 10
100 mg/kg/day Group 2	Mean SD N tCtrl	66.76 11.90 9 1.07
300 mg/kg/day Group 3	Mean SD N tCtrl	61.93 5.95 9 1.00
1000 mg/kg/day Group 4	Mean SD N tCtrl	64.17 8.84 10 1.03

[G] – Anova & Dunnett

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid were established:
Parental NOAEL: at least 1000 mg/kg/day
Reproduction NOAEL: at least 1000 mg/kg/day
Developmental NOAEL: at least 1000 mg/kg/day

Executive summary:

The objectives of this study were to determine the potential toxic effects of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

 

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

 

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the Dose Range Finder.

The study design was as follows:

 

Experimental Design

Group No.	Test Item Identification	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	-	0 (vehicle)	4	0	10	10
2	Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid	100	4	25	10	10
3		300	4	75	10	10
4		1000	4	250	10	10

 

Chemical analyses of formulations were conducted once during the study and confirmed that formulations were prepared accurately and homogenously.

 

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight and food consumption, functional observations, clinical pathology (hematology, coagulation, clinical chemistry and thyroid hormone analysis (T4 in F0-males), macroscopic examination, organ weights and microscopic examination.

 

In addition, the following reproduction/developmental parameters were determined: estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, viability and lactation indices, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopic examination, thyroid hormone analysis (T4 in PND 14-16 pups)).

 

Parental results

At 1000 mg/kg/day, non-adverse lower cholesterol concentration were noted in females.

No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., mortality, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters, thyroid hormone analysis (T4 thyroid hormone levels in F0-males), macroscopic examination, organ weights, and microscopic examination).

 

Reproduction results

No reproductive toxicity was observed up to 1000 mg/kg/day.

No test material-related changes were noted in any of the reproductive parameters investigated in this study (i.e., mating and fertility indices, precoital time, number of implantation sites, estrous cycle, and histopathological examination of reproductive organs).

 

Developmental results

No developmental toxicity was observed up to 1000 mg/kg/day.

No test material-related changes were noted in any of the developmental parameters investigated in this study (i.e., gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) for Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid were established:

Parental NOAEL: at least 1000 mg/kg/day

Reproduction NOAEL: at least 1000 mg/kg/day

Developmental NOAEL: at least 1000 mg/kg/day