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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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

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

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:

Not specified in the study report

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat.
Strain: Crl: WI(Han).
Condition: Outbred, SPF-Quality.
Source of Test System: Charles River Deutschland, Sulzfeld, Germany.
Number of Males: 40
Number of Females: 48 (nulliparous and non-pregnant).
Age at Initiation of Dosing: Males 10 – 12 weeks old; Females 11 – 13 weeks old
Body Weight Range at Initiation of Dosing: 271 – 316 g males; 183 – 242 g females
Number of Acclimation Days: 8
Number of Treatment Days Males: 29; Females: 42 – 64

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.
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.

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 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 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
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated
procedures).
Ventilation: At least 10 air changes per hour.

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

Details on route of administration:

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.

Vehicle:

corn oil

Details on oral 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses will be performed using a validated analytical procedure.
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 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.

Duration of treatment / exposure:

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 up to and including the day before scheduled necropsy.

Frequency of treatment:

Once daily

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 per dose group (10 male/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels for the Main study were based on the results of the Dose Range Finding Study. Up to 1000 mg/kg/day, no toxicity was observed, and this dose level was therefore selected being the high dose for this Main study.

Positive control:

No required for this study.

Examinations

Observations and examinations performed and frequency:

Mortality: 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.
Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.

Clinical Observations: At least once daily, up to the day prior to necropsy. 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. In the data tables, the scored grades will be reported, as well as the percentage of animals affected in summary tables.

Arena Observations: Once before the first administration of the test item and weekly during the Treatment Period.
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.

Individual Body Weights: On Day 1 of treatment (prior to dosing) and weekly thereafter.
In order to monitor the health status animals may be weighed more often.

Food Consumption: Weekly, except for males and females which are housed together for mating
and for females without evidence of mating.
Quantitatively measured per cage.

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

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.

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.

Sacrifice and pathology:

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.

Statistics:

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.

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

Results and discussion

Results of examinations

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.

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

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related gross observations.
All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Details on results:

Wistar Han rats were treated with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, Corn Oil, alone.
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
A lower concentration of cholesterol was noted for females treated at 1000 mg/kg/day. In absence of corroborative histopathological findings this was considered not adverse.

Effect levels

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Any other information on results incl. tables

CLINICAL SIGNS SUMMARY

MALES

		PRE MATING	REPRO PERIOD
SIGN (MAX. GRADE)	WEEK:	1 . . . . . . . . . . . . .	1 . . . . . . . . . . . . .
(LOCATION)	DAY:	12345671234567	12345671234567
GROUP 1 (CONTROL) No clinical signs noted
GROUP 2 (100 MG/KG/DAY) No clinical signs noted
GROUP 3 (300 MG/KG/DAY) No clinical signs noted
GROUP 4 (1000 MG/KG/DAY) No clinical signs noted

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between %% and 15%, …., A=more than 95%)

.: Observation performed, sign not present

 

CINICAL SIGNS SUMMARY

FEMALES

		PRE MATING	REPRO PERIOD
SIGN (MAX. GRADE)	WEEK:	1 . . . . . . . . . . . . .	1 . . . . . . . . . . . . . . . . . . . .4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
(LOCATION)	DAY:	12345671234567	12345671234567123456712345671234567123456712345671
GROUP 1 (CONTROL) Skin / fur             Alopecia (3)	G: %:	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . . . . . 111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 . . . . . . . . . . . . . . . . . . . . .
GROUP 2 (100 MG/KG/DAY) Skin / fur             Piloerection (1)               Alopecia (3)	G: %: G: %:	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111111111111111111 . . . . . . . . . . . . . . . . . . . . . . . . . . . 111111111111111111 . .
GROUP 3 (300 MG/KG/DAY) Skin / fur             Alopecia (3)	G: %:	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . 111111111111111111111111 . . . . . . . . . . . . . . . . . . . . . . 111111111111111111111111 . . . . . . .
GROUP 4 (1000 MG/KG/DAY) Skin / fur             Alopecia (3)	G: %:	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	. . . . . . . . . . . . . . . . . . . . . 1111111111111111111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1111111111111111111 . . . . . . .

G: Median value of the highest individual daily grade

%: Percent of affected animals (0=less that 5%, 1=between 5% and 15%, …., A=more than 95%)

.: Observation performed, sign not present

 

BODY WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAY 1 WEEK 1	MEAN ST.  DEV. N	298 9.9 10	299 11.3 10	292 12.2 10	299 11.1 10
DAY 8 WEEK 2	MEAN ST. DEV. N	320 10.2 10	320 9.9 10	314 13.6 10	322 15.3 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	342 11.4 10	338 10.9 10	331 15.3 10	340 16.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	353 12.0 10	346 10.2 10	340 17.2 10	350 8.8 10
DAY 15 WEEK 3	MEAN ST. DEV. N	368 13.2 10	360 14.5 10	354 17.9 10	363 19.7 10

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAY 1 WEEK 1	MEAN ST.  DEV. N	210 9.6 10	205 12.9 10	209 18.110	210 9.2 10
DAY 8 WEEK 2	MEAN ST. DEV. N	216 9.3 10	209 14.2 10	216 19.7 10	215 10.2 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	221 10.2 10	215 16.9 10	220 19.9	219 10.3 10
DAY 8 WEEK 2	MEAN ST. DEV. N			230 12.9 3	243 -- 1
DAY 15 WEEK 3	MEAN ST. DEV. N			263 -- 1
DAY 22 WEEK 4	MEAN ST. DEV. N			246 -- 1
DAY 29 WEEK 5	MEAN ST. DEV. N			242 -- 1
DAY 36 WEEK 6	MEAN ST. DEV. N			242 -- 1

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

 

BODY WEIGHT GAIN (%) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAY 1 WEEK 1	MEAN ST.  DEV. N	0 0.0 10	0 0.0 10	0 0.0 10	0 0.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	8 1.2 10	7 1.6 10	7 1.3 10	7 1.4 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	15 2.1 10	13 2.4 10	13 1.8 10	14 1.7 10
DAY 8 WEEK 2	MEAN ST. DEV. N	18 2.3 10	16 3.1 10	16 2.1 10	17 2.6 10
DAY 15 WEEK 3	MEAN ST. DEV. N	24 2.2 10	20 4.5 10	21 2.4 10	21 3.0 10

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAY 1 WEEK 1	MEAN ST.  DEV. N	0 0.0 10	0 0.0 10	0 0.0 10	0 0.0 10
DAY 8 WEEK 2	MEAN ST. DEV. N	3 2.3 10	2 1.3 10	4 1.6 10	2 1.8 10
MATING PERIOD
DAY 1 WEEK 1	MEAN ST. DEV. N	5 2.5 10	5 2.6 10	5 2.3 10	4 1.8 10
DAY 8 WEEK 2	MEAN ST. DEV. N			15 4.8 3	14 -- 1
DAY 15 WEEK 3	MEAN ST. DEV. N			29 -- 1
DAY 22 WEEK 4	MEAN ST. DEV. N			21 -- 1
DAY 29 WEEK 5	MEAN ST. DEV. N			19 -- 1
DAY 36 WEEK 6	MEAN ST. DEV. N			19 -- 1

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

 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	23 0.1 2	22 0.0 2	23 0.0 2	23 1.2 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	22 0.4 2	22 0.1 2	22 0.3 2	22 0.8 2
MEAN OF MEANS OVER PRE MATING PERIOD	MEAN	23	22	22	23
MATING PERIOD
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	23 0.6 2	22 0.1 2	24 0.2 2	23 1.4 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	21 0.7 2	21 0.4 2	21 1.1 2	21 0.8 2
MEAN OF MEANS OVER MATING PERIOD	MEAN	22	21	23	22

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
PRE MATING
DAYS 1-8 WEEKS 1-2	MEAN ST. DEV. N (CAGE)	15 0.9 2	14 0.4 2	16 0.4 2	16 0.9 2
DAYS 8-15 WEEKS 2-3	MEAN ST. DEV. N (CAGE)	16 1.2 2	15 0.6 2	16 0.6 2	16 0.7 2
MEAN OF MEANS OVER PRE MATING PERIOD	MEAN	15	15	16	16

 

FUNCTIONAL OBSERVATIONS SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
HEARING SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL L SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
GRIP FORE GRAM	MEAN ST. DEV. N	1042 146 5	1179 240 5	1044 369 5	923 136 5
GRIP HIND GRAM	MEAN ST. DEV. N	575 104 5	542 130 3	579 134 5	442 74 5

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
HEARING SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL L SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
PUPIL R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
STATIC R SCORE 0/1	MEDIAN N	0 5	0 5	0 5	0 5
GRIP FORE GRAM	MEAN ST. DEV. N	1420 148 5	1354 106 5	1334 189 5	1327 255 5
GRIP HIND GRAM	MEAN ST. DEV. N	857 185 5	882 292 5	721 61 5	720 158 5

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

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

 

MOTOR ACTIVITY TEST SUMMARY

MALES

Total Movements	GROUP 1 (CONTROL)	GROUP 2 (100 MG/KG/DAY)	GROUP 3 (300 MG/KG/DAY)	GROUP 4 (1000 MG/KG/DAY)
MEAN N ST. DEV	2896 5 970	2892 5 980	3352 5 672	2865 5 419

* indicates a p-value <0.05, 88 indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal’s total Total Movements over all intervals

Ambulations	GROUP 1 (CONTROL)	GROUP 2 (100 MG/KG/DAY)	GROUP 3 (300 MG/KG/DAY)	GROUP 4 (1000 MG/KG/DAY)
MEAN N ST. DEV	622 5 340	592 5 209	695 5 228	610 5 182

* indicates a p-value <0.05, 88 indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal’s total Ambulations over all intervals

 

MOTOR ACTIVITY TEST SUMMARY

FEMALES

Total Movements	GROUP 1 (CONTROL)	GROUP 2 (100 MG/KG/DAY)	GROUP 3 (300 MG/KG/DAY)	GROUP 4 (1000 MG/KG/DAY)
MEAN N ST. DEV	3686 5 1400	3741 5 646	2833 5 867	4026 5 1483

* indicates a p-value <0.05, 88 indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal’s total Total Movements over all intervals

Ambulations	GROUP 1 (CONTROL)	GROUP 2 (100 MG/KG/DAY)	GROUP 3 (300 MG/KG/DAY)	GROUP 4 (1000 MG/KG/DAY)
MEAN N ST. DEV	817 5 337	745 5 220	586 5 133	890 5 353

* indicates a p-value <0.05, 88 indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal’s total Ambulations over all intervals

 

MACROSCOPIC FINDINGS SUMMARY

MALES

	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT Animals examined Animals without findings   Animals affected   Stomach             Focus/foci Mesenteric lymph node             Focus/foci	10 10   0     0   0	10 10   0     0   0	10 9   1     1   0	10 8   2     0   2

FEMALES

	GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT Animals examined Animals without findings   Animals affected   Liver             Diaphragmatic hernia             Reduced in size             Discolouration Clitoral glands             Discolouration Thymus             Focus/Foci Mesenteric lymph node             Focus/foci	10 10   0     0 0 0   0   0   0	10 9   1     0 0 0   0   1   0	10 7   3     1 1 1   1   0   0	10 9   1     0 0 0   0   0   1

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

 

ORGAN WEIGHTS (GRAM) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	348 13 10	338 12 10	332 18 10	343 20 10
BRAIN (GRAM)	MEAN ST. DEV N	2.02 0.05 5	2.07 0.07 5	2.00 0.03 5	1.98 0.07 5
HEART (GRAM)	MEAN ST. DEV N	0.937 0.053 5	0.992 0.096 5	0.969 0.064 5	0.998 0.090 5
LIVER (GRAM)	MEAN ST. DEV N	8.72 1.08 5	8.49 0.59 5	8.40 0.56 5	8.82 0.78 5
THYROIDS (GRAM)	MEAN ST. DEV N	0.0170 0.0036 10	0.0155 0.0038 10	0.0149 0.0026 10	0.0160 0.0017 10
THYMUS (GRAM)	MEAN ST. DEV N	0.341 0.067 5	0.362 0.101 5	0.367 0.067 5	0.328 0.041 5
KIDNEYS (GRAM)	MEAN ST. DEV N	2.16 0.27 5	2.27 0.12 5	2.28 0.10 5	2.12 0.20 5
ADRENALS (GRAM)	MEAN ST. DEV N	0.055 0.011 5	0.062 0.007 5	0.059 0.007 5	0.059 0.015 5
SPLEEN (GRAM)	MEAN ST. DEV N	0.584 0.093 5	0.546 0.034 5	0.560 0.080 5	0.573 0.097 5
TESTES (GRAM)	MEAN ST. DEV N	3.41 0.19 10	3.51 0.23 10	3.39 0.19 10	3.43 0.17 10
PROSTATE GLAND (GRAM)	MEAN ST. DEV N	0.755 0.132 10	0.739 0.131 10	0.750 0.095 10	0.703 0.120 10
EPIDIDYMIDES (GRAM)	MEAN ST. DEV N	1.050 0.069 10	1.042 0.066 10	1.019 0.052 10	1.053 0.052 10
SEMINAL VESICLES (GRAM)	MEAN ST. DEV N	1.218 0.130 10	1.207 0.184 10	1.181 0.310 10	1.192 0.146 10

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

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY

MALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	348 13 10	338 12 10	332 18 10	343 20 10
BRAIN (%)	MEAN ST. DEV N	0.59 0.03 5	0.61 0.03 5	0.59 0.02 5	0.57 0.05 5
HEART (%)	MEAN ST. DEV N	0.271 0.014 5	0.290 0.022 5	0.287 0.015 5	0.283 0.010 5
LIVER (%)	MEAN ST. DEV N	2.52 0.22 5	2.48 0.14 5	2.49 0.12 5	2.50 0.06 5
THYROIDS (%)	MEAN ST. DEV N	0.0049 0.0009 10	0.0046 0.0011 10	0.0045 0.0009 10	0.0047 0.0006 10
THYMUS (%)	MEAN ST. DEV N	0.098 0.015 5	0.106 0.028 5	0.109 0.024 5	0.093 0.009 5
KIDNEYS (%)	MEAN ST. DEV N	0.62 0.06 5	0.66 0.03 5	0.68 0.06 5	0.60 0.03 5
ADRENALS (%)	MEAN ST. DEV N	0.016 0.003 5	0.018 0.001 5	0.017 0.002 5	0.017 0.005 5
SPLEEN (%)	MEAN ST. DEV N	0.169 0.023 5	0.160 0.012 5	0.165 0.020 5	0.163 0.023 5
TESTES (%)	MEAN ST. DEV N	0.98 0.08 10	1.04 0.08 10	1.02 0.08 10	1.00 0.07 10
PROSTATE GLAND (%)	MEAN ST. DEV N	0.217 0.039 10	0.219 0.042 10	0.225 0.019 10	0.205 0.032 10
EPIDIDYMIDES (%)	MEAN ST. DEV N	0.303 0.026 10	0.308 0.024 10	0.307 0.020 10	0.308 0.027 10
SEMINAL VESICLES (%)	MEAN ST. DEV N	0.351 0.039 10	0.358 0.057 10	0.356 0.095 10	0.349 0.048 10

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

 

ORGAN WEIGHTS (GRAM) SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	289 20 10	273 32 10	276 33 9	282 21 8
BRAIN (GRAM)	MEAN ST. DEV N	1.86 0.04 5	1.89 0.06 5	1.87 0.06 5	1.87 0.03 5
HEART (GRAM)	MEAN ST. DEV N	0.865 0.057 5	0.827 0.062 5	0.833 0.125 5	0.806 0.040 5
LIVER (GRAM)	MEAN ST. DEV N	12.37 1.51 5	11.79 0.96 5	12.36 1.61 5	11.93 1.34 5
THYROIDS (GRAM)	MEAN ST. DEV N	0.0140 0.0024 10	0.0144 0.0024 10	0.0145 0.0026 10	0.0140 0.0020 10
THYMUS (GRAM)	MEAN ST. DEV N	0.196 0.0304 5	0.186 0.035 5	0.198 0.015 5	0.185 0.027 5
KIDNEYS (GRAM)	MEAN ST. DEV N	1.87 0.15 5	1.91 0.09 5	1.92 0.27 5	1.86 0.15 5
ADRENALS (GRAM)	MEAN ST. DEV N	0.082 0.014 5	0.072 0.007 5	0.074 0.015 5	0.074 0.010 5
SPLEEN (GRAM)	MEAN ST. DEV N	0.507 0.074 5	0.500 0.037 5	0.499 0.015 5	0.525 0.050 5
OVARIES (GRAM)	MEAN ST. DEV N	0.126 0.022 5	0.117 0.014 5	0.123 0.020 5	0.097* 0.011 5
UTERUS (GRAM)	MEAN ST. DEV N	0.343 0.027 5	0.322 0.057 5	0.356 0.022 5	0.354 0.040 5

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

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY

FEMALES

		GROUP 1 CONTROL	GROUP 2 100 MG/KG/DAY	GROUP 3 300 MG/KG/DAY	GROUP 4 1000 MG/KG/DAY
END OF TREATMENT
BODY W. (GRAM)	MEAN ST. DEV N	289 20 10	273 32 10	276 33 9	282 21 8
BRAIN (%)	MEAN ST. DEV N	0.66 0.05 5	0.67 0.06 5	0.67 0.07 5	0.69 0.05 5
HEART (%)	MEAN ST. DEV N	0.306 0.021 5	0.291 0.016 5	0.296 0.015 5	0.295 0.015 5
LIVER (%)	MEAN ST. DEV N	4.36 0.39 5	4.14 0.07 5	4.39 0.15 5	4.36 0.35 5
THYROIDS (%)	MEAN ST. DEV N	0.0049 0.0011 10	0.0054 0.0012 10	0.0052 0.0012 9	0.0050 0.0010 8
THYMUS (%)	MEAN ST. DEV N	0.070 0.015 5	0.066 0.015 5	0.071 0.006 5	0.068 0.010 5
KIDNEYS (%)	MEAN ST. DEV N	0.66 0.05 5	0.67 0.03 5	0.68 0.03 5	0.68 0.04 5
ADRENALS (%)	MEAN ST. DEV N	0.029 0.005 5	0.025 0.001 5	0.026 0.003 5	0.027 0.004 5
SPLEEN (%)	MEAN ST. DEV N	0.179 0.025 5	0.176 0.015 5	0.179 0.034 5	0.192 0.008 5
OVARIES (%)	MEAN ST. DEV N	0.045 0.009 5	0.041 0.005 5	0.046 0.012 6	0.036 0.005 5
UTERUS (%)	MEAN ST. DEV N	0.122 0.014 5	0.115 0.028 5	0.129 0.024 5	0.130 0.018 5

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

 

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

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 ester 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.

 

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).

 

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