2-hexyldecanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - October 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid

EC Number:

941-570-9

Molecular formula:

C11H22O2

IUPAC Name:

Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid

Details on test material:

- Name of test material (as cited in study report): Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid
- Substance type: pure active substance
- Physical state: colourless to slight yellow liquid
- Expiration date of the lot/batch: October 2018
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 151 - 186g (males), 119 - 151 g (females)
- Fasting period before study: no
- Housing: kept in groups of 5 animals / sex/ group / cage in IVC cages, type IV, polysulphone cages on Atromin saw fibre bedding (lot no. 02102141114)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1239) ad libitum
- Water: tap water, sulphur acidified to a pH of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals) ad libitum
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2015-03-24 To: 2015-05-11

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The test item formulations were freshly prepared on each administration day before the administration procedure. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): selected based on the test item's characteristics
- Lot/batch no. (if required): MKBP7039V
- Manufacturer: Sigma
- Expiry date: 09/2015

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples of at least 5 mL were retained from all groups once weekly during the treatment period and stored between -15 and -35 °C.
Stability of the dosing formulations was tested once at the beginning of the treatment period (week 1). From the low and high dose group samples of dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C.
In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high and low dose formulations and stored between -15 and -35 °C.
Each sample was retained twice (sample A, sample B, each of at least 25 mL).
All ‘A’ samples of dosing formulations for nominal concentration, stability and homogeneity taken in the first week of the study were analysed on the day of sampling except stability samples which were analysed on next day of sampling (stored between -15 and -35 °C till analysis). All remaining nominal concentration and homogeneity samples taken during the course of the study were analysed at the end of the treatment period.
The ‘B’ samples were retained at the testing laboratory until the analysis has been performed, and discarded after completion of the final study report.
The determination of formulation concentrations of the four main compounds of the test item (2-methyldecanoic acid, 2-ethylnonanoic acid, 2-propyloctanoic acid and 2-butylheptanoic acid) was performed using GC with a FID detection which was sucessfully validated in BSL BIOSERVICE project no. 150020 (non GLP).
Corn oil was used for preparation of calibration standards and quality control samples. The accuracy of prepared stock solutions was demonstrated by comparing the response of at least two separately weighed stock solutions which were diluted in triplicates to a single concentration within the linear range of the method. Only stocks which compare within 5% difference were used to prepare subsequent calibration standards. Alternatively accuracy of prepared calibration solutions was ensured by weighing each calibration sample separately.
Investigation of stability and homogeneity were performed separately for each of the four main components. Each component was quantified by measuring the chromatographic response. The corresponding concentrations were retrieved by applying linear calibration curves.
Results are verfied by reanalysis, if formulation samples do not meet the acceptance criteria:
- Nominal concentration in formulation sample is considered confirmed, if measured concentarions of the test item (using 2-methyldecanoic acid as representative) do not differ by more than 15% from nominal concentration. In case of reanalysis samples are analysed in triplicates.
- The test item in formulation sample is considered stable, if the concentration of the four main components after storage do not differ from start value by more than 15%. In case of reanalysis both stability samples are analysed in triplicates each. Mean of triplicate reanalysis is respected for further evaluation.
- The formulation samples are considered homogenous, if the COV of concentration of the four main components in sample taken from top, middle, and bottom is below or equal 15%. In case of reanalysis all three samples are analysed in triplicates each. Mean of triplicate reanalysis is used for further evaluation.

Analytical method for quantification:
- Vehicle: corn oil
- Working range: 10 - 35 mg/mL
- GC: TRACE 1310 GC, Thermo Fisher Scientific GmbH
- Detector: FID, Thermo Fisher Scientific GmbH
- Software: Chromeleon 7.2, Thermo Fisher Scientific
- Sample storage temperature: room temperature
- Injector: inlet temp.: 250°C, initial flow: 2.0 mL/min constant flow
- Inlet: Split Straight Liner, 4 ID x 6.3 OD x 78.5 mm length
- Column: DA-WAXetr, 50 m x 0.32 mm, ID 1.0 µm film, part.no. 123-7354, Agilent Technologies
- Oven temperature program: initial temp: 50 °C, initial time: 1 min; ramp rate: 25 °C/min; final temp.: 250 °C, final time: 2 min
- Detector: temp.: 250 °C, hydrogen flow: 30 mL/min, air flow: 350 mL/min, Nitrogen make up flow: 30 mL/min
- Calibration standards: approx. 10, 15, 20, 25, 30, 35 mg/mL test item in corn oil
- Quality control samples: 25, 150 and 250 mg/mL test item in corn oil

Evaluation
- Calibration: Calculation of concentrations from reponse of peak area with reference to calibration curve
- Curve fit: linear, weighting 1/conc

Acceptance criteria
- Calibration: deviation from nominal concentrations <= 10%, at least 5 nonzero levels ahould be within these limits, covering concentrations of quality control samples. Values falling outside these limits were excluded from calculation. Regression coefficient > 0.99
- Qualitiy control samples: deviation from nominal concentrations >= 15% for at least 67% (4 of 6) of total umber of quality control samples. 33% of the QC samples could be outside the limit, but not all replicates at the same concentration

Concentrations of formulation samples
Concentration verification: Concentrations analysis of the test item in formulation samples was performed in study weeks 1, 2, 3 and 4 for all dose groups. All samples met the acceptance criteria set for analytical runs.
The mean recoveries observed for the test item (quantified using 2-methyldecanoic acid as representative) were 103% in the low dose (LD), 82% in the medium dose (MD) and 151% of the nominal concentration in the high dose (HD).
The LD group the nominal concentration was confirmed for samples of all study weeks, as measured concentrations were within acceptance criterion of 100% +/- 15% recovery.
For MD level the measured concentrations were with -18% slightly below and for the HD level with +51% clearly above this criterion. These results were observed in samples of all four study weeks. Repeat measurements in triplicate were performed for these samples. Results are considered confirmed by taking into account two additional observations: in homogeneity samples from these study weeks (week 1 and 4) same high recoveries were found. As the calibration samples were prepared individually and for each run separately, a systematic error can be excluded.
Stability of formulation samples was investigated in study week 1 in the LD and HD groups for each component separately. After 6 hours storage at room temperature recovery values compared to starting value were calculated for all four test item components. All samples were stable, concentrations after storage did not differ from start value by more than 15%.

After detailed examination of the analytical method, no explanation can ge given for the deviating concentrations in dose formulation. The method was successfully validated and furthermore showed reliable results for calibration and quality control samples within the course of sample measurements. Moreover, the preparation of formulation samples was carefully examine and gave no indication of an incorrect procedure. However, as even this dose formulation was well tolerated by WISTAR rats (NOAEL = 1000 mg/kg bw/d), the results have no impact on the outcome of the study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

The test item and control formulation were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5 (additional 5 animals per sex in high dose and control group as satellite groups for recovery assessment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): most homogenous variation in body weight throughout the groups of males and females
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): full histopathological evaluation on high dose and control animals, if organs showed gross alterations also in animals of the medium, low dose and the recovery groups

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter
- Cage side observations: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an additional period of 14 days following the last administration. The health condition or the animals was recorded. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing.

BODY WEIGHT: Yes
- Body weight gain was calculated for each animal as the difference in weight measured from one week to the next.
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period

FOOD CONSUMPTION: Yes
- Food consumption was calculated for each animal as the difference in weight measured from one week to the next.
- Time schedule: weekly during treatment and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.
- Dose groups that were examined: all

HAEMATOLOGY AND BLOOD COAGULATION: Yes
- Time schedule for collection of blood: one day after the last administration (main study animals), at the end of the recovery period (recovery animals) prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: ketamine (Pharmanovo, lot no: 25175, expiry date: 06/2016) and xylazin (Rebo Pharm, lot no. 400260/1, expiry date: 01/2017)
- Animals fasted: Yes
- How many animals: all surviving animals, all recovery animals
- Parameters checked: haematocrit value, haemoglobin content, red blood cell count, mean corpuscular volume, mean corpuscolar haemoglobin, mean corpuscolar haemoglobin concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last administration (main study animals), at the end of the recovery period (recovery animals) prior to or as part of the sacrifice of the animals
- Animals fasted: Yes
- How many animals: all surviving animals, all recovery animals
- Parameters checked: alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea, total bilirubin, total bile acids, total cholesterol, glucose, sodium, potassium

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: urine colour/appearance, specific gravity, nitrite, pH-value, protein, glucose, ketone bodies, urobilinogen, bilirubin, erythrocytes, leukocytes

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: once before the first exposure, once in the fourth week of exposure, and in the last week of the recovery period
- Dose groups that were examined: all
- Batteries of functions tested: see Table 1

OTHER:
None

Sacrifice and pathology:

SACRIFICE:
- Anesthesia: ketamine (Pharmanovo, lot no: 25175, expiry date: 06/2016) and xylazin (Rebo Pharm, lot no. 400260/1, expiry date: 01/2017)
- Time schedule: on study day 29 (main study animals), on study day 43 (recovery animals)

GROSS PATHOLOGY: Yes
- Examinations performed: external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents
- Organ wet weights determined: liver, kidneys, adrenals, testes, epididymes, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, spleen, brain, heart, thyroid/parathyroid glands, pituitary gland

HISTOPATHOLOGY: Yes
- Necropsies and tissue-collection for histopathology were performed at the test facility, BSL Bioservice Scientific Laboratories GmbH, Germany. The preparation of the slides (histoprocessing) was performed at the Test Site AnaPath GmbH, Department AnaPath Services, Switzerland. Resulting sections were stained by hematoxylin and eosin. Blocking, embedding, cutting, H&E staining and scientific slide evalution were performed according to the corresponding SOP's of the test sites.
- Organs and tissues listed below (table 2) for all animals of the control and the high dose group sacrificed at the end of the treatment period were examined by light microscopy. Because of possible treatment-related findings noted in the high dose group, liver, thymus and stomach from all animals of the low and mid dose groups and all animals from the recovery groups were examined to establish a no-effect level and to assess the reversibility.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or IDBS E-WorkBook 9.4.0 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred in the control or any of the dose groups during the treatment or recovery period of this study.
During the treatment period, slight to severe salivation was noted occasionally in some males and females of the main study and recovery HD group. Furthermore, moving the bedding was observed in all males and all females of the main study and recovery HD group during the treatment days 6-28 in males and 5-28 in females. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration and just for a short period, these signs were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect.
Low incidences of clinical signs like slight to moderate piloerection and slightly increased spontaneous activity were noted in isolated males and/or females of the LD and HD group main study and recovery animals during the treatment period. As these slight clinical signs were mostly transient and seen irrespective of the groups in isolated animals, they were considered to be incidental. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant effects of test item on body weight and body weight gain were observed in the LD and the MD groups of both males and females during treatment and recovery period. However, overall body weight gain (day 1-28) in male main study animals was observed to be statistically significantly lower in MD and HD group when compared with the controls. In females, mean body weight was marginally higher in the HD group compared to the control group at the end of the treatment period. However, body weight was comparable during the recovery period without achieving statistical significance. Overall mean weight gain (Day 1-28) during the treatment period was slightly and statistically insignificantly higher in the main study female HD group when compared to the controls. During the recovery period, mean daily body weight gain was slightly reduced in the HD group compared to the control group.
As differences were marginal, decrease observed in males and values were within the normal range of variation for animals of this strain and in the absence of major clinical signs and good overall health of the animals, effects on body weight were not considered as an adverse effect of the test item.

FOOD CONSUMPTION
There were no statistically significant effects on food consumption during the treatment period and recovery period in both males and females of the dose groups when compared to the respective controls except statistically significantly lower average daily food consumption was observed in HD recovery females. As no such effect was observed during treatment period, this significant effect during recovery period was considered to be incidental and not related to the treatment. During the treatment period, overall group mean food consumption (Day 1-28) of the main study male was slightly lower when compared with the respective control which was in correlation with respective body weights.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmologic findings were observed in any of the animals of this study.

HAEMATOLOGY
In males sacrificed at the end of treatment period, statistical analysis revealed statistically significantly lower mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), platelet count (PLT), white blood cells (WBC) and higher monocytes values in HD group when compared with the controls. In females sacrificed at the end of treatment period, there was a marginally but statistically significantly lower mean value of mean corpuscular haemoglobin (MCH) observed in the MD and the HD group when compared to the control group. As the respective values were marginally higher or lower and within the normal range of variation for animals of this strain and age, differences are not assumed to be biologically relevant although statistically significant.
At the end of the recovery period, with exception of reticulocytes and platelet count (PLT), no statistically significant difference was observed in males in any of the haematology parameters between the dose groups and the control group. Marginally but statistically significantly higher reticulocytes and platelet count (PLT) values in the male HD group were within the normal range of variation and were not assumed to be biologically relevant.
Statistically significant and marginally lower MCH and MCHC in the female HD group observed at the end of the recovery period was considered incidental with values being in the normal range of variation.
Besides, all haematological parameters and blood coagulation parameters in males and females were in the normal range of variation and no test item-related effects were observed.

CLINICAL CHEMISTRY
In male animals, at the end of the treatment period, alanine aminotransferase (ALAT), total protein (TP) in HD and cholesterol in MD and HD were observed to be marginally but statistically significantly lower when compared to the control group. As all values were within the normal range of variation, this effect was considered to be incidental. In female animals, total protein (TP) and Albumin (Alb) were marginally but statistically significantly lower in the HD group at the end of the treatment period compared to the control group. This effect is not assumed to be biologically relevant, as the difference is marginal and values were within the normal range of variation. In males and females, the observed significant effect on parameters of clinical biochemistry did not persist up to the end of recovery and were not considered to be adverse.

URINALYSIS
Slightly higher leukocyte levels were found in the urine of one male animal of HD recovery group and two animals of control recovery group. No such trend was observed in the female animals and main study males sacrificed at the end of treatment period. In males and females, all other urinary parameters were in the normal range of variation, and no test item-related differences between the dose groups and control group were observed.

NEUROBEHAVIOUR
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period or recovery period.

ORGAN WEIGHTS
At the end of the treatment period, absolute adrenals, thymus, thyroid/parathyroid weights in HD and absolute epididymides weights in LD and HD were statistically significantly lower in male animals than in the respective controls. Absolute spleen weights in MD group were also statistically significantly lower when compared to the control group. The relative (to brain weight) liver and kidney weights in LD and MD, spleen weights in MD, thymus weights in MD and HD, epididymides weights in LD,MD and HD and thyroid/parathyroid weights in LD and HD were statistically significantly lower when compared to the control group. A statistically significantly higher relative (to body weight) brain and testes weights in all dose groups and statistically significantly lower relative (to body weight) thymus and thyroid/parathyroid weights in HD group were observed when compared with the controls. In female animals sacrificed at the end of treatment period, a statistically significantly higher absolute and relative (to body and brain weight) liver weight was found in the female HD group when compared to the controls. There was statistically significantly lower absolute and relative (to body weight) thymus weights observed in HD group when compared with the controls. A statistically significantly lower absolute and relative (to brain and body weight) thyroid/parathyroid weights in LD and statistically significantly higher ovary weights relative to body weights were observed in HD group when compared with the controls. At the end of the recovery period, there were no statistically or biologically significant effects on the absolute and relative organ weights in the male and female dose groups except statistically significantly higher relative (to brain and body weight) ovary weights in HD group females when compared to the respective controls. As no such effect was observed in main study females, this effect on recovery female ovary weight was considered to be incidental and not related to the treatment with the test item. To these achieved statistical significances, no histological correlate was found for the increased or decreased weights. In the light of absence of adverse histopathological findings in the organs up to HD group and observed histopathological lesions recovered after a 14-day withdrawal period and as this increase or decrease in organ weight was minimal, it was not considered to be adverse. At the end of the treatment period and the recovery period, values for the remaining male and female organs from the dose groups were comparable with the control group.

GROSS PATHOLOGY
Predominant macroscopic findings observed in male animals sacrificed at the end of treatment period were yellow spot on right epididymides and large sized mandibular lymphnodes in 1 each male of the HD group. In the male animals sacrificed at the end of recovery period, the yellow spots on epididymides in 2/5 males and discolored dark ileum /payer’s patches were observed in 1/5 males of the control recovery group. In female animals sacrificed at the end of treatment period, dilated right renal pelvis in 1/5 females of the control group and fluid distension in the uterus of 2/5 HD group females was observed. No macroscopic findings observed at necropsy in any of the female animals sacrificed at the end of recovery period. These gross pathological findings were spontaneous in nature and as such not considered to be a systemic effect due to test item administration. Histopathologically also these gross lesions could not be related to treatment with the test item. Spots on epididymides correlated microscopically with the spermatid granulomas of epididymis, the congestion in the ileum, the pelvic dilation of the kidney and the cornual dilation of the uterus related to estrus cyclic changes, respectively. These findings are common in rats and were within the range of normal background lesions which may be recorded in animals of this strain and age and therefore considered not to be treatment-related. Microscopically, enlarged (large sized) mandibular lymph node in HD group male sacrificed at the end of treatment period was a poorly differentiated salivary gland carcinoma. This type of carcinoma occasionally occur in rats, even in the young generation and the neoplasm recorded in this study was deemed to be of a spontaneous in nature.

HISTOPATHOLOGY: NON-NEOPLASTIC
Under the conditions of this study, the microscopic findings which were considered to be attributable to treatment with the test item were recorded in the liver and thymus of both sexes. Hepatocellular hypertrophy was recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). This was characterized mainly by centrilobular hypertrophy, and in some locations of the female liver it looked diffusely hypertrophic. There were neither further indicator of cellular injuries such as necrosis or apoptosis nor indicator of cellular proliferation such as increased mitotic figures or polyploidy, in any animals examined under the condition of this study. Therefore, this finding was considered to be of adaptive character and not to be adverse under the condition of this study. Hepatocellular hypertrophy was no longer present in any recovery animals (see table 1 below).
Increased incidence and/or severity of thymic atrophy/involution were recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). No abnormal histological findings were observed in the other lymphoid organs and tissues including spleen and lymph nodes of animals examined in this study, and therefore, it is unlikely that this was attributed to treatment with the test-item. Rather, it is likely to be a secondary response to the stressful condition due to high-dose exposure of the test item, and it was considered not to be of adverse character. In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups (see table 2 below).
As a result of microscopic examination of testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix and vagina, no treatment-related effects were observed in any animals examined in this study.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.
In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid, in male and female Wistar rats with dose levels of 50, 250, and 1000 mg/kg body weight day revealed no major toxicity or mortality. No mortality or signs of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid were found at the dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of the test item in this study is considered to be 1000 mg/kg bw/day.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid, via oral administration to rats over a period of 28 days.

The substance was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 5 male and 5 female Wistar rats each. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for a period of 14 days following the last administration. The following doses were evaluated: control (0 mg/kg bw), low dose (50 mg/kg bw), medium dose (250 mg/kg bw), high dose (1000 mg/kg bw). The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements. During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly. Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period, multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. Haematological, blood coagulation and clinical biochemistry examinations were made on blood samples obtained from the overnight fasted animals at the terminal sacrifice. A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. At the conclusion of the test, all animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. A full histopathological evaluation of the tissues was performed on high dose and control animals. These examinations were extended to liver, thymus and stomach of animals of all other dosage groups and recovery group as treatment-related changes were observed. All gross lesions macroscopically identified were examined microscopically in all animals. Formulation samples were taken at various intervals for analysis of nominal concentrations, stability and homogeneity and stored between -15 to -35 °C until analysis at the end of the study.

No mortality occurred in the control or any of the dose groups during the treatment and recovery period of this study.

During the treatment period, slight to severe salivation was noted occasionally in some males and females of the main study and recovery HD group. Furthermore, moving the bedding was observed in all males and all females of the main study and recovery HD group during the treatment days 6-28 in males and 5-28 in females. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration and just for a short period, these signs were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No ophthalmologic findings were observed in any of the animals of this study.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period or recovery period. There were no biologically relevant differences in body temperature between the groups.

No statistically significant effects of test item on body weight and body weight gain were observed in the LD and the MD groups of both males and females during treatment and recovery period. However, overall body weight gain (day 1-28) in male main study animals was observed to be statistically significantly lower in MD and HD group when compared with the controls. In females, mean body weight was marginally higher in the HD group compared to the control group at the end of the treatment period. However, body weight was comparable during the recovery period without achieving statistical significance. Overall mean weight gain (Day 1-28) during the treatment period was slightly and statistically insignificantly higher in the main study female HD group when compared to the controls. During the recovery period, mean daily body weight gain was slightly reduced in the HD group compared to the control group. As differences were marginal, decrease observed in males and values were within the normal range of variation for animals of this strain and in the absence of major clinical signs and good overall health of the animals, effects on body weight were not considered as an adverse effect of the test item.

There were no statistically significant effects on food consumption during the treatment period and recovery period in both males and females of the dose groups when compared to the respective controls except statistically significantly lower average daily food consumption was observed in HD recovery females. As no such effect was observed during treatment period, this significant effect during recovery period was considered to be incidental and not related to the treatment.

During the treatment period, overall group mean food consumption (Day 1-28) of the main study male was slightly lower and in females it was slightly higher when compared with the respective controls which was in correlation with respective body weights.

In males and females sacrificed at the end of treatment period and recovery period, no test item-related effect of toxicological relevance was observed for haematology and coagulation parameters. Values for few parameters were marginally higher or lower but within the normal range of variation for animals of this strain and age. These differences are not assumed to be biologically relevant although statistically significant.

In male animals, at the end of the treatment period, alanine aminotransferase (ALAT), total protein (TP) in HD and cholesterol in MD and HD were observed to be marginally but statistically significantly lower when compared to the control group. As all values were within the normal range of variation, this effect was considered to be incidental. In female animals, total protein (TP) and Albumin (Alb) were marginally but statistically significantly lower in the HD group at the end of the treatment period compared to the control group. This effect is not assumed to be biologically relevant, as the difference is marginal and values were within the normal range of variation.

At the end of the recovery period, blood biochemistry values of the male and female HD group were within the normal range of variation for this strain and comparable to the respective controls. Marginally and statistically significantly lower cholesterol in the male recovery HD group was considered to be incidental.

Slightly higher leukocyte levels were found in the urine of one male animal of HD group and two control group animals at the end of the recovery period. No such trend was observed in the female animals and main study males sacrificed at the end of treatment period.

At necropsy, few gross pathological findings were observed and those were considered to be spontaneous in nature and as such not related to the systemic effect of the test item. Histopathologically, also these gross lesions could not be related to treatment with the test item. The microscopic findings which were considered to be attributable to treatment with the test item, were recorded in the liver and thymus of both sexes.

Hepatocellular hypertrophy was recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). This was characterized mainly by centrilobular hypertrophy, and in some locations of the female liver it looked diffusely hypertrophic. There were neither further indicator of cellular injuries such as necrosis or apoptosis nor indicator of cellular proliferation such as increased mitotic figures or polyploidy, in any animals examined. Therefore, this finding was considered to be of adaptive character and not to be adverse. Hepatocellular hypertrophy was no longer present in any recovery animals. Increased incidence and/or severity of thymic atrophy/involution were recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). No abnormal histological findings were observed in the other lymphoid organs and tissues including spleen and lymph nodes of animals examined in this study, and therefore, it is unlikely that this was attributed to treatment with the test-item. Rather, it is likely to be a secondary response to the stressful condition due to high-dose exposure of the test item, and it was considered not to be of adverse character. In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups. As a result of microscopic examination of testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix and vagina, no treatment-related effects were observed in any animals examined in this study. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Statistical analysis of organ weight data from male and female animals sacrificed at the end of treatment or recovery revealed few significant increases or decreases. However, to these achieved statistical significance, no histological correlate was found for the increased or decreased weights. In the light of absence of adverse histopathological findings in the organs up to HD group and observed histopathological lesions recovered after a 14-day withdrawal period and as this increase or decrease in organ weight was minimal, it was not considered to be adverse.

Dose formulation analysis for nominal concentration, stability and homogeneity revealed that all dose formulations were stable and homogenous at the time of application throughout the study period.

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid in male and female Wistar rats with dose levels of 50, 250, and 1000 mg/kg body weight per day revealed no major toxicity or mortality. No mortality or signs of toxicity of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid were found up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of the test item in this study is considered to be 1000 mg/kg bw/day.