Butanedioic acid, 2-hydroxy-, 1,4-bis[5,7,7-trimethyl-2-(1,3,3-trimethylbutyl)octyl] ester

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:

17. March - 02. September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

EC Number:

807-422-1

Cas Number:

66918-01-2

Molecular formula:

C40H78O5

IUPAC Name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

Details on test material:

- Name of test material (as cited in study report): SALACOS 222
- Physical state: liquid
- Analytical purity: > 98%
- Lot/batch No.: 7-2-D
- Expiration date of the lot/batch: 04/10
- Stability under test conditions: stable
- Storage condition of test material: room temperature (20 +/- 5 °C)

Specific details on test material used for the study:

Identification: SALACOS 222
Description: Liquid
Batch Number: 7-2-D
Purity: 99.0%
Expiry Date (Retest Date): 30 April 2010
Storage Conditions: Room temperature (20 ± 5 °C) in the original
container away from direct sunlight

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as a
recommended rodent test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 182 to 197 g (mean 189 g), Females: 138 to 153 g (mean 146 g)
- Fasting period before study: not stated
- Housing: in groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Pelleted standard Kliba Nafag 3433 (batch no.
77/07) rat / mouse maintenance diet (Provimi Kliba
SA, 4303 Kaiseraugst / Switzerland) was available
ad libitum. The feed batch was analyzed for
contaminants.
Community tap-water from Itingen was available ad
libitum in water bottles.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 °C
- Humidity (%): 30 -70%
- Air changes (per hr): 10-15 /h
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 31 Mar 2008 (delivery of animals) To: 05 May 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
SALACOS 222 was weighed into a glass beaker on a tared Mettler balance. A small amount of
vehicle was added. Thereafter the remaining vehicle was added. The mixtures were stirred using
a magnetic stirrer and stored at room temperature (15 - 25 °C).
The test item was weighed into a tared glass container on a suitable precision balance and the
vehicle added to give the appropriate final concentration of the test item in the dose formulation.
The mixtures were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle
was maintained during the daily administration period using a magnetic stirrer.
- DIET PREPARATION

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not stated
- Concentration in vehicle: Group 1: 0 mg/mL/day
Group 2: 10 mg/mL/day
Group 3: 40 mg/mL/day
Group 4: 200 mg/mL/day
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): 18787208
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed using a HPLC coupled to an evaporative light scattering detector
and quantified with the area under the peak provided by the Sponsor.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose range finding study
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups: no satelite groups
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Viability / Mortality
Observations for viability / mortality were recorded twice daily.

General Clinical Observations (Daily)
The animals were observed for clinical signs once before commencement of administration as
well as twice daily on days 1 to 3, and once daily on days 4-28 (treatment period).

Detailed Behavioral Observations (Weekly)
The animals were observed in their home cages, outside their home cages in a standard arena and
in the hand. These observations were performed once before commencement of administration
and once weekly (weeks 1 to 3) thereafter.

Functional Observational Battery
During week 4, relevant parameters (see above) from a modified Irwin screen test were
evaluated in all animals.
The results of the Functional Observational Battery are presented in the summary and individual
tables of the Detailed Clinical Observations (Weekly) under week 4. This method of data
presentation permits a clear evaluation and assessment of weekly clinical signs observed during
the study.
Grip Strength
Forelimb and hind limb grip strength measurements were performed using a push-pull strain
gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular
grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the
animals were held towards the base of the tail and steadily pulled away or towards the ring until
the grip was broken. Each measurement was repeated three times; the means were calculated and
recorded.
Locomotor Activity
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr
Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals
were monitored during the fourth treatment week for a 60-minute period and the total activity of
this time period was recorded.
Low beams count was reported in 10-minute intervals as well as the total activity of the
measuring period.

Food Consumption
The food consumption was recorded once during the acclimatization period and weekly
thereafter, using an on-line electronic recording system consisting of a Mettler balance connected
to the RCC computer.

Body Weights
Body weights were recorded weekly during acclimatization, treatment and recovery periods and
before necropsy, using an on-line electronic recording system consisting of a Mettler balance
connected to the RCC computer.

Clinical Laboratory Investigations
Blood and Urine Sampling:
After 4 Weeks: 05-May-2008
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane
anesthesia. The animals were fasted for approximately 18 hours before blood sampling but
allowed access to water ad libitum. The samples were collected early in the working day to
reduce biological variation caused by circadian rhythms. Blood samples were drawn from the
retro-orbital plexus using a micro-hematocrit glass capillary tube.
The assay was performed at RCC Ltd (Füllinsdorf / Switzerland) under internal laboratory
quality control conditions to assure reliable test results.
In the summary and individual tables, the names of some parameters have been abbreviated.

Hematology
The following hematology parameters were determined:
Complete Blood Cell Count
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium,
high fluorescence)
Leukocyte count, total
Differential leukocyte count:
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
Large unstained cells
Platelet count
Hemoglobin Derivatives
Methemoglobin
Heinz bodies (slides prepared but not
evaluated)
Coagulation
Prothrombin time (= Thromboplastin time) Activated partial Thromboplastin time

Clinical Biochemistry
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Phospholipids
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

Sacrifice and pathology:

Sacrifice:
After 4 Weeks: 05-May-2008
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were
recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed
by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and
fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.
Organ Weights
The organs from animals listed in the above table were weighed before fixation and recorded on
the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body
weight and brain weight.
The terminal body weight was recorded immediately prior to necropsy and the organ to terminal
body weight ratios as well as organ to brain weight ratios were determined.
Histotechnique
All organ and tissue samples (as defined in the table under Necropsy above) were processed,
embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with
hematoxylin and eosin.
Histopathology
Slides of all organs and tissues (as defined in the table under Necropsy above) which were
collected at scheduled sacrifices from all animals of the control and high-dose groups and all
gross lesions from all animals were examined by the study pathologist. Organ and tissue samples
taken from animals which died spontaneously or which were killed in extremis were evaluated
similarly to those organs taken from animals of the high-dose group.

Statistics:

The following statistical methods (see also references) used to analyze grip strength, locomotor
activity, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic
findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if
the variables could be assumed to follow a normal distribution for the comparison of the
treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the
data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

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:

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):

In males, the red blood cell count was significantly elevated at 1000 mg/kg/day (p<0.05) when
compared with the control males. The difference, however, remained well within the ranges of
the historical control data and was considered to be incidental. The mean relative “large
unstained cell” count was significantly lower at 1000 mg/kg/day, but also remained within the
ranges of the historical control data. No differences of toxicological or statistical significance
were noted at 50 mg/kg/day or 200 mg/kg/day.
In females, the mean absolute neutrophil count and mean platelet count at 200 mg/kg/day
showed differences which attained statistical significance (both p<0.05). The differences
(increased and reduced, respectively) remained within the ranges of the historical control values
and were not dose related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males, changes which attained statistical significance included reduced aspartate
aminotransferase activity at 200 mg/kg/day (p<0.05), elevated (but dose-unrelated) increased
activity in alkaline phosphatase activity at 200 mg/kg/day and 1000 mg/kg/day (p<0.01 and
p<0.05, respectively), elevated (but dose-unrelated) increase in the plasma potassium level at 50
mg/kg/day (p<0.01) and elevated plasma phosphorus level at 1000 mg/kg/day (p<0.05). All
differences remained within the ranges of the historical control values or were not dose-related,
and therefore considered to be incidental.
In females, elevated levels of lactate dehydrogenase and creatine kinase, noted at 1000
mg/kg/day, were traced to a single outlying animal and was considered to be due to a hemolytic
sample. These differences were not considered to be of toxicological relevance.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In males treated with 200 mg/kg/day, the mean absolute liver weights were significantly higher
(p<0.05) than those of the controls. This difference was not evident in males at the next highest
dose level and was therefore considered to be unrelated to the treatment with the test item.
In females treated with 50 mg/kg/day, the mean spleen-to-body weight ratio was significantly
lower (p<0.05) than that of the control females. This finding was not evident in females at higher
dose levels and was therefore considered to be incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, no macroscopical findings were noted in any male or female.
At 200 mg/kg/day, reddish foci on the thymus were noted in single rats of each sex (male no 12
and female no 34). In one male (no 12), the testes and epididymides were reduced in size.
At 50 mg/kg/day, unilateral renal pelvis dilation was noted in one male (no 6). In a second male
(no 8), reddish foci on the right testis were evident. One female (no 30) had enlarged dark-red
mandibular lymph nodes.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No findings, considered to distinguish treated rats from controls, were noted.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No findings, considered to distinguish treated rats from controls, were noted.

Other effects:

no effects observed

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

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-effect-level (NOEL) and no-observedadverse-
effect-level (NOAEL) for SALACOS 222 was considered to be above 1000 mg/kg body
weight/day

Executive summary:

General

In this subacute toxicity study, SALACOS 222 was administered daily by oral gavage to SPFbred

Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a

period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded

periodically during pretest and the treatment periods. Functional observational battery,

locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma

chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological

examinations were performed on organs and tissues from all control and high dose animals, and

all gross lesions from all animals.

Mortality / Viability

All animals survived until scheduled necropsy.

Clinical Signs (Daily and Weekly)

No test item-related clinical signs were noted at any dose level during daily observations or

during weekly detailed behavioral observations (weeks 1-3).

Functional Observational Battery

No abnormal clinical findings were evident during the functional observational battery (week 4).

Grip Strength

No test item-related differences in mean fore- or hind-limb grip strength were noted at any dose

level.

Locomotor Activity

The mean locomotor activity values of all rats were generally similar and showed typical intragroup

variations for this parameter.

Food Consumption

The mean daily food consumption values of the test item-treated rats were unaffected when

compared with the controls.

Body Weights

The mean body weights and the mean body weight gain values of the test item-treated males and

females were similar to those of the respective control values.

Hematology

No test item-related changes in the hematology parameters were noted at any dose level.

Clinical Biochemistry

No test item-related differences of toxicological relevance were noted in the clinical

biochemistry parameters at any dose level.

Organ Weights

No test item-related differences in the mean absolute or relative organ weights were noted at any

dose level.

Macroscopic / Microscopic Findings

None of the observed macroscopical changes were considered to be test item related.

No microscopical findings, considered to distinguish test item-treated rats from controls, were

noted in this study.