Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

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:

2017-12-12 to 2019-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

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)

Deviations:

yes

Remarks:

deviations from study plan, see below "Any other information on materials and methods"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

deviations from study plan, see below "Any other information on materials and methods"

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

EC Number:

700-064-6

Cas Number:

2105830-60-0

Molecular formula:

Not applicable as this is a UVCB substance

IUPAC Name:

Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Test material form:

liquid: viscous

Details on test material:

- Name: Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters
- Product Name: Multiester P97-463
- CAS No.: 2105830-60-0
- Batch No.: X24Jan17
- Physical State: viscous liquid
- Colour: amber
- Density: 1.06 g/cm³
- Active Components: 94%
- Expiry Date: 31 December 2018
- Storage Conditions: Room temperature, protected from light and kept in closed container to avoid air contact
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Specific details on test material used for the study:

Active Components: 94 %, were taken into account in formulation preparation
Date of Analysis 26 June 2017

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

PREPARATION
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration animals showed no pathological signs.
All animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).

ANIMALS
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approximately 7-8 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 175 - 195 g (mean: 186.15 g, ± 20 % = 148.92 – 223.38 g)
females: 127 - 148 g (mean: 137.00 g, ± 20 % = 109.60 – 164.40 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.


HUSBANDRY
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of up to 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Manufacturer: Merck Batch No.: S7378385710 Physical State: liquid Storage Conditions: room temperature Expiry Date: 05 December 2018

Details on oral exposure:

Experimental Groups and Doses
According to the results of a previous dose range finding study (BSL Munich Study No. 175736, non GLP) and in consultation with the sponsor the following doses
were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 28 days.
C 0 mg/kg bw
LD 100 mg/kg bw
MD 300 mg/kg bw
HD 1000 mg/kg bw
Dose formulation was prepared based on active ingredient content within the test item (94 %)

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

Administration of Doses
The test item formulation or vehicle was 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.

C 0 Concentration [mg/mL]
LD 26.6 Concentration [mg/mL] = corresponding to 25 mg/mL of active ingredient in the dosing formulation
MD 79.8 Concentration [mg/mL] = corresponding to 75 mg/mL of active ingredient in the dosing formulation
HD 266 Concentration [mg/mL] = corresponding to 250 mg/mL of active ingredient in the dosing formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before start of the treatment period, stability and homogeneity of the test item formulation samples in PEG 400 were tested at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 175740). In week 1, homogeneity was tested on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
For Dose Formulation Analysis samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1, 2, 3 and 4 of the treatment. However, only samples taken from week 2 and 4 were analysed (20 samples). Mean concentration over all sampling locations were reported.
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 175741) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 other: mg/kg bw/day

Remarks:

dose formulation was prepared based on active ingredient content within the test item (94 %)

Doses / concentrations 3

Dose / conc.:

300 other: mg/kg bw/day

Remarks:

dose formulation was prepared based on active ingredient content within the test item (94 %)

Doses / concentrations 4

Dose / conc.:

1 000 other: mg/kg bw/day

Remarks:

dose formulation was prepared based on active ingredient content within the test item (94 %)

No. of animals per sex per dose:

40 animals (20 males and 20 females) were included in the study (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

The test item is orally administered daily in graduated doses to several groups of test animals, one dose level per group, for a period of 28 days. During the period of
administration, the animals are observed precisely each day for signs of toxicity. Animals which die or are euthanised for animal welfare reasons during the test are
examined macroscopically and histopathologically. At the end of the test all surviving animals are euthanised and then examined macroscopically and histopathologically.
As a specific endpoint for neurological effects a battery of functional observation tests is performed.

Examinations

Observations and examinations performed and frequency:

BODY WEIGHT AND FOOD CONSUMPTION
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Food consumption was measured weekly during the treatment period.

CLINICAL OBSERVATIONS
All animals were observed for clinical signs during the entire treatment period of 28 days.
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.
The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering 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 were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

FUNCTIONAL OBSERVATIONS
Once before the first exposure and once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.

BLOOD SAMPLING FOR CONFIRMATION OF EXPOSURE
For confirmation of exposure, at mid of the study (day 15 or 16) blood were sampled at 3 time points (pre-value before application, 30 min and 2 hours post treatment) from the jugular vein of all surviving animals. Approx. 250 µL blood was sampled in K3 EDTA coated tubes.
The samples were placed on ice and were then centrifuged for 10 min at 4 °C at approx. 1000 g. After centrifugation approx. 100 µL plasma was transferred into a tube labeled with “Plasma”, study no., animal ID number and the time point of blood collection (a total of 120 tubes). All plasma samples were stored at ≤ -70 °C until analysis.
As requested by the sponsor the samples were not analysed and will be discarded after finalisation of the study.

Sacrifice and pathology:

HAEMATOLOGY
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value, haemoglobin content, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells.

BLOOD COAGULATION
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time and activated partial thromboplastin time.

CLINICLA BIOCHEMISTRY
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea, total bilirubin, total bile acids, total cholesterol, glucose, sodium and potassium.

URINALYSIS
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL): specific gravity, nitritwe, pH-value, protein, glucose, ketone bodies, urobilinogen, bilirubin, erythroctes and leukocytes. Additionally, urine colour/ appearance were recorded. 

PATHOLOGY
One day after the last administration (study day 29) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine and xylazin)
and subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORAN WEIGHT
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenal glands, thyroid/ parathyroid glands, testes, spleen, epididymides brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of all sacrificed animals was recorded as soon as possible.
Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
The brain weight of animal no. 18 was weighed at test site 1 after fixation in formalin and resulted in 2.1124 g.

The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, parathyroid glands, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.

HISTOPATHOLOGY
The afore-listed organs (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, parathyroid glands, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and animals no. 21 and 36 that died prematurely during the course of treatment.
Due to test item-related morphologic changes detected in the liver (both sexes) and thymus (females) of high dose animals these organs were examined also from the low and medium dose groups.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The Study phase from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study .

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 were performed for each gender by comparing values of dosed with control animals using either a parametric
one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity
and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered
as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see Details on results

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see Details on results

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

see Details on results

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Urinalysis findings:

no effects observed

Description (incidence and severity):

see Details on results

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

see Details on results

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

see Details on results

Other effects:

not examined

Details on results:

MORTALITY
Female control animal no. 21 was euthanized in a bad health condition for animal welfare reasons on study day 16.
Markedly reduced spontaneous activity and apathy were noted on study day 16. Eyes were half-closed and abnormal breathing was observed in this animal.
Female animal no. 36 of the HD group was found dead on study day 10. Prior to death no abnormal clinical signs were noted.

CLINICAL OBSERVATIONS
Moving the bedding and salivation were observed in dosed animals and also in few control animals.
Moving the bedding was noted in all HD animals from day 3 or 4 onwards and in all MD and LD animals from day 16, 17 or 18 onwards.
Salivation was observed in females of the LD group and in MD and HD groups, occasionally or in all of the animals per group but only on single or few days.
In one HD male this symptom was seen for approx. 2 weeks. Moving the bedding and salivation were noted in 2 or 1 control animals, respectively.
These symptoms indicate a local reaction of the formulation after oral gavaging and are not considered to be test item related.
Mild symptom of crust in male MD animal no. 14 is not considered to be treatment related. Piloerection was observed in
HD animal no. 37 during the last 3 days coinciding with kyphosis and slight body weight loss on the last treatment day.

FUNCTIONAL OBSERVATION BATTERY
Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters had no effect on neurobehavioural parameters of the Irwin Test examined during the last treatment week. No opthalmoscopic abnormalities were observed in this study.

BODY WEIGHT
Daily treatment with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters did not affect body weight development in male animals of this study.
Statistically significantly lower body weight gain of male MD animals during the 3rd treatment week is not considered toxicologically relevant due to the slightness and the lack of dose-dependency. At the end of the treatment period, a tendency towards attenuated body weight development could be observed in female animals of LD, MD and HD groups (approx. 8 %, 4 % and 6 % below controls, respectively). This was related to statistically significantly lower body weight gain observed in female animals of all dose groups during the first treatment week.
 
FOOD CONSUMPTION
Throughout the treatment period no differences of biological or statistical significance were observed in weekly food consumption between dose groups and control group in this study.

HAEMATOLOGY AND BLOOD COAGULATION
Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters slightly affected red blood cell parameters determined at the end of the treatment period of this study. At 1000 mg/kg/d MCV of red blood cells was statistically significantly lower in male – but not female animals when compared to controls (approx. 7 % lower than controls). This coincided with a very slight but statistically significantly higher mean cell haemoglobin concentration in these animals (approx. 5 % above controls). Although a relation to the test item cannot be excluded, in absence of any associated findings and due to the slightness this is not assumed to be toxicologically relevant.
In male animals of this dose group reticulocytes were statistically significantly lower than in controls (1.4 % vs. 2.2 % in controls).
Due to the slightness this is not considered to be biologically relevant.
In female animals of the HD group a slight but statistically significantly lower RBC count was noted (7.4 x 109 cells/µL vs. 8.1 x 109 cells/µL).
In accordance statistically significantly lower haemoglobin and hematocrit values were noted in female animals of this group (approx. 10 or 12 % below controls, respectively). Mean haematocrit – but not hemoglobin and RBC values were also slightly but statistically significantly lower in MD and LD group females when compared to controls (11 % and 9 % below controls, respectively). Although an effect of the test item seems likely, this is not considered adverse.
Slight but statistically longer mean PT was found in female animals of the MD group (approx. 23 sec vs. 19 sec in controls). Due to the slightness and lack of dose dependency this slightly longer PT was not assumed to be toxicologically relevant.

CLINICAL BIOCHEMISTRY
Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters had no toxicologically relevant effect on parameters of clinical biochemistry determined at the end of the treatment period. Statistically significantly lower serum level of ALAT, ASAT, Urea and TBA found in male animals of the HD group is not considered to be of toxicologically relevance as only increases in these parameters are usually associated with pathological conditions. ALAT and ASAT levels were also slightly lower in males of the MD group.
Serum urea level was also slightly low in female animals at 1000 mg/kg/d. Mean serum glucose level was statistically significantly higher in female animals of the HD group when compared to control (approx. 72 % higher). Although a test item related effect cannot be excluded in the absence of correlated findings (e.g. histomorphological changes in pancreas or liver) this difference is not considered toxicologically relevant.

URINALYSIS
Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters had no effect on parameters of clinical biochemistry determined at the end of the treatment period.
 
PATHOLOGY
A fluid-filled thoracic cavity was found in female animal no. 36 that was found dead on study day 10.
The general condition of the animal was autolytic and the lung had an abnormal red surface.
The thoracic cavity of female control animal no. 21 that was euthanized in a bad health condition was filled with a clear fluid.
A cyst was found on the left ureter of this animal.
At scheduled necropsy only incidental macroscopic findings were noted.
Red coloured jejunum and ileum was observed in male MD group animal no. 12. Jejunum was also red coloured in male MD animal no. 15 and in male animal no. 6 of the LD group. The ureters of control female animal no. 25 were fluid-filled.
The uterus of animal no. 30 of the LD group was fluid-filled.

ORGAN WEIGHT
Statistically significantly lower thymus weight (absolute and normalized to body weight) was found in female animals of the HD group when compared to controls (35 and 33 % below controls, respectively). A tendency towards lower thymus weights were also observed in male animals of this group (16 % below controls). Slightly but statistically significantly higher normalised (to body weight) kidney weight was found in female animals of the HD group (10 % above controls). This was not associated with any histopathological findings of nephropathy in these animals and is not considered toxicologically relevant.
Absolute and normalized liver weight was statistically significantly higher in females of the HD group when compared with controls (27 and 33 % above controls, respectively). A tendency towards higher liver weight in males of the MD and HD groups (approx. 11 %, respectively).
A tendency towards lower spleen weight was observed in male and female animals of the HD group (approx. 10 and 13 % below controls, respectively).
Slightly lower thyroid gland weight was seen in LD, MD and HD groups of males (16, 11 and 10 % above controls, respectively) and females (35, 26 and 22 % above controls, respectively). Above-mentioned changes are not considered toxicologically relevant due to their slight degree.

HISTOPATHOLOGY
Moribund female no. 21 presented microscopically a slight mucosal/submucosal perforation in the esophagus. Along with the clear fluid observed at necropsy in the thoracic cavity, this finding was considered to be indicative for traumatical injury that may occur during the application procedure and, therefore, was considered to be not test item related.
Microscopic findings related to the premature death of male no. 36 consisted of minimal congestion and alveolar edema in the lung.
At 1000 mg/kg bw, minimal degrees of mainly centrilobular hepatocellular hypertrophy were recorded in the liver of two male animals.
Minimal to slight hepatocellular hypertrophy was recorded also in some high dose females.
The severity grade of thymus atrophy increased in female animals treated with 1000 mg/kg bw.
The mean severity grade of fatty replacement in the bone marrow increased slightly in high dose animals. In absence of any indicators of bone marrow injury, this finding was considered to be most likely incidental.
All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age and in this study type.

DOSE FORMULATION ANALYSIS
Concentration analysis of the formulation samples was determined at three levels, 25 mg/mL, 75 mg/mL and 250 mg/mL in the study performed in week 2 and week 4. The mean recoveries of triplicate samples analyzed in week 2 and week 4 for the LD group were 91.3 % and 87.6 % of the nominal value, 97.7 % and 88.9 % for the MD group and 109.4 % and 98.7 % of the nominal value for HD group, respectively.
The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 89.5 %, 93.3 %, and 104.1 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 20 %. Homogeneity of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in the study performed in week 2 and week 4. The coefficients of variation of the different sampling locations (top, middle and bottom) were 3.0 % in LD group, 6.7 % in MD group and 7.3 % in HD group. All samples were homogenous, as COV was below or equal 15 %.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of adverse findings

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in male and female Wistar rats with dose
levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made: In the absence of adverse findings the NOAEL for general systemic toxicity could be established at 1000 mg/kg bw.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters via oral administration to rats over a period of 28 days.

The test item 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 PEG 400(polyethylene glycol), the vehicle used in this study.

The 4 groups comprised of 5 male and 5 female Wistar rats.

The following doses were evaluated:

Control:                 0         mg/kg body weight

Low Dose:             100   mg/kg body weight

Medium Dose:       300  mg/kg body weight

High Dose:            1000 mg/kg body weight

The test item formulation was prepared and administered within the time frame of stability.

The test item was suspended in PEG 400 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.

Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice from all surviving animals.

Urinalysis was performed on samples collected at terminal sacrifice from all surviving animals.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Due to test item-related morphologic changes detected in the liver (both sexes) and thymus (females) of high dose animals these organs were examined also from the low and medium dose groups. Any gross lesion macroscopically identified was examined microscopically in all animals.

Summary Results

No test item-related mortality was observed in this study. Mortality occurred in one female animal of the control group and HD group, respectively.

The cause of morbidity or death of both females was considered to be most likely due to traumatic injury of the esophagus/thoracic cavity that may occur during the application procedure and, therefore, was considered to be not test item related.

In the remaining animals there were no clinical signs of systemic toxicity and no effect of treatment with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters on neurobehavioural parameters were observed. Body weight development was not affected in male and only very slightly negatively in female animals at all dose levels.

 

Slight non-adverse effects on the red blood cell system were observed at the end of the treatment period in male (slightly lower mean cell volume without decrease in haemoglobin) and in female animals (mild anemia) at 1000 mg/kg/d. No toxicologically relevant differences in coagulation parameters and parameters of clinical biochemistry were observed at the end of the treatment period. Moreover, urinary parameters were unaffected. At scheduled necropsy only incidental macroscopic findings were noted.

Histopathologically, the treatment withCyclohexene-1,2-dicarboxylic acid, methyl-, castor oil alkyl esterinduced minimal to slight hepatocellular hypertrophy in the liver of high dose animals that correlated with increased liver weights recorded in high dose females. This hepatocellular hypertrophy was deemed to be not adverse in the absence of any further lesion (e.g. Kupffer’s cell proliferation, increased apoptosis, necrosis, fibrosis etc.) and hence was considered to represent an adaptive change. A further treatment-related finding consisted of an increased mean severity grade of thymus atrophy correlating with decreased thymus weights in high dose females. This finding was considered to be secondary to stress and to be not adverse. Further slight differences in organ weights (i.e. lower spleen weight, higher kidney weight, lower thyroid weight were not correlated with histopathological findings and are not considered biologically relevant.

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

In the absence of adverse findings the NOAEL for general systemic toxicity could be established at 1000 mg/kg bw.