Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The Study Director signed the protocol on 16 Jun 2014, and dosing was initiated on 01 Jul 2014. The in-life phase of the study was completed on 31 Oct 2014. The experimental start date was 18 Jun 2014, and the experimental completion date was 20 Apr 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an established protocol and consistent with Good Laboratory Practice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Noted deviations from the study protocol had no impact on the outcome or integrity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): ALCHISOR CAL 145
- Substance type: Product
- Physical state: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test animals: The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies. On 10 Jun 2014, 52 male and 52 female Han Wistar rats were received from Charles River UK, Margate, Kent. At the initiation of dosing the animals were 8-9 weeks old and weighed between 212-300 g (males) and 147-194 g (females). The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

Environmental conditions: Temperatures of 18-24°C with a relative humidity of 40-75% were maintained. The target temperature and humidity ranges were 19-23oC and 40-70% respectively. A 12 hour light/12 hour dark cycle and ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% HPMC (Methocel E4M), 0.1% Tween 80 in Milli-Q water
Details on oral exposure:
The test and control items were administered to the appropriate animals by once daily oral gavage for 90 days. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a syringe with attached gavage cannula. The first day of dosing was designated as Day 1.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item dosing formulations were prepared, based on a method established at the Test Facility (under Study Numbers 432356 and 431130), at appropriate concentrations to meet dosage level requirements. The required amount of test item was weighed and transferred to a pre-labelled container. The appropriate amount of vehicle (control item) was weighed and added to the container and the solution was stirred magnetically until a visibly homogeneous formulation was obtained.
The dosing formulations were prepared weekly stored in a refrigerator set to maintain 2-8C, and dispensed daily. The dosing formulations were stirred continuously during dosing.
Any residual volumes were discarded. Details of the preparation and dispensing of the test item have been retained in the Study Records.
Dose formulation samples were collected for analysis during Week 1, 6 and 12, and were submitted for analysis within 1 week of preparation. All samples to be analysed were transferred in ambient conditions to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure (AP No. 432356).
Duplicate top, middle and bottom (middle only for Group 1, concentration only) samples
(1 mL) for each sampling time point were sent to the analytical laboratory at the Test Facility. Triplicate top, middle and bottom samples were retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result within or equal to ± 20%. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of <= 10% for each group.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily oral gavage
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
10 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
30 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
100 mg/mL
Basis:
nominal in water
No. of animals per sex per dose:
10 animals per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The objective of this study was to determine the potential toxicity of Alkenes, C13-C14, hydroformylation products, distn. residues (CAS/No. 90622-29-0) when given by oral gavage for at least 90 days to rats and to evaluate the potential reversibility of any findings after 28 days.
Alkenes, C13-C14, hydroformylation products, distn residues (CAS/No. 90622-29-0) was administered using 0.5% HPMC (Methocel E4M), 0.1% Tween 80 in Milli-Q water as the vehicle at a constant dose volume of 10 mL/kg. Dose levels were 0 (control), 20, 30, and 100 mg/mL (corresponding to dosage levels of 0, 100, 300, and 1000 mg/kg/day, respectively). Five males and five females at each of the control and high-dose levels, a total of 20 animals, were designated as recovery animals.

Examinations

Observations and examinations performed and frequency:
All animals were checked twice daily, once in the morning and once in the afternoon throughout the study for general health/mortality and moribundity. All animals were removed from their cages for examination once a week from Week -2 and on the first day of scheduled necropsy. All animals were observed regularly throughout the day on dosing days, with particular attention paid during and for the first hour after dosing and at least once daily on non-dosing days during the dosing period for reaction to treatment. In addition animals were observed at least once daily during the recovery period. The onset, intensity and duration of any signs were recorded.
Body weights were recorded for each animal twice during pretreatment, daily during the dosing period and twice weekly during the recovery period.
Animals were individually weighed. A weight was recorded on the first day of scheduled necropsy.
Food consumption was quantitatively measured twice during pretreatment and as follows during the dosing period: Weekly in Weeks 1 to 12 and over 6 days in Week 13 for main study animals: the first 5 animals per sex in Group 1 and all animals in Groups 2 and 3; Weekly in Weeks 1-13 and over 3 days in Week 14 for main study animals: the last 5 animals per sex in Group 1, all animals in Group 3 and all recovery study animals. Food consumption was measured weekly during the recovery period.
Water consumption was monitored throughout the study by visual inspection of the water bottles.
All animals were subject to an ophthalmic examination once during pretreatment, Control and High Dose animals were examined in Week 13, and all recovery animals were examined during Week 16 and Week 18.
Detailed functional observations (cageside observations, observations in a standardized arena; functional tests; grip strength; pain perception; landing food splay; and motor activity) were performed for all animals once during pretreatment period, once during Week 12 of the dosing period and once for all recovery animals during Week 18.
Sacrifice and pathology:
All animals were weighed, and euthanised by exposure to a rising concentration of carbon dioxide, followed by exsanguination. The animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day. Animals were not fasted before their scheduled necropsy.
All main study and recovery animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Scheduled necropsy examinations were conducted under the supervision of a veterinarian with appropriate training and experience in animal anatomy and gross pathology. See the following summary for additional pathology measures.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. For more details, see the following summary for statistical methods.

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 specified
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight was increased in males at 300 and 1000 mg/kg/day and females at 1000 mg/kg/day at end of treatment, with no correlating gross or microscopic findings. After a 28 day recovery period, the increase was resolved, indicating reversibility.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no unscheduled deaths during the study. There were no adverse clinical signs, body weight and food consumption changes, ophthalmoscopic and functional observation battery findings or haematology, coagulation and urinalysis parameters that were considered to be attributable to treatment.
Lower globulin level in females at 1000 mg/kg/day after treatment and recovery period was considered to not be adverse.
In organ weights, liver weight was increased in males at 300 and 1000 mg/kg/day and females at 1000 mg/kg/day at the end of treatment period, with no correlating gross or microscopic findings. Following a 28 day recovery period, this increase was no longer observed, indicating reversibility.
No test item-related macroscopic or microscopic findings were noted among either main study or recovery animals.
In conclusion, administration of Alkenes, C13-C14, hydroformylation products, distn. residues (CAS/No. 90622-29-0) by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. Based on the results, due to reversible changes in the liver weights in males and females the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RESULTS

Dose Formulation Analyses

The results of all samples were found to be within or equal to the acceptance criteria of±10% of their theoretical concentrations (individual values within or equal to±20%) of their theoretical concentrations except for Week 1, Group 2 (-13.9%) and Group 3 (623%).  Therefore, as part of the investigation, the samples were re-diluted and reanalysed and the results were within the acceptance criteria.  The investigation demonstrated that Week 1, Group 2 and Group 3 were likely to be within specification.

For homogeneity, the RSD of concentrations for all samples in each group tested was within the acceptance criteria of ≤ 10% except for Week 1, Group 2 (25.4%).  As part of the investigation, the samples were re-diluted and reanalysed and the results were within the acceptance criteria (1.6%).  One sample had a result that was found to be out of the 20% acceptance criteria with a result of ‑56.6% where originally analysed and -58.8% from nominal when re-diluted.  This sample was rejected as an outlier. 

Mortality

There were no unscheduled deaths during the course of this study.

Clinical Observations

There were no clinical signs that were considered to be attributable to administration of thetest item,Alkenes,C13-C14, hydroformylation products, distn. residues (CAS/No. 90622-29-0).

Excess salivation was recorded at 15 minutes following dosing, in eight males and five females receivingAlkenes,C13-C14, hydroformylation products, distn. residues at 1000 mg/kg/day on up to 4 occasions during the study.  Only for one of these animals salivation was observed up to 2 hours post dosing.  There were incidences of ploughing in a bedding recorded in six females at 1000 mg/kg/day immediately post dosing.

Excess salivation and ploughing may have been observed due to the taste of the test item.

The other clinical observations recorded were considered incidental, of the nature commonly observed in this strain and age of rats, therefore, were considered unrelated to administration ofAlkenes,C13-C14, hydroformylation products, distn. residues.

Body Weights and Body Weight Changes

Notest item‑related changes in body weight occurred during the study.

Food Consumption

Notest item‑related changes in food consumption occurred during this study.

Ophthalmic Examinations

There were no ophthalmoscopic findings that could be attributed to treatment with the test item.  The majority of the findings recorded during Weeks 13, 16 and 18 among animals treated with the test item had also been observed at the pretreatment examination, and thus were not considered to be treatment-related. 

Functional Observations Battery

There were no differences between treated groups andcontrols in clinical signs seen during the functional observation battery recordings, or in the values recorded for foot splay, grip strength, tail flick and body temperature at either the pretreatment or the Weeks 12 and 18 observations.

Although there were apparent separate occasions when differences were noted between motor activity of control and treated animals in Week 12 and Week 18, in the absence of changes in any other functional observation battery parameters and endpoints and a clear dose relationship, such differences are not considered to be of toxicological significance and related to treatment.

Haematology and Coagulation

                  Main study animals

There were no differences in haematology and coagulation parameters that were considered to be related to treatment.

Activated partial thromboplastin time was shorter than control values in males at 1000 mg/kg/day and longer in females at 300 mg/kg/day.  Also prothrombin time was longer in females at 300 mg/kg/day.  Fibrinogen level was lower in females at 100 and 300 mg/kg/day, however was not affected at 1000 mg/kg/day.  Due to lack of changes in the related parameters or lack of clear dose relationship, these differences are not considered to be toxicologically significant.

Haematocrit and mean cell volume were higher in females at 300 mg/kg/day and lower in females treated at 1000 mg/kg/day.  Mean cell volume was also higher in females at 100 mg/kg/day and mean cell haemoglobin concentration was lower in these animals.  Reticulocytes count was lower in females at 100 mg/kg/day.  Basophils count in was statistically significantly higher than control values in females at 1000 mg/kg/day.  Due to lack of clear dose relationship or lack of changes in the related parameters, these differences were considered not to be toxicologically significant.

                  Recovery animals

Monocytes count was lower in males treated at 1000 mg/kg/day following 4 weeks recovery period.  Platelets count and large unclassified cells were lower and neutrophils were higher in females treated at 1000 mg/kg/day following 4 weeks recovery period.  As these differences were not observed at the end of the treatment period, they are considered not to be toxicologically significant.

Clinical Chemistry

Main study animals

There were no adverse effects on clinical chemistry parameters that were considered to be related totreatment.

Cholesterol and globulin levels were lower for females at 300 and 1000 mg/kg/day when compared to the controls, with statistical significance achieved.  Albumin was higher in females at 1000 mg/kg/day.  Albumin /globulin ratio was higher in females at 100, 300 and 1000 mg/kg/day. 

Creatinine level was noted to be slightly higher in males at 300 mg/kg/day.  Glucose level was noted to be slightly higher in males at 100 and 300 mg/kg/day.  Urea was higher in males at 100 mg/kg/day.  Cholesterol and calcium levels were considered to be lower for males in the same groups.  Calcium and phosphate levels were lower in females at 100 and 300 mg/kg/day.  Alkaline phosphatase activity was noted to be slightly lower in males at 1000 mg/kg/day.  Due to the small magnitude of change and lack of a dose relationship this difference is not considered to be toxicologically significant.

Recovery animals

There were no adverse effects on clinical chemistry parameters that were considered to be related to treatment.

Globulin level in females at 1000 mg/kg/day was lower when compared to the controls following 4 week recovery period, similarly as at the end of the treatment period.  As these were not associated with any histopathology findings they were considered to not be treated as adverse. 

Albumin level in males at 1000 mg/kg/day was lower when compared to the controls following 4 week recovery period.  As these differences were not observed at the end of the treatment period, they are considered not to be toxicologically significant.

Urinalysis

There were no differences in urinalysis parameters in either Week 13 or 17.

Gross Pathology

                  Terminal Euthanasia Animals (Day 91/95/96)

No test item-related gross findings were noted. 

An increased incidence of dark discoloration in the lung was noted in animals from Groups 2 and 3, with frequent histologic correlation to lung congestion.  However the incidence of histologic lung congestion was similar in these groups and in control animals.  Other gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Alkenes, C13-C14, hydroformylation products, distn. residues.

                  Recovery Euthanasia Animals (Day 123)

No test item-related gross findings were noted. 

The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Alkenes, C13-C14, hydroformylation products, distn. residues. 

Organ Weights

Terminal Euthanasia Animals (Day 91/95/96)

Test article-related organ weight changes are summarized in the following table.

Summary of Organ Weight Data – Terminal Euthanasia (Day 91/95/96)

 

Males

Females

Group

2

3

4

2

3

4

Dose (mg/kg/day)

100

300

1000

100

300

1000

No. Animals per Group

10

10

10

10

10

10

Liver (No. Weighed)a

10

10

10

10

10

10

     Absolute value

3.8%

8.5%

11.2%

2.6%

-1.9%

10.2%

     % of body weight

5.8%

7.0%

10.0%

2.6%

-0.6%

8.0%

a   All values expressed as percent difference of control group means.

     Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group, refer to data tables for actual significance levels and tests used.

The liver weights were higher in males received Alkenes, C13-C14, hydroformylation products, distn. residues at 300 and 1000 mg/kg/day ; when compared with controls with statistical significance achieved for relative weights.  Very slight increase was also observed in males at 100 mg/kg/day.  Absolute and relative liver weights were higher in females at 1000 mg/kg/day, when compared with controls with statistical significance achieved.  Liver weights were lower for females at 300 mg/kg/day. No histopathological findings correlating with liver weight changes were observed.

No other test item-related organ weight changes were noted.  There were other isolated organ weight values that were statistically different from their respective controls.  There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant.  Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of Alkenes, C13-C14, hydroformylation products, distn. residues.

Recovery Euthanasia Animals (Day 123)

No test item-related organ weight changes were noted in the liver or other organs. 

There were isolated organ weight values that were statistically different from their respective controls (increased relative ovarian in females at 1000 mg/kg/day).  There were, however, no correlating data to suggest these values were toxicologically relevant.  Thus, the organ weight differences observed were considered incidental and/or related to differences of sexual maturity and unrelated to administration of Alkenes, C13-C14, hydroformylation products, distn. residues. 

Histopathology

Terminal and Recovery Euthanasia (Day 91/95/96 and 123)

No test item-related microscopic findings were noted. 

The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of Alkenes, C13-C14, hydroformylation products, distn. residues.

Applicant's summary and conclusion

Conclusions:
Administration of Alkenes, C13-C14, hydroformylation products, distn. residues (CAS/No. 90622-29-0) by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. Based on the results, due to reversible changes in the liver weights in males and females the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

SUMMARY

The objective of this study was todetermine the potential toxicity of Alkenes, C13-C14, hydroformylation products,distn.residues(CAS/No. 90622-29-0)when givenby oral gavage for at least 90 daysto rats andto evaluate the potential reversibility of any findings after 28 days.

The study design was as detailed in the following table.

Experimental Design

Group No.

Test Item

Dosage Level (mg/kg/day)

 

Dosage Volume

(mL/kg)

Dosage Concentration

(mg/mL)

Number of Animals

Main Study

Recovery Animals

Males

Females

Males

Females

1

Control

0

10

0

10

10

5

5

2

Test Item

100

10

10

10

10

-

-

3

Test Item

300

10

30

10

10

-

-

4

Test Item

1000

10

100

10

10

5

5

Test Item = Alkenes, C13-C14, hydroformylation products,distn.residues(CAS/No. 90622-29-0).

 

Alkenes, C13-C14, hydroformylation products,distn. residues was administered using 0.5% HPMC (MethocelE4M), 0.1% Tween 80 inMilli-Q water as the vehicle at a constant dose volume of 10 mL/kg.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ophthalmology, functional observations and motor activity, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathological examinations.

There were no unscheduled deaths during the study. There were no adverse clinical signs, body weight and food consumption changes, ophthalmoscopic and functional observation battery findings or haematology, coagulation and urinalysis parameters that were considered to be attributable to treatment.

Lower globulin level in females at 1000 mg/kg/day after treatment and recovery period was considered to not be adverse.

In organ weights, liver weight was increased in males at 300 and 1000 mg/kg/day and females at 1000 mg/kg/day at the end of treatment period, with no correlating gross or microscopic findings. Following a 28 day recovery period, this increase was no longer observed, indicating reversibility.

No test item-related macroscopic or microscopic findings were noted among either main study or recovery animals. 

In conclusion, administration of Alkenes, C13-C14, hydroformylation products, distn.residues by once daily oral gavage was well tolerated in rats at levels of 100, 300 and 1000 mg/kg/day. Based on the results, due to reversible changes in the liver weights in males and females the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.