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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012-11-08 to 2013-06-21
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
• OECD 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents”, 1998. • OECD 416, “Two-Generation Reproduction Toxicity Study”, 2001.(only for clinical pathology and histopathological evaluations of male reproductive organs including sperm parameters)
Deviations:
no
Principles of method if other than guideline:
The purpose of this repeat dose toxicity study was to assess the systemic toxicity potential of the test item, Acetalization product between glucose and C16/18 (even numbered)-alcohol in Wistar rats when administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects following 28-days recovery period.
This study was designed to provide information on major systemic toxic effects, target organs, the possibility of accumulation, additional parameters (clinical pathology and histopathological evaluations to evaluate effects of the test item on male reproductive organs) and an estimate of a No Observed Adverse Effect Level (NOAEL)/No Observed Effect Level (NOEL).
Detailed testicular histopathological examination was conducted in order to identify possible treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Intact epididymis was also examined to include the caput, corpus, and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for possible
leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. As described in paragraphs 41 to 44 (OECD n°416).
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles of Good Laboratory Practice [C (97) 186/Final]
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
Test Item: Acetalization product between glucose and C16/18 (even numbered)-alcohol

Chemical Name (IUPAC): Acetalization product between glucose and C16/18 (even numbered)-alcohol

Lot No.: T22335

Batch Manufactured and Supplied by (Name and Address): SEPPIC, 127 chemin de la poudrerie, 81105 Castres, France

Manufactured Date: 06.June.2012

Retest date: 06.June.2015

Purity as per Certificate of Analysis: >99% Dry extract

Physical Appearance: Solid

Recommended Storage Condition: Ambient (+15 to +25ºC)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Wistar rats – HsdCpb: WU rats conventionally bred (In-house random bred)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 2 hours) each day for a period of 90 consecutive days. Similarly, vehicle was administered by oral gavage to rats in vehicle control and vehicle control recovery groups for 90 consecutive days. The vehicle or the dose formulations were not administered to the rats in the recovery groups for 28 Days following the 90-day treatment period.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of dose formulation (i.e. at 10, 30 and 100 mg/mL) analysis on Day 1 and during the second and third month of the treatment showed that, the mean analysed concentration of the active ingredient was within the acceptable limits (15% and RSD was less than 10%) of variation and was homogeneously mixed in the vehicle. The test item was not detected in the vehicle control group samples analysed during different intervals of the treatment period.
Duration of treatment / exposure:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 2 hours) each day for a period of 90 consecutive days. Similarly, vehicle was administered by oral gavage to rats in vehicle control and vehicle control recovery groups for 90 consecutive days. The vehicle or the dose formulations were not administered to the rats in the recovery groups for 28 Days following the 90-day treatment period.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Frequency of treatment:
Daily for 90 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
Each main group comprised of 10 rats/sex and recovery groups 5 rats/sex as described below:


Group No. Dose (mg/kg/day) No. of Animals Dose volume (mL/kg) Terminal sacrifice
G1 Vehicle control* 10M + 10F 10 Day 91
G2 100 10M + 10F 10 Day 91
G3 300 10M + 10F 10 Day 91
G4 1000 10M + 10F 10 Day 91
G1R Vehicle control* 5M + 5F 10 Day 119
G4R 1000 5M + 5F 10 Day 119
M: Males; F: Females; * The vehicle was Olive oil
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 50 males and 50 female rats were randomly allocated to four main groups and two recovery groups, each main group comprised of 10 rats/sex and recovery groups 5 rats/sex as described below:


Group No. Dose (mg/kg/day) No. of Animals Dose volume (mL/kg) Terminal sacrifice
G1 Vehicle control* 10M + 10F 10 Day 91
G2 100 10M + 10F 10 Day 91
G3 300 10M + 10F 10 Day 91
G4 1000 10M + 10F 10 Day 91
G1R Vehicle control* 5M + 5F 10 Day 119
G4R 1000 5M + 5F 10 Day 119
M: Males; F: Females; * The vehicle was Olive oil
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
Each rat was observed twice daily for mortality and morbidity during the treatment and recovery period. Routine observations for checking general clinical signs were performed once during pre-dose and two-three times during post-dose to observe the test item-related clinical sign of salivation during the treatment period and once daily during the recovery period.
Sacrifice and pathology:
All the animals of main and recovery groups were subjected to detailed necropsy and findings were recorded. The rats to be sacrificed at term were fasted overnight (water allowed), weighed, exsanguinated under isoflurane anaesthesia and subjected to detailed necropsy by a Pathologist, as per the random numbers generated for terminal sacrifice (main and recovery).
Other examinations:
Toxic effects, target organs, the possibility of accumulation, additional parameters (clinical pathology and histopathological evaluations to evaluate effects of the test item on male reproductive organs
Statistics:
The Data captured using Provantis™ for the parameters namely body weights, food consumption and organ weights were analyzed using built-in statistical tests. Derived data like net body weight change and organ weight ratios was also analyzed using above mentioned methods.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables like neurological observations (neuromuscular observation and body temperature) and clinical pathology (haematology, coagulation and clinical chemistry) data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment, ‘F’ test when found significant.
In the case of recovery groups, data was analysed using the methods stated above. Comparison of means between treatment recovery group(s) and control recovery group was performed.
All analyses and comparisons were evaluated at the 5 % (P ≤ 0.05) level. Statistically significant differences (P ≤ 0.05), indicated by the aforementioned tests was designated by the following superscripts throughout the report:
+/- : Significantly higher (+) /lower (-) than the vehicle control group
a+/a- : Significantly higher (+) /lower (-) than the vehicle control recovery group

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
• No mortalities were observed in males and females of main toxicity and recovery groups at all dose levels. • The salivation (slight to moderate) was observed in both sexes at all the test item treated groups during the course of the treatment period. This clinical sign of salivation was observed at 5-10 minutes post-dose and was persisted up to approximately 45 minutes post-dose only. The incidences of salivation were more in the high dose group rats (1000 mg/kg/day) and these were not observed consistently in same rats. The observed clinical sign of salivation was considered as transient and test item-related non-adverse effect. There were no other clinical signs observed in any of the test item treated groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
• No mortalities were observed in males and females of main toxicity and recovery groups at all dose levels. • The salivation (slight to moderate) was observed in both sexes at all the test item treated groups during the course of the treatment period. This clinical sign of salivation was observed at 5-10 minutes post-dose and was persisted up to approximately 45 minutes post-dose only. The incidences of salivation were more in the high dose group rats (1000 mg/kg/day) and these were not observed consistently in same rats. The observed clinical sign of salivation was considered as transient and test item-related non-adverse effect. There were no other clinical signs observed in any of the test item treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
• Body weights, net body weight gains were unaffected by the treatment at all dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption were unaffected by the treatment at all dose levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
• No eye abnormalities were found in the ophthalmological examination conducted at the end of the treatment and recovery period.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in hematology, coagulation parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in clinical chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in urinalysis parameters.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related neurological abnormalities observed at at the end of the treatment and recovery period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related changes in organ weights/ratios and sperm parameters.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross changes among the doses tested.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic changes among the doses tested.
Details on results:
CLINICAL SIGNS AND MORTALITY
The clinical sign of salivation (slight to moderate) was observed in both sexes from Day 25 onwards in all the test item treated groups during the course of the treatment period. This clinical sign of salivation was observed at 5-10 minutes post-dose and was persisted up to approximately 45 minutes post-dose only. The incidences of salivation were more in the high dose group rats (1000 mg/kg/day). The observed clinical signs were intermittent, as they were not observed in the same rat consistently during the whole course of in-life phase. The observed clinical sign of salivation was considered as transient and test item-related non-adverse effect of the test item.

There was no mortality observed during the course of the experiment.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights and net body weight gains were unaffected by the treatment at all the tested dose groups throughout the treatment period in both main and recovery group males and females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption was unaffected by the treatment in males at all the tested dose groups throughout the treatment period in both main and recovery group.
However, in females the following statistically significant changes were observed in the mean food consumption as compared to the vehicle control group:
- at 100 mg/kg Bwt/day, lower food consumption was observed in females during Days 57 -64,
- at 300 mg/kg Bwt/day, lower food consumption was observed in females during Days 22-29, 36-43, 57-64, 64-71 and 78-85.
- at 1000 mg/kg Bwt/day, lower food consumption was observed in females during Days 57-64, 64-71 and 78-85.
- at the high dose recovery group females, higher food consumption was observed during Days 64-71, 71- 78, 85-90, 90-97, 104-111 and 111-118 as compared to the vehicle control recovery group.
The few minor changes observed in food consumption was considered as incidental and not test item-related findings as there decreases were not associated with any changes in absolute mean body weights.

OPHTHALMOSCOPIC EXAMINATION
No eye abnormalities were found in the ophthalmological examination conducted during the acclimatization, end of the treatment and recovery period.

HAEMATOLOGY
No changes of toxicological relevance were observed in hematology parameters of both sexes. Increase in total leukocyte and lymphocyte counts noted at 1000 mg/kg/day dose recovery males were considered to be incidental, as the change was not apparent in main group rats. In addition, the magnitude of change was comparable with historical control data. (Refer Tables 1 to 4, Appendices 1 to 4 of the attached report)

No test item-related effects were noted in PT and APTT values in any of the treated groups. A decrease in APTT value noted at 1000 mg/kg/day dose recovery males was considered incidental as the change had no toxicologic relevance. (Refer Tables 1 to 4, Appendices 1 to 4 of the attached report)

CLINICAL CHEMISTRY
There were no toxicologically significant changes in biochemical parameters, in any tested groups. Increased total cholesterol (1000 mg/kg males), glucose/potassium (1000 mg/kg recovery males), and decreased GGT, ALP, total cholesterol and calcium levels (1000 mg/kg recovery males) were considered incidental, as the alterations were either comparable with in-house historical control data or lack biological significance. (Refer Tables 5 to 8, Appendices 5 to 8 of the attached report)

URINALYSIS
There were no test item-related changes in urine parameters at all the doses tested.

NEUROBEHAVIOUR
Neuromuscular Observations: There were no test item-related variations in hind limbs footsplay and fore limbs and hind limbs grip strength in both sexes.

ORGAN WEIGHTS
There were no significant changes attributed to treatment in terminal fasting body weights, organ weights and organ to body weight ratios in both the sex groups at any tested doses when compared to the vehicle control group.
The weight changes involving thymus (1000 mg/kg recovery males; ≥ 100 mg/kg females), pituitary (1000 mg/kg recovery females) and uterus (≥ 300 mg/kg females) were considered to be toxicologically not relevant, as the alterations did not show corresponding histological findings and/or were within the range of laboratory historical control data. (Refer Tables 13 to 20, Appendices 13 to 20 of the attached report)

SPERM EVALUATION (Refer Table 21, Appendices 21 to 24 of the attached report)
Sperm motility was examined for all the dose groups. There were no significant differences in percentages of progressive motile sperms and motile sperms between the groups except for the G2 group where a statistically but not biological significant decrease was observed. This observation was considered as incidental.
Sperm morphology was examined for all the dose groups. The Significantly but not statistically increased percentage of abnormal sperms noted at 1000 mg/kg/day dose was considered incidental, as the change was not dose-related and the level of abnormal sperm was near the control group values. The commonly observed abnormality across groups was headless sperms.

Cauda epididymal sperm count was done for all the dose groups. The non statistical decreased values for number of sperms per cauda epididymis and number of sperms per gram of cauda epididymis at 1000 mg/kg/day dose were due to inter-individual variations and were considered to be of no biological significance, as the alterations were not dose-related. Further, the cauda epididymal weight did not differ significantly among the treated groups.
Detergent and homogenization resistant testicular spermatic counts were performed at all the dose levels. Significantly decreased values observed at 1000 mg/kg/day involving number of spermatid per testis and number of spermatid per gram of testis were considered incidental in the absence of dose-relationship and due to an individual lower count (animal Ro3440). There were no significant differences in testicular weights between the groups. All sperm parameters evaluated remained unaffected by dosage as high as 1000 mg/kg/day.

GROSS PATHOLOGY
No changes attributed to test item were noted in treated rats of both the sexes.
Dilatation of uterus observed in a female at 100 mg/kg/day dose was microscopically confirmed and considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic changes attributed to treatment in males and females. The microscopic findings observed in few organs in some of the animals were considered as incidental background findings and hence they were not related to test item-administration. (Refer Table 23, Appendices 29 to 32 of the attached report)

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
Acetalization product between glucose and C16/18 (even numbered)-alcohol
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
CONCLUSION
The repeated administration of test item Acetalization product between glucose and C16/18 (even numbered)-alcohol to Wistar rats through oral gavage for 90 consecutive days at the dose levels of 100, 300 and 1000 mg/kgBwt/day did not cause any toxicological effects on general health, mortality, functional observational battery, growth and food consumption. No test item-related changes observed in haematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights, organ weights/ratios and sperm parameters and gross and histopathological changes.
The slight salivation which was observed in all rats at all the doses was considered transient and test item-related non-adverse effect. Hence, under the test conditions, the evaluated “No Observed Adverse Effect Level (NOAEL)” for test item Acetalization product between glucose and C16/18 (even numbered)-alcohol is 1000 mg/kg Body weight/day under the test conditions and doses employed.
Executive summary:

The purpose of this repeat dose toxicity study was to assess the systemic toxicity potential of the test item, Acetalization product between glucose and C16/18 (even numbered)-alcohol in Wistar rats when administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects following 28-days recovery period. This study was intended to provide information on major systemic toxic effects, target organs, the possibility of accumulation, additional parameters (clinical pathology and histopathological evaluations to evaluate effects of the test item on male reproductive organs) and an estimate of a No Observed Adverse Effect Level (NOAEL).

A total of 50 males and 50 female rats were randomly allocated to four main groups and two recovery groups, each main group comprised of 10 rats/sex and recovery groups 5 rats/sex as described below:

Group numbers

Dose (mg/kg/day)

Number of Animals

Dose volume (mL/kg)

Terminal sacrifice

G1

Vehicle control*

10M + 10F

10

Day 91

G2

100

10M + 10F

10

Day 91

G3

300

10M + 10F

10

Day 91

G4

1000

10M + 10F

10

Day 91

G1R

Vehicle control*

5M + 5F

10

Day 119

G4R

1000

5M + 5F

10

Day 119

M: Males;  F: Females;  * The vehicle was Olive oil

The vehicle or test item suspensions were not administered to the recovery groups (G1R and G4R) for 28 days following the 90-days dosing period. All rats were observed for clinical signs and mortality daily. Body weights and food consumption were measured during the course of in-life phase of the experiment. The functional observation tests were performed during 13thweek of in-life phase. The blood samples were collected for clinical pathology at termination. All the rats were sacrificed at the end of the treatment for main groups and at the end of recovery groups for recovery groups and subjected to gross examination. The study plan specified organs were collected, weighed and preserved.

Histopathological examination was restricted to the preserved organs from vehicle control (G1) and high dose (G4) group animals (with qualitative assessment of stages of spermatogenesis), as there were no identifiable target organs/tissues in the high dose group rats. In addition, all the gross lesions from all the animals were examined microscopically.

Detailed testicular histopathological examination was conducted in order to identify possible treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Intact epididymis was also examined to include the caput, corpus, and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for possible leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm.As described in paragraphs 41 to 44 (OECD n°416).

The identity of the Acetalization product between glucose and C16/18 (even numbered)-alcohol was provided by the study Sponsor by a Certificate of Analysis. The correct identity and purity of the test item are the responsibility of the Sponsor. The test item was not authenticated at the test facility.

Summary of Results:  

·        No mortalities were observed in males and females of main toxicity and recovery groups at all dose levels.

·        The salivation (slight to moderate) was observed in both sexes at all the test item treated groups during the course of the treatment period. This clinical sign of salivation was observed at 5-10 minutes post-dose and was persisted up to approximately 45 minutes post-dose only. The incidences of salivation were more in the high dose group rats (1000 mg/kg/day) and these were not observed consistently in same rats.The observed clinical sign of salivation was considered as transient andtest item-related non-adverse effect. There were no other clinical signs observed in any of the test item treated groups.

·        Body weights, net body weight gains and food consumption were unaffected by the treatment at all dose levels.

·        No eye abnormalities were found in the ophthalmological examination conducted at the end of the treatment and recovery period.

·        There were no test item-related neurological abnormalities observed at at the end of the treatment and recovery period.

·        There were no test item-related changes in hematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights, organ weights/ratios and sperm parameters.

·        There were no test item-related gross and microscopic changes among the doses tested.

Conclusion:

The repeated administration of test item Acetalization product between glucose and C16/18 (even numbered)-alcohol to Wistar rats through oral gavage for 90 consecutive days at the dose levels of 100, 300 and 1000 mg/kg Bwt/day did not cause any toxicological effects on general health, mortality, functional observational battery, growth and food consumption. No test item-related changes were observed in haematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights, organ weights/ratios and sperm parameters and gross and histopathological changes.The salivation (slight/moderate) which was observed in all rats at all the doseswas considered transient andtest item-related non-adverse effect. Hence, under the test conditions, the evaluated “No Observed Adverse Effect Level (NOAEL)” for test item Acetalization product between glucose and C16/18 (even numbered)-alcohol is 1000 mg/kg Body weight/day under the test conditions and doses employed.