Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
On Day 20, the examination for general clinical signs was done prior to the test item administra tion for all rats and post dose was carried out 5 to 6 hours after the dose administration instead of 0.5 to 3 hours after dose administartion.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Phosphoric acid, dodecyl ester, potassium salt
EC Number:
254-414-3
EC Name:
Phosphoric acid, dodecyl ester, potassium salt
Cas Number:
39322-78-6
Molecular formula:
not applicable
IUPAC Name:
Reaction mass of potassium didodecylphosphate and dipotassium dodecylphosphate and water
Details on test material:
Chemical name (IUPAC): Reaction mass of potassium didodecylphosphate and dipotassium do decylphosphate and water
CAS No.: 39322-78-6
Manufactured date: 23.10.2015
Expiry date: 22.10.2017

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is one of the standard test systems used to assess the toxicity and also acceptable to the regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 210.29 g to 235.98 g; Females: 141.66 g to 175.47 g
- Fasting period before study: Not applicable
- Housing: Housed two rats/sex/cage except for the last cage of recovery groups wherein one rat/cage
was housed
- Diet (e.g. ad libitum): Teklad certified (2014C) global 14% protein rodent maintenance diet - pellet (c
ertified) manufactured by Envigo,
PO Box 44220, Madison, WI 53744-4220, were provided ad libitum to rats.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed
to UV rays in Aquaguard on-line
water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India was provided.
- Acclimation period: 5 days
DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants in the food or w
ater that were expected to interfere with the
results of this study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 48 to 67%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 01 September 2016 To: 02 January 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of test substance administration was through oral gavage. This route has been chosen because it is the potential route of human exposure.
Vehicle:
other: Milli-Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared at intervals of 3 to 4 days and used within the established
stability period of 4 days.
Required quantities of the test item was weighed in a beaker (previously pre-calibrated to desired volume) for each dose levels separately and small volume of vehicle - Milli-Q water (hot Milli-Q water maintained at approximately 80 - 86.7 °C) was added and mixed by using glass rod till a uniform formulation was obtained. The resulting pre-mix was made up to the pre-calibrated mark using the vehicle to get the final desired concentration and mixed well.
The homogeneity of the dosing formulations during administration/sampling was maintained by constant stirring using magnetic stirrer.

DIET:
Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet – pellet (Certified) manufactured by Envigo, PO Box 44220, Madison, WI 53744-4220, were provided ad libitum to rats.

VEHICLE: Milli-Q water
- Justification for use and choice of vehicle (if other than water):
As per the information provided by the sponsor, water was used as a vehicle in the dose range finding study, hence Milli-Q water was used as vehicle for
dose formulation preparation.
- Concentration in vehicle: Not applicable
- Amount of vehicle (if gavage): 10 mL/kg Body weight per day
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Leomin PN pa in the dose formulations was determined using Liquid Chromatograph with Mass Spectrometer (LC-MS/MS)
Duration of treatment / exposure:
The dose formulations were administered by oral gavage to rats of the respective groups once daily at approximately the same time each day (varied by ± 3 hours) for 90 consecutive days. Similarly, vehicle was administered by oral gavage to rats in vehicle control 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 90-day treatment period.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was calculated for individual animal on the first day of the treatment and was adjusted according to the most recent body weights recorded at different intervals of the study.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females for main groups
5 males and 5 females for recovery groups (control recovery and high dose recovery)
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels of 50, 250 and 1000 mg/kg bwt/day have been selected in consultation with the sponsor based on a previously performed dose range finding study along with a concurrent control group.
- Rationale for animal assignment (if not random): Selected randomly as per body weight stratification
- Rationale for selecting recovery groups: control and high dose recovery
- Post-exposure recovery period in recovery groups: 28 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily twice (pre and post dose)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
FOOD CONSUMPTION: Yes
- Time schedule: Weekly
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At termination
- Dose groups that were examined: Control and High dose groups
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 100
- Parameters checked in table [No.1, 2, 3, 4 (Pathology report)] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At terminatin
- Animals fasted: Yes
- How many animals: 100
- Parameters checked in table [No.5, 6, 7, 8 (Pathology report)] were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: At termination
- Animals fasted: Yes
- Parameters checked in table [No. 9, 10, 11, 12 (Pathology report)] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Dose groups that were examined: At prior to terminatin of main and recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity / :
IMMUNOLOGY: No
Sacrifice and pathology:
Gross pathology:
At the end of the treatment (main groups) and recovery period (recovery groups), all rats in the study were subjected to necropsy. All the rats were fasted overnight (water allowed) prior to necropsy, weighed, anaesthetized with isoflurane and exsanguinated. All the rats were subjected to detailed necropsy by a veterinary pathologist and findings were recorded.

Statistics:
Data captured using ProvantisTM: Parameters such as body weight, body weight gains (derived data), food consumption (derived data), terminal fasting bodyweight, organ weights and their ratios data (derived data), laboratory investigations - Haematology (except coagulation parameters PT and APTT which was entered retrospectively in ProvantisTM and analysed) and Clinical Chemistry was a nalysed using ProvantisTM built-in statistical tests.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using the licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables like neurological examinations (neuromuscular parameters, body weight and body temperature) 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 was found to be significant. The data from the vehicle control group (G1) was compared with the data from the treatment groups (G2, G3 & G4).
In the case of recovery groups, data was analysed using the methods stated above. Comparison of mean between treatment recovery group(s) and control recovery group was performed. When analysis was done outside provantis the comparison of means between the control recovery and high dose recovery group was done using two sampled ‘t’ test. All analyses and comparisons were evaluated at the 5% (P < 0.05) level. Statistically significant differences (P < 0.05), indicated by the aforementioned tests were designated by the following symbols 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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Few statistical significant changes in body weights and body weight gains observed are provided below:
- Decrease in the mean body weights in female rats at 1000 mg/kg/day (Days 36, 43 and 50) which were less than 10%.
- Decrease in the body weight gains in male rats (Days 15-22, 36-43) and female rats (Days 1-8, 22-29) at 1000 mg/kg/day.
- Increase in the body weight gains in male rats at 50 and 250 mg/kg/day (Days 64-71)
The above few sporadic changes in mean body weights and gains which were randomly observed during the treatment period were considered incidental and not related to test item. Hence, the body weights and body weight gains were unaffected at all the doses tested.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
• Decreased absolute and relative reticulocyte counts at 1000 mg/kg/day dose in both sexes
• Increased total leucocyte count, neutrophils, lymphocytes and monocytes at 250 mg/kg/day dose group females and at 1000 mg/kg/day dose in both sexes.
• All the above test item-related changes were reversible after 28 days recovery period.

There were no test substance-related changes in coagulation parameters at all the doses tested.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
• Increased ALP and AST activity at1000 mg/kg/day dose in both sexes and increased ALP activity at 250 mg/kg/day dose females.
• Increased triglyceride concentration at 1000 mg/kg/day dose in both sexes.
• At 1000 mg/kg/day dose, decreased total protein and globulin in both sexes, decreased albumin concentration in females and increased albumin globulin ratio in males. These changes were associated with decreased food intake.
• All the above test item-related changes were reversible after 28 days recovery period.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
• Decreased terminal fasting body weight at 1000 mg/kg/day dose females.
• Increased relative (body weight ratios) weight of liver at 1000 mg/kg/day dose in both sexes. This change was microscopically associated with hepatocellular hypertrophy.
• Decreased absolute weight of epididymides at 1000 mg/kg/day dose in males.
• All the above test item-related changes were reversible after 28 days recovery period.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
• Hepatocellular hypertrophy of liver observed at 1000 mg/kg/day dose in both sexes was mainly seen in centrilobular area and was considered as test substance related adaptive change. Hepatocellular hypertrophy correlated with increase in relative weight of liver. This change was reversible after 28 days recovery period.
• Hypertrophy of the zona glomerulosa layer of adrenals was observed at 1000 mg/kg/day dose groups and was considered as test substance related reversible change
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
In view of the results observed, the evaluated “No Observed Adverse Effect Level (NOAEL)” for the test substance Leomin PN pa when administered orally for 90 consecutive days to Wistar rats is 1000 mg/kg body weight under the test conditions and doses employed.
Details on results:
In view of the results observed, the evaluated “No Observed Adverse Effect Level (NOAEL)” for the test substance Leomin PN pa when administered orally for 90 consecutive days to Wistar rats is 1000 mg/kg body weight under the test conditions and doses employed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Complete reversal of observed changes post recovery of 28 days

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL was established at 1000 mg/kg bw/day.
Executive summary:

A 90-day study was conducted to determine the oral repeated dose toxicity of the test substance, mono- and di- C12 PSE, K+, according to OECD Guideline 408, in compliance with GLP. Wistar rats were administered the test substance at doses of 50, 250, or 1000 mg/kg/day at an equivolume of 10 mL/kg/day via gavage for 90 d. Milli-Q water was used as a vehicle. The vehicle or dose formulations were not administered to the recovery groups for 28 days following the 90-day dosing period. The Leomin PN pa formulations at 2 and 125 mg/mL concentration levels in the vehicle were found to be stable for 4 days at room temperature. In this study, the dose formulations were analysed three times. Clinical signs, body weights, food consumption, ophthalmological examinations, neurological examinations, clinical laboratory investigations of blood and urine, gross pathology, organ weights, and histopathological evaluation were performed. The liver and adrenals were considered as target organs and were examined in the lower dose groups and recovery groups.No mortalities, clinical signs were observed. No effect on ophthalmological examination was observed. No test substance-related neurological abnormalities were observed at all the dose levels in both the sexes. No test substance-related variations on the body weights were observed, while food consumption was significantly lower and is considered treatment-related in both the sexes. Treatment-related changes were observed in the haematology parameters which includes, decreased absolute and relative reticulocyte counts at 1000 mg/kg/day dose in both sexes, increased total leucocyte count, neutrophils, lymphocytes, and monocytes at 250 mg/kg/day dose group females and 1000 mg/kg/day dose in both sexes. Changes in clinical chemistry parameters were observed, which include, increased ALP and AST activity at 1000 mg/kg/day dose in both sexes and increased ALP activity at 250 mg/kg/day dose females. Increased triglyceride concentration at 1000 mg/kg/day dose in both sexes. At 1000 mg/kg/day dose, decreased total protein and globulin in both sexes, decreased albumin concentration in females, and increased albumin globulin ratio in males. These changes were associated with decreased food intake. All the haematological and clinical chemistry-related changes were reversible after 28 days recovery period. There were no test substance-related changes in urinalysis parameters at all the doses tested. Decreased terminal fasting body weight at 1000 mg/kg/day dose females. Increased relative (bodyweight ratios) weight of liver at 1000 mg/kg/day dose in both sexes. This change was microscopically associated with hepatocellular hypertrophy. The decreased absolute weight of epididymides at 1000 mg/kg/day dose in males. All these changes were reversible after 28 days recovery period. There were no test substance-related gross pathological changes were observed at all the doses tested. In histopathological evaluation, hepatocellular hypertrophy of liver observed at 1000 mg/kg/day dose in both sexes was mainly seen in centrilobular area and was considered as test substance-related adaptive change. Hepatocellular hypertrophy correlated with an increase in the relative weight of the liver. This change was reversible after 28 days recovery period. Hypertrophy of the zona glomerulosa layer of adrenals was observed at 1000 mg/kg/day dose groups and was considered as test substance-related reversible changes. Under the study conditions, the NOAEL was established at 1000 mg/kg bw/day (Ramesh, 2017).