Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 February 2008 to 19 December 2008 (Revised Final Report 14 May 2009)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted in compliance with the United States Environmental Protection Agency (EPA) TSCA Good Laboratory Practice Standards (40 CFR Part 792), 18 September 1989; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice [C (97) 186/Final]. The protocol was designed to be in general accordance with the OECD Guidelines for Testing Chemicals, Health Effects Test Guidelines, Section 408, September 1998, and the European Chemicals Bureau test method B.26

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD Guidelines for Testing Chemicals, Health Effects Test Guidelines, Section 408, September 1998.
Deviations:
yes
Remarks:
Functional Observational Battery (FOB) was performed 2 days closer to termination than scheduled by protocol. This deviation did not negatively impact the quality or integrity of the data nor the outcome of the study.
Principles of method if other than guideline:
The protocol was designed to be in general accordance with the OECD Guidelines for Testing Chemicals, Health Effects Test Guidelines, Section 408, September 1998, and the European Chemicals Bureau test method B.26
GLP compliance:
yes
Remarks:
Conducted in compliance with the United States Environmental Protection Agency (EPA) TSCA Good Laboratory Practice Standards (40 CFR Part 792), 18 September 1989; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laborato
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Clear colorless liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Each animal was uniquely identified by a Monel® metal ear tag displaying the permanent identification number. Individual body weights and food consumption were recorded and detailed physical examinations were performed periodically during the pretest period. Ophthalmic examination data were also recorded for pretest animals prior to assignment of animals to treatment groups. The animal facilities were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
TEST ANIMALS
- Source: Forty-five male and 45 female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks old at the initiation of dose administration.
- Weight at study initiation: body weights ranged from 182 g to 236 g for males and from 143 g to 184 g for females at the time of randomization.
- Housing:housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
- Diet (e.g. ad libitum): ad libitum - PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer.
- Water (e.g. ad libitum):ad libitum - Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system
- Acclimation period:15 days
- Municipal water and feed were analyzed for contaminants and no contaminants were present at concentrations sufficient to interfere with the objectives of this study.
ENVIRONMENTAL CONDITIONS
Room temperature and humidity controls were set to maintain daily averages of 71 ± 5°F (22 ± 3°C) and 50 ± 20% relative humidity. They were controlled, monitored, and recorded approximately hourly using the Metasys® DDC Electronic Environmental control system. Actual mean daily temperature ranged from 69.4°F to 72.4°F (20.8°C to 22.4°C) and mean daily relative humidity ranged from 34.4% to 62.3% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

IN-LIFE DATES: Date(s) Event(s)
4 March 2008 Experimental starting date (animal receipt)
14 March 2008 Assignment to study groups
19 March 2008 Experimental start date (initiation of dose administration; study day 0)
18 and 19 June 2008 Scheduled necropsy (study week 13)
7 August 2008 Experimental termination (completion) date (last histopathological examination)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DIET:
The selected route of administration for this study was oral (diet) ingestion since this route is an acceptable and standard method of dosing animals in toxicity studies.
The appropriate amount of the test substance for each group was weighed into a tared weighing vessel. A premix of test substance in a sub-sample of the total diet was prepared using a Hobart mixer (5 kg capacity) and blended for 3 to 5 minutes. The premix was then added to the remaining diet in a Hobart mixer (25 kg capacity) and mixed for an additional 10 minutes to obtain the appropriate dietary concentration. Concentrations were prepared from lowest to highest. The test substance diet formulations were prepared approximately weekly as single formulations for each dosage level and stored at room temperature. Prior to the initiation of dose administration, duplicate samples (approximately 100 g each) for homogeneity and stability determinations were collected on 3 March 2008 from the top, middle and bottom strata of the 0.1% and 1.0 % w/w test diet formulations and stored at room temperature for 10 or 15 days. Samples (approximately 100 g each) for concentration analyses were collected weekly for the first 4 weeks and monthly thereafter from each test diet formulation (including the basal diet administered to the control group).



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet concentrations were analyses by chromatographic test substance peak detection.
Duration of treatment / exposure:
91-92 days
Frequency of treatment:
Daily by dosed feed
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 3000, and 10,000 ppm
Basis:
other: 0, 0.1, 0.3 and 1.0 (w/w) in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dosage levels were selected based on results from previous studies with a similar test substance including an oral gavage developmental toxicity study which considered the NOAEL as 300 mg/kg/day for both maternal and developmental toxicity, a 28-day oral gavage toxicity study which showed minimal toxicity at dosage levels of 15, 150 and 1000 mg/kg/day, and two 13-week dietary studies which demonstrated marked intolerance at 0.3 - 1.0% w/w test substance in the diet.
- Rationale for animal assignment (if not random): random - Computerized randomization printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification in a block design. The animals were then arranged into groups according to the printout. Individual body weights at randomization were within ± 20% of the mean for each sex. Each group (Groups 1-4) consisted of 10 males and 10 females. These animals were then randomized into 3 study replicates to allow for the reasonable conduct of the functional observational battery and locomotor activity assessments. Each dose group and sex was approximately equally represented within each study replicate.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for
mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, except on days when detailed physical examinations were performed. The absence or presence of findings were recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals weekly and reported beginning approximately 1 week prior to test substance administration and on the day of scheduled necropsies. A separate computer protocol was used to record any observations noted outside of the above-specified intervals.

BODY WEIGHT: Yes
- Time schedule for examinations:Individual body weights were recorded at least weekly during pretest and throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:2 wks pretest and wk 12
- Dose groups that were examined:all

HAEMATOLOGY: Yes
- Time schedule for collection of blood:day of necropsy week 13
- Anaesthetic used for blood collection: Yes - anesthetized by inhalation of isoflurane.
- Animals fasted: Yes
- How many animals: 10/sex/dose level
- Parameters checked in Report Summary Data Table 16 were examined.
HEMATOLOGY AND COAGULATION
Total leukocyte count (White Cells)
Erythrocyte count (Red Cells)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin
(MCH)
Mean corpuscular hemoglobin
concentration (MCHC)
Platelet count (Platelet)
Prothrombin time (ProTime)
Activated partial thromboplastin time
(APTT)
Reticulocyte count
Percent (Reticulocyte)
Absolute (Retic Absolute)
Differential leukocyte count
Percent and absolute
-Neutrophil
-Lymphocyte
-Monocyte
-Eosinophil
-Basophil
-Large unstained cell
Platelet estimatea
Red cell morphology
(RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:day of necropsy week 13
- Animals fasted: Yes
- How many animals:10/sex/dose/level
- Parameters checked in Report Summary Data Table 17 were examined.
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio)
[by calculation]
Total bilirubin (Total Bili)
Urea nitrogen
Creatinine
Alkaline phosphatase
(AlkalinePhos ‘tse)
Alanine aminotransferase
(Alanine Transfer)
Aspartate aminotransferase
(AspartatTransfer)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Triglycerides (Triglyceride)

URINALYSIS: Yes
- Time schedule for collection of urine:day of necropsy week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked inReport Summary Data Table 18 were examined.
Specific gravity (SG)
pH
Urobilinogen (URO)
Total volume (TVOL)
Color (COL)
Clarity (CLA)
Protein (PRO)
Glucose (GLU)
Ketones (KET)
Bilirubin (BIL)
Occult blood (BLD)
Leukocytes (LEU)
Nitrites (NIT)
Microscopy of sediment
[Tabular abbreviations appear
on individual tables]

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:study week 12
- Dose groups that were examined:all
- Battery of functions tested: sensory activity / grip strength / motor activity / home cage observations / open field observations (evaluated over a 2-minute observation period) / physiological observations.


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - A complete necropsy was conducted for all animals. Animals were euthanized by carbon dioxide inhalation and exsanguinated. The necropsies included an examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. Clinical findings that were confirmed macroscopically were designated CEO (correlates with externally observed). The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except asnoted).
HISTOPATHOLOGY: Yes - Adrenals (2), Aorta, Bone with marrow (Femur and Sternum), Bone marrow smear (from femur)a, Brain (Cerebrum Level 1, Cerebrum Level 2, Cerebellum with medulla/pons), Cervix, Epididymides (2)b, Eyes with optic nerve (2)c, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Mandibular and Mesenteric), Nasal cavityd, Ovaries (2) with oviducts, Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pituitary, Prostate, Salivary glands (mandibular (2)), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary glandf, Spinal cord (cervical, thoracic,
lumbar), Spleen, Testes (2)b, Thymus, Thyroid (with parathyroids), Trachea, Urinary bladder, Uterus, Vagina and All Gross lesions and masses (when possible).
a - Bone marrow smears were obtained at scheduled necropsy, but not placed in formalin; slides were not examined
b - Fixed in Bouin’s solution
c - Fixed in Davidson’s solution
d - Levels I and III according to the method of Young (Young, 1981) were examined microscopically
e - Examined microscopically if in the plane of section and in all cases when a gross lesion of the organ is present
f - For females; a corresponding section of skin was taken from the same anatomical area for males
Microscopic examination was performed on all tissues listed from all animals in the control (basal diet) and 1% w/w Isodecyl benzoate-treated groups, and gross lesions from all animals were examined. The kidneys from all males in the 0.1% and 0.3% w/w Isodecyl benzoate-treated groups and the liver from males in the 0.3% w/w and females in the 0.1% and 0.3% w/w Isodecyl benzoate-treated groups were processed to slides but were not examined by the pathologist based on the findings from the control (basal diet) and 1% w/w Isodecyl benzoate -treated groups.
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.) and the number of animals (N) used to calculate the mean.
Body weight, body weight change, food consumption, continuous functional observational battery (FOB), clinical pathology, and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (pAll RANOVA statistical analyses for total and ambulatory locomotor activity counts were conducted by BioSTAT Consultants, Inc. using SAS version 9.1 (SAS Institute, Inc. 2002-2003). Each analysis endpoint was analyzed, by session, with a repeated measure analysis of variance (RANOVA). Factors in the model included treatment group (TRT), time interval (TIME), and the interaction of time interval and treatment group (TRT*TIME). The SAS® procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike’s Information Criterion (AIC). The interaction terms TRT*SEX and TRT*TIME*SEX were evaluated at the 0.5 significance level. If either interaction term was significant the analyses described below were conducted separately for each sex.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
>= 619 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
>= 736 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no-observed-effect level (NOEL) for oral (diet) administration of Isodecyl benzoate to Crl:CD(SD) rats for 91 or 92 consecutive days was 1.0% w/w which was approximately equivalent to 619 mg/kg/day for males and 736 mg/kg/day for females.
Executive summary:

SUMMARY

OBJECTIVE

The objective of the study was to evaluate the possible toxic effects of ISODECYL BENZOATE (JAYFLEX®MB 10) when administered as dietary admixes to rats for 90 days. This included evaluation of potential neurotoxicity by Functional Observation Battery and Motor Activity assessment.

 

STUDY DESIGN

ISODECYL BENZOATE was administered ad libitum via the basal diet for a minimum of 91 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 1000, 3000 and 10,000 ppm administered as 0.1%, 0.3% and 1.0% w/w of ISODECYL BENZOATE in the basal diet, respectively. A concurrent control group (Group 1) received the basal diet on a comparable regimen. Each group consisted of10 animals/sex. Following 91 or 92 days of test diet administration, all rats were euthanized.

All animals were observed twice daily for mortality and moribundity. Clinical observations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity data were recorded for all animals near the end of the test diet administration period. Ophthalmic examinations were performed during study weeks -2 and 12. Clinical pathology evaluations (hematology, coagulation, serum chemistry and urinalysis) were performed on all animals on the day of necropsy (study week 13). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy (study week 13). Selected tissues were examined microscopically from all animals in Groups 1 and 4 and all gross lesions were examined microscopically from all animals.

 

RESULTS

There were no test substance-related effects on clinical observations, body weights, food consumption, functional observational battery parameters, locomotor activity, clinical pathology parameters (hematology, coagulation, serum chemistry or urinalysis), ophthalmic examinations, organ weights, macroscopic or microscopic examinations.

 

CONCLUSIONS

Based on the results of this study, orally (diet) administered ISODECYL BENZOATE for 91 or 92 days was well tolerated in Crl:CD(SD) rats at dosage levels of 0.1%, 0.3% and 1.0% w/w ISODECYL BENZOATE (1000, 3000 and 10,000 ppm, respectively). There were no test substance-related effects on clinical observations, body weights, food consumption, functional observational battery parameters, locomotor activity, clinical pathology parameters (hematology, coagulation, serum chemistry or urinalysis), ophthalmic examinations, organ weights, and macroscopic or microscopic examinations. Therefore, the no-observed-effect level (NOEL) for oral (diet) administration of ISODECYL BENZOATE to Crl:CD(SD) rats for 91 or 92 consecutive days was 1.0% w/w which was approximately equivalent to 619 mg/kg/day for males and 736 mg/kg/day for females.