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Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
28-Day Dietary
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Treatment Period: 28 days; Recovery Period: 14 days
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Adopted by the Council on October 03, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide
Cas Number:
2101947-22-0
Molecular formula:
C14H21NO2
IUPAC Name:
2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RDAC475-59
- Expiration date of the lot/batch: October 2019
- Purity test date: 96.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (room) temperature
- Stability under test conditions: 8 days
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar rat was selected as the test system because it is a readily available laboratory rodent species. It has been historically shown to be a suitable model for repeated dose toxicity studies. The OECD and other regulatory authorities also recommend it. The results of this study are believed to be of value in predicting the toxicity of the test item to human beings.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility (ABF), Jai Research Foundation, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 to 6 weeks old
- Weight at study initiation:
- Fasting period before study: Not applicable
- Housing: Housed in groups of 2 to 3 rats/cage/sex during the study period in sterilised solid floor polypropylene rat cages (size: 410 × 282 × 150 mm)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: Microbial and chemical contaminants are checked at every 6 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 64 to 66%
- Air changes (per hr): 20 to 21 per hour
- Photoperiod (hrs dark / hrs light): 12 h lighting and 12 h darkness; light hours were 06.00 - 18.00 h

IN-LIFE DATES: From: March 09, 2018 To: April 27, 2018

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
It was selected as it is a potential route of human exposure.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Teklad Certified Global 16% Protein Rodent Diet, Envigo, USA
- Storage temperature of food: Experimental room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability, active ingredient (a.i.) concentration and homogeneity of diet formulations were analysed at Department of Chemistry, using a validated analytical method (JRF study number 228-2-13-18800).

Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except control (only middle layer). The sampling was done before initiation of the treatment on day 1 and last week of treatment on day 22 for the diet formulation analyses. One set of samples was used for analyses and the other set of samples was stored under refrigerated conditions. The second set of samples will be disposed of during report finalisation. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analyses was ±20% from nominal value and % CV < 10.

Instrumental parameters for method of analyses:
Instrument : GC-Perkin Elmer, Clarus 500
Detector : Flame Ionization Detector (FID)
Column : DB-5 (30 m × 0.25 mm) 0.25 µm film thickness
Carrier Gas : N2
Injection Volume (µL) : 0.5
Injector Temperature : 250 °C
Detector Temperature : 250 °C
Flow Rate (mL/minute) : 3.0
Split Ratio : 20.0
Detector H2 Flow (mL/minute): 40.0
Detector Air Flow (mL/minute): 400.0
Oven Temperature : 150 °C (hold 3 minute) to 300 °C @ 45 °C/minute (hold 9 minute)
Duration of treatment / exposure:
The duration of exposure was 28 consecutive days. The first day of exposure was designated as day 1 for each rat. Rats allocated to recovery groups were kept untreated for a period of 14 days, to assess reversibility, persistence or progression of any toxicity after the treatment for 28 days.
Frequency of treatment:
Daily, up to 28 consecutive days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control group
Dose / conc.:
800 ppm
Remarks:
Low dose group of test item
Dose / conc.:
2 000 ppm
Remarks:
Mid dose group of test item
Dose / conc.:
5 000 ppm
Remarks:
High dose group of test item
Dose / conc.:
0 ppm
Remarks:
Recovery control group
Dose / conc.:
5 000 ppm
Remarks:
High dose recovery group of test item
No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Three dose levels (low dose - 800, mid dose - 2000 and high dose - 5000) in ppm in diet were selected based on the result of a 14-day dose range finding study (JRF study number 410-1-02-18798) and in consultation with the Sponsor.
Results of DRF study:
Statistically, significant decrease in terminal body weight (8 to 10% compared to control) was observed in the high dose group of both sexes which could be considered as a treatment related effect.
Statistically, significant increase in absolute and relative weights of liver were observed in mid dose and high dose groups of both sexes in a dose dependent manner which could be considered as a treatment related effect.
In females, a statistically significant decrease was observed in absolute weight of spleen (high dose group) and thymus (mid dose and high dose groups) when compared with that of the control group. Though the effect was not observed in males, it was considered as related to test item treatment, due to dose dependency.

- Rationale for animal assignment (if not random): Randomisation
- Rationale for selecting satellite groups: As per the Guidline to assess reversibility, persistence or progression of any toxicity after the treatment for 28 days.
- Post-exposure recovery period in satellite groups: 14-days
- Section schedule rationale (if not random): Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: At beginning of the treatment and at weekly intervals, thereafter and on the day of necropsy

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and pre-sacrifice
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At terminal and recovery sacrifices
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At terminal and recovery sacrifices
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At terminal and recovery sacrifices
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and at weekly intervals
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity / Foots play

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (table 4)

HISTOPATHOLOGY: Yes (table 4)
Statistics:
Data were recorded in standard formats and were summarised in tabular form. Data were processed to get group means and standard deviations with significance among the control and treatment groups using a validated statistical software. All parameters characterised by continuous data such as body weight, body weight change, food consumption, NBO parameters (urination, defecation and rear), FOB parameters (motor activity, grip strength and foot splay), organ weight, relative organ weight and clinical pathology (haematology, clinical chemistry and some of the urinalysis parameters) data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad, S.C. and Weil, C.S., 2007). When, data do not meet the homogeneity of variance, F-test was performed followed by t-tests to calculate significance. All analyses and comparisons were evaluated at the 5% (P≤0.05) and 1% (P≤0.01) levels.

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):
The statistically significant decrease in mean body weight change was observed at week 1, 2 and 4 in male rats of high dose group when compared with that of the control group. A similar effect was also observed at week 1 in male rats of high dose recovery group when compared with that of the control recovery group.

The decrease observed in body weight change of the high dose group could not be considered as treatment related effect due to the absence of other supporting finding (food consumption) and marginal changes (8 to 11%) from control.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In low dose group male rats, statistically, a significant decrease was observed in haemoglobin and haematocrit. These effects could not be considered as treatment related due to the lack of consistency between sexes and lack of dose dependency.
Statistically, a significant decrease was observed in haemoglobin and haematocrit in high dose recovery group of both sexes. Additionally, in high dose recovery female rats, statistically, a significant decrease was noted in RBC. As consistency between sexes was observed and some of the values of treated groups were below historical ranges, these effects could be consided as delayed onset, but could not be considered as adverse as % decrease compared to control was below 10% (Hall and Everds, 2013).
Statistically, a significant decrease was observed in MCHC and neutrophil in high dose recovery group male rats, these effects were not considered as effect of test item treatment due to lack of consistency between sexes and lack of similar effect at the end of treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In high dose female rats, statistically, a significant increase was observed in total protein and globulin and decrease was noted in albumin: globulin ratio. The effect could be related to test item treatment and considered as non-adverse as all individual values of total protein, globulin, and albumin: globulin ratios were within historical control ranges.
Statistically, a significant increase was noted in cholesterol in female rats of the high dose group. The effect could be related to test item treatment as values of 4/5 female rats were higher than historical range.
Statistically, a significance decrease was observed in ALP (low dose and high dose group male rats) and increase was noted in calcium (high dose group male rats). These alterations were not considered as an effect of test item treatment due to lack of consistency between sexes and/or dose dependency.
Effects observed at the end of treatment (increase in globulin, total protein and cholesterol, and decrease in albumin: globulin ratio in high dose female rats) were recovered after 14 days of recovery period.
In high dose recovery groups, statistically, a significant decrease was observed in bile acids (male rats) and inorganic phosphorus (female rats), these alterations were not considered as related to test item treatment due to lack of consistency between sexes and absence of similar effect at the end of treatment.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Rear counts in female rats of low dose, mid dose and high dose groups was increased statistically at week 3 when compared with that of the control recovery group which could not be considered as treatment related effect due to the absence of dose dependency and absence of similar effects in male rats and recovery group of female rats.
Statistically, a significant increase in ambulatory and total activity at interval 0-10 minutes and increase in fine and total activity at interval 21-30 minutes was observed in female rats of high dose recovery group when compared with that of the control recovery group which could not be considered as treatment related effect due to the absence of similar effects in male rats and main group of female rats.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically, a significant decrease was noted in terminal body weight in high dose group male rats.
Statistically, a significant increase was noted in absolute and relative liver weights in the high dose group of both sexes (relative weight 35 to 37% higher than control). Statistically, a significant increase in relative liver weight (6 to 19% higher than control) was also noted in the mid dose group (both sexes) and low dose group (female rats) with reduction in severity compared to the high dose group. These effects were supported by histopathological lesions observed in liver (hepatocellular hypertrophy).
An increase without statistical significance was observed in relative weight of thyroid with parathyroid in mid dose and high dose group male rats. The effect was well supported by histopathological lesions observed in the thyroid (follicular hypertrophy).
Though the effect of increase in relative liver weight was continued in both the sexes with statistical significance, after recovery period, marked reduction in severity was observed.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of the liver revealed hypertrophy of hepatocytes (periportal/diffuse) in all rats of the high dose group of both sexes. The lesion was also observed in the mid dose group (both sexes: male 3/5 and female 2/5) and low dose group (one female rat) with dose dependent reduction in incidence and severity. These lesions were considered as an effect of the test item and it was supported by an increase in relative and absolute weight of the liver.
The severity and incidence of hypertrophy of hepatocytes and increase in liver weight in mid dose and low dose groups was very low when compared with that of the high dose group. Hence, effects observed in the mid dose and low dose groups could be considered as adaptive and non-adverse.
Microscopic examination of the thyroid revealed follicular hypertrophy in male rats of high dose (5/5) and mid dose (2/5) groups with dose dependent reduction in incidence. It was supported by an increase in relative weight of thyroid with parathyroid. The effect observed in thyroid was considered as adaptive response to the enalarged liver, and prominency of the effect in male rats was also reported earlier (Zabka et al, 2011).
Other microscopic lesions observed in various organs were at lower rate of occurrence. Further, these lesions were mostly minimal to mild in nature, non specific, insignificant and were not dose related. Hence, these lesions were considered to be spontaneous or incidental in nature and not treatment related.
Follicular hypertrophy noted in male rats, was recovered in high dose recovery male rats. Similarly, hepatocellular hypertrophy was recovered in female rats. Though hepatocellular hypertrophy was continued after recovery period in treated male rats, marked reduction in incidence and severity was observed.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it is concluded that FRET 15-0735 produced hepatocyte hypertrophy (in all rats) and an increase in liver weight (35 to 37% higher than control) at a dose level of 5000 ppm in diet, and did not produce any severe toxicity or adverse effect up to the dose level of 2000 ppm in diet after the 28 day dietary administration in Wistar rats. The NOAEL (No Observed Adverse Effect Level) for FRET 15-0735 of both male and female rats was found to be 2000 ppm in diet (corresponding to 199.67 ± 35.87 mg/kg b. wt./day for male and corresponding to 208.33 ± 27.80 mg/kg b. wt./day for female) under the conditions and procedures followed in this study.
Executive summary:

STUDY TYPE  :  Repeated Dose Dietary Toxicity - Rat (Wistar); OECD 407

 

TEST ITEM      :  FRET 15-0735[96.4%]

CITATION       :  Kunjan N. Shah.Repeated Dose 28-Day Dietary Toxicity Study of FRET 15-0735 in Wistar Rats. Jai Research Foundation, India. Laboratory report number: 410-1-02-18799; MM/DD/YYYY.

 

SPONSOR        :  International Flavors & Fragrances Inc., USA.

 

EXECUTIVE SUMMARY:The objective of this study was to evaluate the potential toxicity of FRET 15-0735 in Wistar rats following dietary administration for a period of 28 consecutive days. The results of this study provide information on any systemic adverse effects, target organs and an estimation of the No-Observed -Adverse-Effect Level (NOAEL) and/or No-Observed-Effect-Level (NOEL).

Method:

Test System

Wistar (RccHan: WIST) Rat

Route of Admnistration

Oral through diet

Dose Levels

G1 (control) and G5 (control recovery): 0 ppm in diet,

G2 (low dose): 800 ppm in diet, G3 (mid dose): 2000 ppm in diet,

G4 (high dose) and G6 (high dose recovery): 5000 ppm in diet

Duration of Study

Treatment period: 28 days; Recovery period: 14 days

N° of Rats

5 rats/sex/group

Based on results of stability (JRF study number: 228-2-13-18800), the test item was found stable up to 8 days in formulated diet. Three samples (from upper, middle and lower levels) of formulated diet from each concentration and one from control were collected on days 1 and 22 of treatment for homogeneity and active ingredient (a.i.) concentration analysis. Each rat was observed twice daily for clinical signs, morbidity and mortality during study period. Body weight was recorded and body weight change compared to pre-treatment (PT) body weight was calculated at weekly intervals. Food consumption was also calculated at weekly intervals. Neurobehavioral observations (NBO) were performed prior to the initiation of treatment and weekly intervals, thereafter. Functional Observational Battery (FOB) was performed during 4thweek of treatment (G1 to G4) and 2ndweek of recovery (G5 and G6) periods. Ophthalmological examination was performed prior to the initiation of treatment and prior to terminal (G1 to G4) and recovery sacrifices (G5 and G6).


 

All rats were sacrificed by carbon dioxide asphyxiation and subjected to a gross pathological examination at the end of the treatment and recovery periods. Clinical pathology was performed at the end of the treatment and recovery periodson whole blood, plasma, serum and urine samples. Absolute organ weights were recorded and relative organ weights were calculated for the adrenals, testes, ovaries with oviduct, epididymides, uterus with cervix, kidneys, brain, heart, spleen, liver, thymus, pituitary, prostate + seminal vesicle with coagulation glands, and thyroid with parathyroid. Microscopic examination was performed for the preserved organs and tissues in control and high dose groups. In addition, liver from low, mid and recovery groups of both sexes and thyroid from low, mid and recovery groups of male rats were examined microscopically. The statistical analysis was carried out on all parametric data by using validated statistical software.

Results:

The a.i. analysis results of diet formulation samples collected on day 1 (low dose - 108.95%, mid dose – 100.64%, and high dose - 110.24%) and on day 22 (low dose - 86.94%, mid dose - 86.53%, and high dose - 88.59%) were within acceptable range of ±20% of nominal concentration.

No mortality or morbidity was observed in any group of male or female rats during the study period.

All rats from all groups were found normal during the study period.

Ophthalmological examination did not reveal any abnormality in any of the rats.

No treatment related changes were observedinneurobehavioral observationsor functional observational battery in rats from treatment groups.

No treatment related significant changes were observed in mean body weight, mean body weight change or mean food consumption in rats from treatment groups.

The actual test item intake (mean ± SD) corresponding to low, mid and high dose levels for male and female rats are summarized below:

Group

Dose (ppm in diet)

Test Item Intake (mg/kg b. wt./day)

Male

Female

G2

800

75.35 ± 12.41

84.13 ± 8.79

G3

2000

199.67 ± 35.87

208.33 ± 27.80

G4

5000

478.32 ± 100.26

499.27 ± 68.70

G6

5000

517.23 ± 86.68

479.79 ± 93.29

Test item treatment at high dose lead to delayed onset of decrease in red cell mass (RBC, haemoglobin and haematocrit). As the % decrease compared to control was below 10%, the effect was not considered as adverse. 

Statistically, significant increase was noted in cholesterol in female rats of the high dose group. The effect could be related to test item treatment as values of 4/5 female rats were higher than historical range.

 

External and internal examination of terminally and recovery sacrificed rats of either sex across various groups (G1 to G6) did not reveal any abnormality.

Statistically, significant increase was noted in absolute and relative liver weights in the high dose group of both sexes (relative weight 35 to 37% higher than control). Statistically, significant increase in relative liver weight (6 to 19% higher than control) was also noted in the mid dose group (both sexes) and low dose group (female rats) with reduction in severity compared to the high dose group.

Microscopic examination of liver revealed hypertrophy of hepatocytes (periportal/diffuse) in all rats of the high dose group of both sexes. The lesion was also observed in the mid dose group (both sexes: male 3/5 and female 2/5) and low dose group (one female rat) with dose dependent reduction in incidence and severity.Microscopic examination of thyroid revealed follicular hypertrophy in male rats of high dose (5/5) and mid dose (2/5) groups with dose dependent reduction in incidence. It was supported by increase in the relative weight of thyroid with parathyroid. The effect observed in thyroid was considered as adaptive response to the enalarged liver, and prominency of the effect in male rats was also reported earlier.

The severity and incidence of hypertrophy of hepatocytes and increase in liver weight in mid dose and low dose groups was very low when compared with that of the high dose group. Hence, effects observed in mid dose and low dose groups could be considered as adaptive and non-adverse.

Conclusion:

Based on the results of this study, it is concluded that FRET 15-0735 producedhepatocyte hypertrophy (in all rats) and anincrease in liver weight (35 to 37% higher than control)at a dose level of 5000 ppm in diet, and did not produce any severe toxicity or adverse effect up to the dose level of 2000 ppm in diet after the 28 day dietary administration in Wistar rats. The NOAEL (No Observed Adverse Effect Level) for FRET 15-0735 of both male and female rats was found to be 2000 ppm in diet (corresponding to 199.67 ± 35.87 mg/kg b. wt./day for male and corresponding to 208.33 ± 27.80 mg/kg b. wt./day for female) under the conditions and procedures followed in this study.

COMPLIANCE: Signed and dated GLP and Quality Assurance statements are provided.There was no deviation from regulatory requirements.