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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-24 to 2013-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(22 March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 Guideline from the Office of Prevention, Pesticides and Toxic Substances, July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Benzylamine

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., NM Horst / The Netherlands
- Age at study initiation: 11 weeks old
- Weight at study initiation: Males: 288 to 334 g; Females: 183 to 219 g
- Fasting period before study: None
- Housing: In groups of three to four animals
- Diet: Pelleted standard Harlan Teklad 2018C (batch nos. 80/11 and 43/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70%
- Air changes: Air-conditioned with 10 - 15 air changes per hour
- Photoperiod: 12-hour fluorescent light / 12-hour dark cycle

IN-LIFE DATES: From: 2012-07-26 To: Males: 2012-08-30 Females: 2012-09-11

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Common non hazard vehicle
- Lot/batch no.: Batch 1: 499150899, Batch 2: 292189296
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle to confirm the stability for 4 hours. Towards the end of the study, samples were taken from the middle to confirm concentration.The aliquots for analysis of dose formulations were
frozen (-20 ± 5 °C) and delivered on dry ice to Harlan Laboratories Ltd. (Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.The samples were analyzed by GC following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard.
Duration of treatment / exposure:
Males: 28 days
Females: Approximately 6 weeks
Frequency of treatment:
Once daily (within four hours)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 75, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study (report number D55063) the dose levels of 30, 75, and 150 mg/kg bw/day were selected for the present study.
- Rationale for animal assignment: The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Once daily, during acclimatization and up to the day of necropsy. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter, detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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
Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 – 14, after pairing period days 1 - 2
Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14

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 data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy from 5 males randomly selected from each group and 5 lactating females from each group (day 5 post partum)
- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 rats per dose and sex
- Parameters checked:
Erythrocyte count, Hemoglobin, Hemoglobin concentration distribution width, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Leukocyte count total, Differential leukocyte count, Platelet count,
Reticulocyte count

Coagulation
Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy from 5 males randomly selected from each group and 5 lactating females from each group (day 5 post partum)
- Animals fasted: Yes, for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 rats per dose and sex
- Parameters checked: Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study
- Dose groups that were examined: Males shortly before the scheduled sacrifice and females on day 3 post partum relevant parameters were performed with 5 P generation males and 5 P generation females randomly selected from each group.
- Battery of functions tested: Sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All parent generation animals were exsanguinated. All parent animals was examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system.

HISTOPATHOLOGY: Yes

Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined microscopically. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically.
In addition, because of possible test item related findings that were noted during the initial histopathologic examination in animals of the high dose group, the stomach including forestomach and glandular stomach from 5 males and 5 females in each of the low and intermediate groups (groups 2 and 3, respectively) were additionally examined to establish a no-effect level.

ORGAN WEIGHT:
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.

Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Uterus (including cervix), Prostate, Liver, Thymus, Spleen, Thyroid (after fixation), Ovaries (weighed as pairs), Seminal vesicles (inclusive coagulating gland)

Statistics:
The following statistical methods were used to analyze food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Details on results:
Parent Animals

CLINICAL SIGNS AND MORTALITY

One female treated at 150 mg/kg bw/day was killed in extremis on day 21 of its gestation period. Upon microscopic examination, the irritant property of the test item was considered to be the cause of animal’s morbidity. There were no further unplanned deaths during the course of this study.

In females treated at 150 mg/kg bw/day, effects on breathing were noted. Slight breathing noises were noted during the last 1 or 2 days of the lactation period in two females. Also, slight breathing noises accompanied by gasping were noted in the female that was killed in extremis, on the day of sacrifice. These findings were considered to be test item-related. Furthermore, in one female treated at 150 mg/kg bw/day, slightly ruffled fur was noted on days 11 and 12 of the prepairing period. Thereafter, this animal was free from clinical signs until the end of the study.
Incidentally, in one female treated at 75 mg/kg bw/day, slight hair loss was noted towards the end of the prepairing period and the two first days of pairing. From day 3 of pairing onwards, this finding was no longer present. The remainder of females from this group was free from clinical signs.
No clinical signs were noted in males of all dose levels during the whole course of the study and in females up to the dose of 30 mg/kg bw/day.

No findings at detailed weekly clinical observation were noted in males and females at any dose level.


BODY WEIGHT AND WEIGHT GAIN

Body Weights of Males (Pre-pairing, Pairing and After Pairing Periods)
During the prepairing period, there was a slight, but statistically significant decrease in body weight gain in males treated at 150 mg/kg bw/day. No further statistically significant differences were noted thereafter and no effect on mean body weights was observed, therefore this effect at the beginning of treatment was considered not to be adverse. In males treated at 30 and 75 mg/kg bw/day, no effects of the test item were noted. The overall differences in mean body weight gain at the dose levels of 0, 30, 75 and 150 mg/kg bw/day were: +15%, +15%, +14% and +13% during the pre-pairing period and +6%, +7%, +6% and +7% during the pairing period and +1%, +1%, +1% and ±0% during the after pairing period, respectively (percentages refer to the body weight gain within the period).

Body Weights of Females (Pre-pairing, Pairing, Gestation and Lactation Periods)
No test item-related effects on mean body weight and mean body weight gain were noted. The overall differences in mean body weight gain at the dose levels of 0, 30, 75 and 150 mg/kg bw/day were: +8%, +8%, +10% and +8% during the pre-pairing period, +52%, +51%, +58% and +50% during the gestation period and +8%, +4%, +6% and +5% during the lactation period, respectively (percentages refer to the body weight gain within the period).


FOOD CONSUMPTION

Food Consumption of Males (Pre-Pairing and After-Pairing Period)
There were no statistically significant effects on mean food consumption at any dose level and in any study phase. However, in males treated at 150 mg/kg bw/day, there was a slight reduction in mean food consumption when compared with controls (-4.2% during prepairing and -3.4% during after pairing). Since mean body weight gain was statistically significantly decreased during the prepairing period, the slight reduction in mean food consumption was considered to be test item-related but not adverse.

Food Consumption of Females (Pre-pairing, Gestation and Lactation Periods)
There were no effects on mean food consumption at any dose level.



HAEMATOLOGY

The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.
In females at 30 and 75 mg/kg bw/day, reticulocyte counts were slightly, but statistically significantly decreased. However, in the absence of a dose relationship and since the values were well within the range of historical control data, this finding was considered to be incidental.


CLINICAL CHEMISTRY

In females at 150 mg/kg bw/day, triglyceride levels were statistically significantly increased (approximately 1.7-fold). Triglyceride values were also higher in males at 150 mg/kg bw/day (approximately 1.7-fold), but not to a statistically significant degree. Although a compoundrelated effect cannot be excluded, this finding was not considered to be adverse since all other parameters remained normal. At 75 mg/kg bw/day in males, statistically significantly increased glucose levels were noted. However, in the absence of a dose relationship, this was considered to be incidental.



NEUROBEHAVIOUR

Functional Observational Battery
No relevant findings were noted during the functional observational battery in males or females at any dose level.

One male treated at 75 mg/kg bw/day showed an increased number of rearings. Due to the isolated occurrence of this finding and its absence in female animals or in the high dose group males, this finding was considered to be incidental. No further observations were made in males.

Findings in females consisted of vocalization in two control animals and in four animals treated at 75 mg/kg bw/day. Furthermore, one control female and two females treated at 150 mg/kg bw/day showed slightly ruffled fur. One female treated at 30 mg/kg bw/day was slightly defensive/fearful during lifting. Due to the absence of dose relationship or presence of similar findings in controls, the findings were considered not to be test item-related.

Locomotor Activity
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor.
Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.


ORGAN WEIGHTS

In males and females no test item-related effects on organ weights were noted.
Absolute testis weights in males at 75 and 150 mg/kg bw/day were statistically significantly higher, confirmed by the values relative to body and brain weight. However, all absolute and relative testes weights were in the range of the historical control data (3.39 g – 3.92 g absolute testes weights, 0.92 % – 1.12 % relative to the body weight, 155.90 % – 193.13 % relative to brain weights). Testes weights from control animals were at the lower end of the historical control data range and the values of the dose group at the upper end. Due to these facts, and since no histopathological correlation was noted, the higher testes weights were considered to be a result of biological variability and not a test item-related effect.
Absolute thyroid weights and thyroid to brain weight ratios were statistically significantly lower in males at 150 mg/kg bw/day. Since this difference occurred in one sex only, and in the absence of histopathological changes, this was considered to be incidental. The same applies to the organ weights showing statistical significant changes only based on relative, but not on absolute weights, and in which no histopathological alterations were noted (brain, liver, epididymis).
No relevant effects on organ weights in females were noted at any dose level. The slightly increased values of relative (to body weight) adrenal weights were well in the range of the historical control data (0.031 g - 0.041 g). The lower relative (to brain) spleen weights noted at 150 mg/kg bw/day were borderline to the historical control data (32.25 % – 40.21 %). In the absence of histopathological findings and since no effects occurred in males, this finding was also considered to be incidental.


GROSS PATHOLOGY

In female no. 83 of the high dose group (150 mg/kg bw/day) that was killed in extremis during the treatment period, gray white and foamy abnormal contents in the stomach, bilateral pale discoloration of the kidney, reddish and pale discoloration of the liver surface, and fetus contents in both uterine horns were recorded. Gastric abnormal contents were considered to represent the stagnation of the dosing solution that arose from animal’s morbidity.
In the survivors, gross lesions attributable to treatment with the test item were recorded in the stomach (nodules, foci, crateriform retractions, discolorations) of three males and three females treated at 150 mg/kg bw/day.
All other gross lesions recorded in the survivors were within the range of normal background alterations which may be recorded in animals of this strain and age.


HISTOPATHOLOGY

Degenerative, necrotic, inflammatory and/or reactive changes were observed in the forestomach and/or glandular stomach of both sexes in all test item treatment groups (i.e. at 30, 75 and 150 mg/kg bw/day). They consisted of mucosal degeneration with or without pustules, ulcer, inflammatory cell infiltrate with or without edema, reactive squamous hyperplasia, and hyperkeratosis and/or parakeratosis with occasional dyskeratosis in the forestomach, and erosion with or without regenerated mucosa and/or increased inflammatory cell infiltrate in the glandular stomach. These histologic changes were considered to be treatment-related adverse effects which were attributable to the irritant property of the test item.
In the high dose female that was killed in extremis, mucosal degeneration as well as inflammatory cell infiltrate with edema, hyperkeratosis and reactive squamous hyperplasia were observed in the forestomach. These lesions that were attributable to the irritant property of the test item were considered to be the cause of animal’s morbidity. Tracheal mucosal abrasion was also recorded. However, this lesion was considered to be incidental, caused by the regurgitation of gastric contents, which occasionally happen in morbid animals. Furthermore tubular dilatation in the kidney and congestion of the liver were recorded in this animal, but these were also considered to be non-specific alterations which are often observed in morbid animals.
No microscopic alteration that could be attributed to treatment with the test item was observed by detailed histopathologic evaluation of the testes.


OTHER FINDINGS
Analysis of Dose Formulations
The application formulations investigated during the study were found to comprise Benzylamine in the range of 80.2% to 92.4% of the nominal concentration and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Benzylamine in the preparations was approved because single results found did not deviate more than 2.9% (acceptance criterion: <15%) from the corresponding mean.
In addition, the test item was found to be stable in application formulations when kept 4 hours at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
In conclusion, the results indicate the accurate use of the test item Benzylamine and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance – related adverse effects were observed.
Dose descriptor:
NOAEL
Remarks:
local effets
Effect level:
< 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the histopathological findings at the stomach, a no-observed-adverse effect level (NOAEL) was not obtained under the condition of this study. The gastric lesions were attributed to the irritant properties of the test item.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Under the conditions of this study, no test substance - related adverse effects on reproduction (fertility and/ or development) were noted.

Applicant's summary and conclusion