[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days with two weeks recovery period was found to be 1000 mg/kg bw/day.

Repeated inhalation study:

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7) which is reported as 0.02302689mmHg at 25 C. Thus, exposure to inhalable dust, mist and vapour of the [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile . Therefore this study is considered for waiver.

Repeated dermal study;

The acute toxicity value for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Repeated dose 28-day oral toxicity study with test chemical by daily gavage in the rat followed by a 2 week recovery period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

1)In order to meet the regulatory requirement for testing in a rodent species;
2)Widely used in as a species of choice for pre-clinical toxicological studies.
3)This strain is widely used throughout the industry in the non-clinical laboratory studies.
4)This study is intended to provide information on the health hazards likely to arise from exposure to the test item.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Inbred
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation:
Male Mean: 159.40 g (= 100 %)
Minimum : 149.3 g (- 6.33 %)
Maximum : 178.1 g (+ 11.73 %)
Total No. of animals : 36
Female Mean : 141.35 g (= 100 %)
Minimum : 132.2 g (- 6.48 %)
Maximum : 159.7 g (+ 12.98 %)
Total No. of animals : 36

- Fasting period before study:
- Housing: The rats were housed in polycarbonate cages with paddy as bedding.
After allocation to respective dose groups rats were housed 2/sex/cage
- Diet (e.g. ad libitum): Rodent feed supplied ad libitum from individual feeders on cage top.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages.
- Acclimation period: 5 days prior to dosing

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants, which were reasonably expected to be in the dietary materials capable of interfering with the conduct of this study.Water was from a local source and passed through the reverse osmosis membrane before use. There were no known contaminants, which were reasonably expected to be in the water capable of interfering with the conduct of this study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range, 20.6 °C to 22.6 °C)
- Humidity (%): 30% to 70% (actual range, 44.6% to 58.4%).
- Air changes (per hr): ten air changes per hour of 100% fresh air that has been passed through the HEPA filters
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room

IN-LIFE DATES: From: To:30-04-2018 to 20.06-2018

Route of administration:

oral: gavage

Details on route of administration:

1) The dosage can be accurately administered;
2) It is the proposed route for clinical use;

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

Preparation: The test item was suspended in distilled water for preparation of solution(s).
The solution(s) of test chemical were made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered. The concentration of the test item was varied so as to maintain the dose volume constant at or upto 10 ml/kg body weight.
- VEHICLE
- Concentration in vehicle: 0, 25, 50 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days consecutively and 2 week recovery period.

Frequency of treatment:

Animals were observed twice daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group (G1)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group (G2)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (G3)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (G4)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group (G5)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group (G6)

No. of animals per sex per dose:

Control (0 mg/kg bw/day): 6 males and 6 females
Control Recovery (0 mg/kg bw/day): 6 males and 6 females
Low dose (250 mg/Kg/Day): 6 males and 6 females.
Mid Dose (500 mg/Kg/Day): 6 males and 6 females
High Dose (1000 mg/Kg/Day): 6 males and 6 females
High Dose Recovery(1000 mg/Kg/Day): 6 males and 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

Details on study design
- Dose selection rationale: The doses are selected in agreement with Sponsor.
- Rationale for animal assignment (if not random): A total of 72 animals (36 males + 36 females) were selected and randomly distributed into six groups with 6 animals/sex/group for main groups and 6 animals /sex /group for reversal group. At the commencement of the study the weight variation of animals used were minimal and did not exceed ± 20 % of the mean weight of each sex.
- Rationale for selecting satellite groups: Two additional dose levels were added to the study as control 0 mg/kg (Rev.) and test item 1000 mg/kg (Rev.), in order to study the reversibility or delayed occurrence of symptoms, if any.
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No Data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made for all animals from treatment groups to assess the behavioral status of each animal. Detailed clinical observations were performed once before the start of dose administration and once a week thereafter until scheduled sacrifice.
a) Home Cage Observations:
In home cage, rats were observed for behavior, alterations, vocalizations, respiration and palpebral closer.
1) Behavior in Home cage:
The behavior of the rat was observed in home cage upon initial approach by the observer and description of behavior in home cage was recorded as:
1 = apparently sleeping.
2 = Awake, but immobile; apparently normal posture.
3 = Engaged in apparently normal movement such as rearing, drinking, or grooming.
4 = Immobile, with unusual posture or tonic convulsion (lying on side with legs extended, flattened body or arched back. (opisthotonos or emprosthonos).
5 = Unusual behaviour or muscular patterns (stereotyped behaviour such as head bobbing or weaving, circling, repetitive licking or grooming, bizarre behaviour such as self mutilation, writhing or retropulsion, unusual muscular patterns such as tremors, spasms or clonic convulsion).
2) Alterations Home cage:
The alterations of the rat were observed in home cage upon initial approach by the observer and description of alterations in home cage was recorded as:
1 = No alterations in behaviour or posture (Normal).
2 = Stereotyped behaviour pattern (head bobbing or weaving, circling, repetitive licking or grooming).
3 = Bizarre behaviours such as self-mutilation, writhing or retropulsion.
4 = Twitches or tremors in the limbs or repetitive movements of the mouth or jaws.
5 = Whole body tremors or spasms.
6 = Unusual posture (opisthotonos, emprosthotonos, tonic extension, head tilt, straub tail).
7 = Tonic-clonic seizure.
3) Vocalizations:
The occurrence of spontaneous or unprovoked vocalization was recorded as:
1= No vocalization/ Normal
2= Vocalization noted
If vocalization observed the actual number was recorded.
4) Respiration:
The observations for respiration were recorded as:
1 = Normal
2 = Abnormal
The type of abnormal respiration e.g. bradypnea, hyperpnea, dyspnea, rals was recorded in the clinical signs.
5) Palpebral closer:
The degree of closure of the eyelids was recorded as:
1 = Eyelids wide open (Normal).
2 = Eyelids slightly closed.
3 = Eyelids dropping, approximately half-closed.
4 = Eyelids completely shut.
b) Handling Observations:
After completing home cage observations, the rat was observed for reaction to removal, reaction to handling, urination, defecation, prominence of eye, lacrimation, salivation, piloerection, examination of mucous membrane, examination of skin / fur, examination of natural orifices and animal appearance.
1) Reaction to Removal:
The reaction of the rat removed from home cage was recorded as:
1 = Sits quietly and is easily removed.
2 = Vocalization without resistance to being picked up.
3 = Runs around cage with or without vocalization, or freezes or rears, following the
investigator’s hand.
4 = Tail and throat rattles, may attack.
2) Reaction to handling:
The reaction of the rat to handling from home cage was recorded as:
1 = Quiet with no resistance.
2 = Vocalization without resistance.
3 = Tense or rigid.
4 = Squirming and twisting, may attempt to bite.
3) Urination:
The frequency of urination was recorded as:
0 = No urination during the observation period.
1 = Urine present; quantity is not excessive.
2 = the amount of urination is excessive.
4) Defecation:
The frequency of defecation was recorded as:
0 = No defecation during the observation period.
1 = Fecal boluses have normal consistency.
2 = Soft or liquid feces.
5) Prominence of Eye:
The eyes were examined for prominence of eye and observation was recorded as:
1 = Normal
2 = Exophthalmos
3 = Enophthalmos
6) Lacrimation:
The degree of lacrimation was recorded as:
1 = No excess lacrimation (normal).
2 = Excess moisture at the margin of the eyelid.
3 = Persistent dampness at the margin of the eyelid
4 = Dampness extends beyond the margin of the eyelid.
7) Salivation:
The degree of salivation was recorded as:
1 = No excess salivation (normal).
2 = Margin of mouth wet.
3 = Wet zone ¼ to ½ of submandibular area.
4 = Wet zone extends to the entire submandibular area.
8) Piloerection:
Piloerection was differentiated from a scruffy or ungroomed coat by patting the back of the animal in a rostral to caudal direction. Piloerection was considered in case the animal hairs were erect after patting. The observation for piloerection was recorded as:
0 = Absent
1 = Present
9) Examination of Mucous Membrane:
The observation for visual mucous membrane was recorded as:
1 = Normal
2 = Abnormal (discolouration)
10) Examination of Skin / Fur:
The observation for skin / fur examination was recorded as:
1 = Normal
2 = Abnormal
11) Examination of Natural Orifices:
The observation for natural orifices examination was recorded as:
1 = Normal
2 = Abnormal
12) Animal Appearance:
The observation for appearance of animal was recorded as:
1 = Clean and groomed.
2 = Unkempt (with scruffy and ungroomed coat)
3 = Stained by urine and/or feces.
c) Open field observation:
The animal was placed in the open field and its appearance and behavior were observed. The following observations were made and recorded:
1) Stereotype Behaviour:
The stereotype behaviour can be defined as the pronounced repetition of specific gesture or movements i.e. presence of excessive or repetitive behaviour that appears purposeless to the observer. The observation was recorded as:
0 = Absent
1 = Excessive grooming / licking / head bobbing or weaving
2 = Circling movements
2) Bizarre Behaviour:
The bizarre behaviour includes any unusual behaviour that will not be normally observed in the test species. The observation was recorded as:
0 = Absent
1 = Retropulsion
2 = Biting of cage
3 = Biting to other animal(s)
4 = Self destructive biting or mutilation
3) Rearing (Rears):
The number of times the rat raises both forelimbs off the surfaces is considered as rearing. The number of these actions was counted and total number of rearing was recorded.
4) Movements:
In the open field, each animal was observed for presence of clonic and tonic movements.
4.1) Clonic Movements:
The observation for clonic movement was recorded as:
0 = None /Normal
1 = repetitive mouth/jaw motion, such as, Chewing, clones of jaw
2 = Mild clonic tremors of whole body
3 = Repetitive clonic tremors/ seizure of whole body
4.2) Tonic Movements:
The observation for tonic movement was recorded as:
0 = None /Normal
1 = Contractions of limbs
2 =Unusual posture (Opisthotonos, Emprosthotonous, tonic extension, head tilt, straub tail).
3= seizure
5) Gait Pattern:
The gait pattern was evaluated by observing the movement of the rat in the open field and the observation was recorded as:
1 = Normal
2 = Ataxic
3 = Hind limbs or forelimbs show exaggerated or overcompensated movements, drag or appear splayed.
4 = Spastic
5 = Duck walk
6 = Scissor
7 = Hunched back
6) Mobility Score:
A measure of the ability of the animal to locomote despite gait abnormalities was recorded. The ranking of the degree of impairment of locomotion was recorded as:
1 = Normal
2 = Slightly impaired
3 = Totally impaired
7) Severity of Gait:
The severity of the gait abnormalities is graded and documented as follows:
1 = Slight gait abnormality
2 = Moderate gait abnormality
3 = Extreme gait abnormality
8) Pupillary response:
The animal eyes were briefly covered for 30 seconds with hand/cloth and then the penlight was pointed and the response to penlight was recorded as:
1 = Response
2 = No response

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

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
The quantity of feed consumed by control and different treatment groups was recorded on commencement of treatment and weekly thereafter until scheduled sacrifice and the feed consumption per animal was calculated for each group.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice. Eye examination was carried out by using a HEINE mini 2000 ophthalmoscope were employed for evaluation after the induction of mydriasis with 1% solution of tropicamide sulfate
- Dose groups that were examined

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: Functional Observation Battery:
Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42, sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment were conducted for all the animals (Group I, II, III, IV, V and VI).
Sensory Reactivity Observations:
Towards the end of the exposure period, sensory reactivity to stimuli of different types (e.g., auditory, visual and proprioceptive stimuli) was conducted for all the animals.
1) Arousal level:
The activity/arousal level of the animal was described during the observation period and is documented as follows:
1 = Very low (stuporous, comatose)
2 = Low (sluggish, some exploratory movement possible).
3 = Apparently normal (alert with exploratory movement).
4 = High (sudden startle, darting or freezing without apparent stimuli).
5 = Very high (sudden bouts of running, freezing with spontaneous vocalization).
2) Sensory Activity:
Sensory activity was assessed by following methods:
2.1) Assessment of Visual Response: A blunt probe was held approximately 4 cm away from front of the face / eye and moved away steadily and reaction was documented.
2.2) Touch Response: Avoiding the animal’s field of vision, the rump was gently touched with blunt probe. The animals reaction to this stimulus was observed and was documented.
2.3) Auditory Response: Using a clicker approximately 5 cm above the back of the animal sudden sound was made. The animals reaction to this stimulus was observed and was documented.
2.4) Tail Pinch Response: The reaction to a tail pinch was rated (The tail pinch was applied by the blunt forceps at approximately 3 cm from the tip of tail. The reaction to tail pinch was observed and was documented.
All above four responses were graded as follows:
1 = No reaction.
2 = orientating response: Slowly turns towards the stimulus or walks away.
3 = Startle response or freezing reaction.
4 = More energetic response than “2” or “3”.
5 = Jumps at or away, attack or bites.
3) Visual Placing Response:
The animal was removed from its cage and held at the base of the tail (holding the tail more distally can strip off the skin) then slowly lowered forward towards the edge of the observation area or another raised edge (such as the rim of an overturned cage). The visual placing response is graded and documented:
1 = Early extension of forelimbs to reach for the screen.
2 = Extends limbs only after contact with the vibrissae or nose.
3 = No extension even after contact with nose.
4) Air righting response:
Holding the animal in a supine position, it was dropped from approximately 30cm and the righting response was rated:
1 = Lands with all feet on the ground.
2 = Uncoordinated landing or lands on side.
3 = Lands on back.
Grip Strength:
Grip strength of fore limbs was measured with a digital grip strength meter (Columbus Instruments International Corporation, Ohio, USA) to determine the ability of the rat to grasp and hold on the mesh platform. The grip strength of each rat was measured in Kilogram (Kg) for 3 consecutive times and average of the three grip strength values was calculated.
Motor Activity:
Motor activity of each animal was monitored using an automated animal activity measuring system (Columbus Instruments, OPTO-M3, Ohio, USA). Animals were monitored for three consecutive 10 minutes intervals allowing for examination of both exploratory and acclimation activity levels. During this period, total and ambulatory activity of the animal was recorded. Stereotypic activity was calculated by subtracting ambulatory activity from total activity.
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

The rats were sacrificed by CO2 asphyxiation.
Sacrifice - after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI).
Organ Weights
Liver, Kidneys, Adrenals, Epididymides, Prostate + Seminal Vesicle with Coagulation gland as whole, Thymus, Spleen, Brain, Heart, Uterus, Testes/Ovaries were dissected free of fat and weighed. The paired organs were weighed together.
Gross Pathology
All the rats surviving at the end of the treatment were sacrificed and gross lesions were noted.
Tissue Preservation and Histopathology
From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin.
Procedure for preparation of slides of tissues of various organs from the rats of various dose groups were performed as per the standard operating procedures of Indian Institute of Toxicology, Pune.
Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination.Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Muscles - Skeletal muscle, Oesophagus, Ovaries, Pancreas, Pharyngeal Lymphnodes, Pituitary, Prostate, Rectum, Sciatic Nerve, Seminal Vesicles with coagulation gland, Skin with Mammary Gland, Spleen, Spinal Cord (Cervical, mid thoracic and lumbar), Sternum with bone marrow, Stomach, Testes, Thymus, Trachea, Thyroid, Urinary Bladder, Uterus, Vagina.

Other examinations:

The rats were sacrificed by CO2 asphyxiation.
Sacrifice - after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI).

Statistics:

Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data does not meet the homogeneity of variance, Student’s t-test were performed to calculate significance.

Significance was calculated at 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical toxicity were seen in all male and females of all the treated and control groups.
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer. All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Grip strenght and motor activity values of the control and treated groups were normal.

Mortality:

no mortality observed

Description (incidence):

All animals from control and different dose groups survived throughout the dosing period of 28 days and the post-dosing recovery period of 14 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Male and Female -
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Male and Female -
Animals from control and different dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.
Animals from control reversal and high reversal dose groups exhibited normal feed consumption at the end of the recovery period of 14 days.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination.

Haematological findings:

no effects observed

Description (incidence and severity):

Male and Female -
Haematological investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the decrease in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.


Male :
MCHC : Decreased values were obtained for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose group, sacrificed on day 29 (p<0.05).
Female :
No significant changes were observed in the values of different parameters studied in animals from different dose groups when compared with that of controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male and Female -
Biochemical investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.
Male :
Creatinine : Elevated levels were observed in animals from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05),
Sodium : Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Bile Acids : Elevated levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05),
Total Protein : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05),
Alanine Aminotransferase : Decreased levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Calcium : Decreased levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Albumin : Decreased levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05) and
Calcium and Chloride : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).





Female :
Total Bilirubin : Elevated levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Sodium : Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Bile Acids : Elevated levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Glucose : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Calcium : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Male and Female -
No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 24, 25, 26 and 43) in male and female animals from different dose groups as compared to control group animals.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

1) Behavior in Home cage:
The behavior of the rat was observed in home cage upon initial approach by the observer and description of behavior in home cage was recorded as:

1 = apparently sleeping.
2 = Awake, but immobile; apparently normal posture.
3 = Engaged in apparently normal movement such as rearing, drinking, or grooming.
4 = Immobile, with unusual posture or tonic convulsion (lying on side with legs extended, flattened body or arched back. (opisthotonos or emprosthonos).
5 = Unusual behaviour or muscular patterns (stereotyped behaviour such as head bobbing or weaving, circling, repetitive licking or grooming, bizarre behaviour such as self mutilation, writhing or retropulsion, unusual muscular patterns such as tremors, spasms or clonic convulsion).
2) Alterations Home cage:
The alterations of the rat were observed in home cage upon initial approach by the observer and description of alterations in home cage was recorded as:

1 = No alterations in behaviour or posture (Normal).
2 = Stereotyped behaviour pattern (head bobbing or weaving, circling, repetitive licking or grooming).
3 = Bizarre behaviours such as self-mutilation, writhing or retropulsion.
4 = Twitches or tremors in the limbs or repetitive movements of the mouth or jaws.
5 = Whole body tremors or spasms.
6 = Unusual posture (opisthotonos, emprosthotonos, tonic extension, head tilt, straub tail).
7 = Tonic-clonic seizure.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Male -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 1000 mg/kg dose group revealed increased relative weights of testes (p<0.05).
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group was found to be comparable.

Female -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from different dose groups was found to be comparable.
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group revealed decreased relative weights of heart and thymus (p<0.05).

Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathological examination on male and female animals from control and different dose groups did not reveal any abnormality.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No treatment related histopathological changes were evident in male and female animals from control and high dose groups.
Incidental, congenital and physiological histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, multifocal periportal mononuclear cells infiltration in liver; minimal, focal to multifocal tubular vacuolation and/or mineralization and/or tubular dilatation in the kidneys; minimal, diffuse dilatation of zona reticularis and/or vacuolation of zona fasiculata in the adrenals; minimal, diffuse luminal dilatation and/or endometrial gland dilatation in the uterus; ultimobranchial cyst and/or presence of Ectopic thymus in thyroid; presence of Rathke’s pouch in pituitary; Mucification of epithelium in vagina; presence of seminal coagulum in the urinary bladder in male and female animals from control and high dose group.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

The NOAEL for the test chemical after dosing for 28 days with two weeks recovery period was found to be 1000 mg/kg bw/day.

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

        

APPENDIX NO.I

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : I

Dose  : 0 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
1	149.3	151.0	171.6	206.9	240.4	244.5
2	152.8	156.5	173.2	204.8	236.1	240.9
3	154.6	158.2	180.5	204.6	230.8	235.8
4	161.0	163.6	188.3	202.0	236.5	243.8
5	164.7	168.0	211.6	244.7	276.3	285.6
6	173.7	175.8	213.3	247.3	261.9	276.4

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : I

Dose  : 0 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
7	132.2	134.1	165.5	176.5	194.8	198.9
8	134.9	136.2	144.8	153.9	163.9	167.4
9	138.7	139.8	157.5	180.7	190.1	201.7
10	140.1	141.4	164.6	176.4	199.2	203.6
11	146.2	147.9	159.4	178.6	191.5	208.6
12	153.5	154.9	171.1	188.6	207.6	211.0

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : II

Dose : 0 mg/kg (Reversal)

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4	5	6
13	149.9	154.4	174.7	186.9	207.9	217.2	230.6	245.8
14	151.4	153.2	172.8	195.9	217.2	226.6	244.9	262.2
15	156.0	159.1	185.8	201.6	232.6	244.3	261.7	276.9
16	161.0	165.7	188.6	212.1	232.0	242.8	266.8	280.4
17	164.7	169.1	202.3	227.9	246.3	248.5	270.8	288.8
18	174.9	178.3	218.3	245.6	270.8	279.4	294.4	310.3

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : II

Dose : 0 mg/kg (Reversal)

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4	5	6
19	132.3	135.3	161.2	176.4	185.5	196.5	209.0	223.1
20	133.9	135.8	155.4	175.1	201.1	209.5	221.2	234.4
21	139.1	142.4	171.3	190.1	213.7	217.6	229.2	242.7
22	140.7	143.7	153.3	169.3	184.3	192.2	206.4	222.7
23	148.8	151.6	185.0	207.9	223.2	226.0	238.0	252.2
24	154.6	156.2	167.2	194.5	204.9	212.4	223.9	236.6

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : III

Dose  : 250 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
25	151.1	153.5	178.5	196.4	227.2	230.7
26	152.6	156.9	182.7	203.2	233.4	236.7
27	153.2	157.5	180.9	198.8	230.0	234.5
28	161.9	164.4	192.2	225.1	256.0	260.1
29	166.6	168.9	200.9	222.1	252.6	278.4
30	175.8	177.6	205.2	230.4	261.4	282.8

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : III

Dose  : 250 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
31	132.6	135.8	162.7	186.5	203.2	211.4
32	133.9	135.9	155.5	173.9	182.7	186.5
33	135.2	137.3	147.2	161.6	177.1	181.4
34	143.0	150.7	177.9	195.3	207.3	213.2
35	149.0	151.2	168.9	182.3	200.9	204.3
36	156.0	157.7	186.9	207.8	219.1	225.0

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : IV

Dose  : 500 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
37	151.1	156.7	181.9	206.6	223.8	226.0
38	153.2	155.0	183.3	200.7	228.2	234.9
39	153.5	157.3	186.3	211.8	238.0	243.0
40	156.1	161.6	189.2	208.8	234.1	239.8
41	168.0	172.4	212.4	251.2	275.6	277.1
42	177.0	179.3	214.5	254.5	286.5	306.7

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : IV

Dose  : 500 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
43	133.0	135.8	148.8	157.6	182.2	184.9
44	134.1	135.0	147.0	162.9	187.9	191.7
45	136.2	138.3	171.4	184.8	195.1	201.7
46	139.2	140.1	161.4	182.8	196.3	200.2
47	150.9	150.3	158.2	193.6	217.7	222.8
48	159.1	161.2	181.7	198.8	212.8	212.8

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : V

Dose  : 1000 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
49	151.0	154.4	183.5	217.1	252.0	254.4
50	152.6	157.4	179.6	193.1	217.8	221.4
51	154.1	156.1	191.3	211.0	227.8	231.9
52	157.0	160.7	187.7	208.7	231.3	234.0
53	162.1	166.4	205.4	219.2	256.4	260.5
54	178.1	179.5	201.2	224.5	250.0	255.6

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : V

Dose  : 1000 mg/kg

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4
55	133.0	133.5	163.9	188.1	203.2	209.8
56	134.3	138.8	154.2	170.6	185.0	189.5
57	136.8	137.0	147.2	174.9	188.0	191.7
58	139.8	141.0	162.0	181.8	191.7	194.2
59	144.6	149.6	165.2	186.3	197.0	200.2
60	159.7	162.3	187.0	204.1	218.1	227.3

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Male

Group : VI

Dose : 1000 mg/kg (Reversal)

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4	5	6
61	151.3	153.2	180.7	206.6	226.9	235.4	253.9	270.7
62	152.9	156.2	185.6	214.7	256.8	273.2	289.7	302.9
63	154.3	159.9	200.9	230.8	263.6	277.7	296.0	310.9
64	159.0	162.6	197.5	220.3	245.7	256.5	275.6	291.0
65	162.2	165.4	192.2	212.3	237.9	240.6	258.8	272.5
66	169.6	173.2	212.9	225.6	261.5	279.1	298.2	317.9

APPENDIX NO.I (Contd.)

 

 

INDIVIDUAL ANIMAL - BODY WEIGHT (g)

 

Laboratory Test Item Code : TAS/122/071

Test System : Sprague Dawley Rat

Sex    : Female

Group : VI

Dose : 1000 mg/kg (Reversal)

Animal	Weeks
Number	Day 0	Day 1	1	2	3	4	5	6
67	134.0	135.4	167.4	197.5	210.3	217.8	230.6	246.9
68	134.3	135.1	169.3	195.4	222.6	235.9	247.1	257.1
69	137.8	138.6	167.6	194.2	204.6	214.7	226.2	239.5
70	139.9	142.6	158.4	185.1	198.3	213.3	228.5	241.5
71	145.0	145.8	163.8	181.3	194.2	206.5	218.5	231.6
72	152.3	155.8	177.9	189.3	204.5	213.3	226.4	239.1

Conclusions:

Based on all the findings, the No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days with two weeks recovery period was found to be 1000 mg/kg bw/day.

Executive summary:

The present study aimed with repeated dose 28-day oral toxicity study with test chemical  by daily gavage in the rat  followed by a 2 week recovery period. In the present study, the animals randomly selected and assigned to group G1, G2, G3, G4 and G5, Group G1 (nominal) and G2(actual dose received) were control animals, Group G3, G4 and G5 received daily dose of 250, 500 and 1000mg/kg (actual dose received) of test chemical whereas. Group G6 received nominal dose of 1000mg/kg of test chemical. The treated animals observed daily for the clinical signs of toxicity; observation for bahviour in home cage and score accordingly; body weight, food consumption, opthalmic examination and neurobehavioural examination (grip strenght and motor activity). After 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI). All rats were sacrificed by CO2 asphyxiation.

The results of the study revealed, No mortality in both male and females during the study. No clinical toxicity were seen in all male and females of all the treated and control groups. In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer. All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Grip strenght and motor activity values of the control and treated groups were normal. Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days. No change in feed consumption were seen in control and different dose groups at the end of the dosing period of 28 days. No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination. No change in haematological parameters were observed only Decreased values of MCHC ( mean corpuscular hemoglobin concentration) were obtained for males from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose group, sacrificed on day 29 (p<0.05). Different Clinical biochemitry parameters studied and compared with respective controls, however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent. No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 24, 25, 26 and 43) in male and female animals from different dose groups as compared to control group animals.

Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. The changes in organ weight were, increased relative weights of testes at end of 29 day study in males and decreased relative weights of heart and thymus at teh end of recovery period at 43 days ini females.

Gross pathological examination on male and female animals from control and different dose groups did not reveal any abnormality.

 

No treatment related histopathological changes were evident in male and female animals from control and high dose groups.However, incidental, congenital and physiological histopathological changes were recorded but it was unrelated to the test chemical treatment.

Based on all the findings, the No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days with two weeks recovery period was found to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1 rating study report

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the test chemicals was reviewed to determine the toxic nature of [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7) . The studies are as mentioned below:

Repeated dose toxicity: Oral

The present study aimed with repeated dose 28-day oral toxicity study with test chemical  by daily gavage in the rat  followed by a 2 week recovery period. In the present study, the animals randomly selected and assigned to group G1, G2, G3, G4 and G5, Group G1 (nominal) and G2(actual dose received) were control animals, Group G3, G4 and G5 received daily dose of 250, 500 and 1000mg/kg (actual dose received) of test chemical whereas. Group G6 received nominal dose of 1000mg/kg of test chemical. The treated animals observed daily for the clinical signs of toxicity; observation for behavior in home cage and score accordingly; body weight, food consumption, ophthalmic examination and neurobehavioral examination (grip strength and motor activity). After 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI). All rats were sacrificed by CO2 asphyxiation. The results of the study revealed, No mortality in both male and females during the study. No clinical toxicity were seen in all male and females of all the treated and control groups. In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer. All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Grip strength and motor activity values of the control and treated groups were normal. Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days. No change in feed consumption were seen in control and different dose groups at the end of the dosing period of 28 days. No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination. No change in haematological parameters were observed only Decreased values of MCHC ( mean corpuscular hemoglobin concentration) were obtained for males from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose group, sacrificed on day 29 (p<0.05). Different Clinical biochemistry parameters studied and compared with respective controls, however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent. No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 24, 25, 26 and 43) in male and female animals from different dose groups as compared to control group animals. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. The changes in organ weight were, increased relative weights of testes at end of 29 day study in males and decreased relative weights of heart and thymus at the end of recovery period at 43 days ini females. Gross pathological examination on male and female animals from control and different dose groups did not reveal any abnormality. No treatment related histopathological changes were evident in male and female animals from control and high dose groups .However, incidental, congenital and physiological histopathological changes were recorded but it was unrelated to the test chemical treatment. Based on all the findings, the No Observed Adverse Effect Level (NOAEL) for the test chemical in Sprague Dawley Rats, via oral gavage, for 28 days with two weeks recovery period was found to be 1000 mg/kg bw/day.

Repeated inhalation study:

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7) which is reported as 0.02302689mmHg at 25 C. Thus, exposure to inhalable dust, mist and vapour of the [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile . Therefore this study is considered for waiver.

Repeated dermal study;

The acute toxicity value for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile (54079-53-7) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that for [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Based on the data available and applying the weight of evidence approach, the test chemical [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino -2-methylphenyl]methylene]malononitrile (54079-53-7) is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino -2-methylphenyl]methylene]malononitrile (54079-53-7) is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.