Reaction mass of Diethyl[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]ammonium and acetate

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The No Observed Adverse Effect level (NOAEL) for the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate in male and female rats is considered to be non toxic upto a dose level of 40 mg/Kg bw.

Repeated dose toxicity: Inhalation

Reaction mass of Ethanaminium, N-[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl- & acetate has very low vapor pressure (9.77E-014 Pa.), so the potential for the generation of inhalable vapours is very low. Also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point was considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value forReaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate(as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose & carcinogenicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Limit test:

no

Species:

other: 1 Rat, 2. Mice, 3. Rats

Strain:

other: 1. Fischer 344, 2. B6C3F1, 3. Albino

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

1. TEST ANIMALS
- Source: National Center for Toxicological Research
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were housed in barrier housed conditions containing hardwood chips as cage bedding in filter topped cages
- Diet (e.g. ad libitum): Feed ad libitum
- Water (e.g. ad libitum): Drinkng water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± -16.6 °C
- Humidity (%): 50 ±5 %
- Air changes (per hr): 14-16 changes of air/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark cycle

IN-LIFE DATES: From: To: No data

2. TEST ANIMALS
- Source: The animals were derived from specific-pathogen-free (SPF) mice of the NCTR breeding colony
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 8-15 g
- Fasting period before study: No data
- Housing: The animals were housed four animals/cage in barrier-type animal holding room with hardwood chips as the bedding material and filter top cages
- Diet (e.g. ad libitum): Feed ad libitum
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± -16.6 °C
- Humidity (%): 50 ±5 %
- Air changes (per hr): 14-16 changes of air/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark cycle

IN-LIFE DATES: From: To: No data

3. No data

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed

Details on oral exposure:

1. PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in ethanol and sprayed directly into the feed in a sanitized chamber at dose levels of 0, 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and Females: 0, 40, 100 or 200 mg/Kg bw). Ethanol was subsequently removed from the prepared feed during a 30 min blending process using a vacuum

DIET PREPARATION
- Rate of preparation of diet (frequency): Mixing of the test chemical into the feed was done on a weekly basis and feed was used in less than 30 days after mixing.
- Mixing appropriate amounts with (Type of food): Purina 5010M autoclavable, Purina MIlles, Inc. Richmong IN, USA
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: Males: 0, 30, 80 or 160 mg/Kg bw
Females: 0, 40, 100 or 200 mg/Kg bw
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

2. PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose levels of 0, 100, 300 or 600 ppm (Males: 0, 75-100, 225-250, 450-475 mg/Kg bw/week and Females: 0, 100, 250-275 or 500 mg/Kg bw/week)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: Males: 0, 75-100, 225-250, 450-475 mg/Kg bw/week and Females: 0, 100, 250-275 or 500 mg/Kg bw/week
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

3. PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed to give a dietary level of 0.1%

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0 or 100 mg/Kg bw/day
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

1. 80 days
2. 12, 18 or 24 months
3. 20-21 months

Frequency of treatment:

Daily

Remarks:

Males: 0, 30, 80 or 160 mg/Kg bw
Females: 0, 40, 100 or 200 mg/Kg bw / 1

Remarks:

Males: 0, 75-100, 225-250, 450-475 mg/Kg bw/week

Females: 0, 100, 250-275 or 500 mg/Kg bw/week / 2

Remarks:

0 or 100 mg/Kg/day / 3

No. of animals per sex per dose:

1. Total: 1140
0 mg/Kg bw: 210 males and 210 females
30 (males) and 40 (females) mg/Kg bw: 120 males and 120 females
100 (males) and 80 (females) mg/Kg bw: 120 males and 120 females
200 (males) and 160 (females) mg/Kg bw: 120 males and 120 females

2. Total: 1440
0 mg/Kg bw: 120 males and 120 females
75-100 (males) and 100 (females) mg/Kg bw: 120 males and 120 females
225-250 (males) and 250-275 (females) mg/Kg bw: 120 males and 120 females
450-475 (males) and 500 (females) mg/Kg bw: 480 males and 480 females

Control animals:

yes, concurrent vehicle

Details on study design:

1.
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): F0 animals were randomly divided into dose groups
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

2. No data

Positive control:

No data

Observations and examinations performed and frequency:

1. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly
- Cage side observations checked in table [No.?] were included. Mortality and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data - Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

2. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly
- Cage side observations checked in table [No.?] were included. Mortality and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At scheduled sacrifice
- Animals fasted: No data
- How many animals: All animals
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data - Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

3. Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. Mortality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data - Animals fasted: No data - Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

1. GROSS PATHOLOGY: Yes, the animals were subjected to complete necropsy
HISTOPATHOLOGY: Yes

2. GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, the animals were subjected to histopathological examination

3. GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

No data

Statistics:

Please refer below section

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Other effects:

not specified

Details on results:

1. Clinical signs and mortality:
Clinical signs: 253 rats were found to be in the moribund stage throughout the study period.

Mortality: At the end of 24 months, mortality was approximately 33% for both males and females in the control group and approximately 66% for females and 48% for males in the highest 200 or 160 mg/Kg bw dosed group for males and females respectively. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw.

The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels.

Body weight and weight gain:
Female: The body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups.

Males: At 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk.

Food consumption and compound intake: Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for
the females and 25-30 g food/kg body weight for the males.

Based on the consumption rates, the dosage of GV remained relatively constant following the rapid growth stage in the first 20 wk.

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology : No data

Clinical chemistry: No data

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No data

Gross pathology: No data

Histopathology: No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the
160 mg/Kg bw group.

The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls.

The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered GV in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months.

Incidences of leukemia were high in all groups of female rats fed GV for 24 months and statistical analysis showed no significant differences overall or in the dose groups.

In the males fed GV for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed GV for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively.

No incidence of mononuclear cell leukaemia show a dose response in male rats fed GV for either 18 or 24 months.

No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. In non-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes.

2. Clinical signs and mortality

Clinical signs: No data

Mortality: Mortality rates in males and females was very low up to approximately 450 days, after which time a dose-response trend was noted in both the males (p = 0.01288) and females (p = 0.00005). The dose response was more pronounced in the females. At the end of the 2- year dosing, mortality in the female control group was approximately 13%, and 28, 27, and 64% in the 100, 250-275 and 500 ppm dose groups, respectively. The males' mortality rates at the 0, 75-100, 225-250, and 450-475 ppm-dose levels were 13, 14, 20, and 23%, respectively. When each dose level was compared with the control, all dose levels in the females were significantly different, i.e., at 100 mg/Kg bw/week , p = 0.00088, at 250-275 mg/Kg bw/week, p = 0.00079, and at 500 mg/Kg bw/week, p = 0.00005. The dose response in mortality is likely due to the toxic effects of the gentian violet and not related to nutritional factors.

Body weight and weight gain: The body weights increased gradually throughout the study in all dose groups at essentially the same rate as the control group. There appeared to be no dose effect on body weight gain.

Food consumption and compound intake: The average food consumption remained essentially the same for each of the dose groups and the controls. When the consumption was calculated on the basis of body weight, the food consumed started out at about 1.3 g food/g body wt/week for females and 1.1 g for males, then gradually decreased during the initial 30 to 40 weeks to about 0.90 and 0.80 g for the females and males, respectively

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: No data

Clinical chemistry: There were no observed effects at 12 months. At 18 months there was a small increase in direct bilirubin; however, this was probably caused by interference of the blue pigment in the serum.

At 24 months, positive effects were noted for AST-GOT, ALT-GPT, serum cholesterol, α1 serum protein, α2 serum protein, and triglycerides. All of these effects related to abnormalities of the liver. In addition, most of the statistically positive trends were in the females at 24 months and at 500 mg/Kg bw/week.

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No data

Gross pathology: No data

Histopathology: Histopathological examination revealed several liver neoplasms (hepatocellular carcinoma). A slight dose response at 18 months for the liver neoplasms was noted in females and no other dose responses were noted in the 12- or 18- month sacrifice groups. Essentially, all dose-related lesions were noted in the 24-month sacrifice groups.

Malignant liver neoplasms occurred with an incidence of 4, 5, 32, and 77% in the female controls, 100, 250-275, and 500- mg/Kg bw/week-dose groups, respectively, by 24 months. A background incidence of 15% was noted in the control males by 24 months compared to an incidence of 17, 18, and 35% in the 75-100, 225-250, and 450-475 mg/Kg bw/week-dose groups, respectively. The incidence in the females by 18 months was 2, 0, 4, and 13% for the controls, 100, 250-275, and 500 mg/Kg bw/week dose groups, respectively. The incidence of liver neoplasms in males by 18 months did not rise above the background levels of 10% observed in the controls.

The males demonstrated a lower susceptibility than females to liver carcinogenicity from gentian violet. While the overall comparisons for both malignant tumors alone and malignant plus benign tumors showed a significant positive trend for mortality, prevalence, and onset in both sexes, the pairwise comparisons of doses to controls showed less positive trends in the males. For malignant liver neoplasms, positive increases from control were noted only in prevalence and onset at 500 mg/Kg in females and 450-475 mg/Kg in males. Mortality at this dose level showed a borderline p value of 0.02. The results for malignant plus benign lesions were essentially the same as malignant only except positive increases from control were noted for prevalence and onset in the 250-275 mg/Kg in females and 225-250 mg/Kg in males also.

Dose responses were noted in females for erythropoiesis in the spleen with 7,16, 20, and 44% responding for the 0-, 100, 250-275, and 500-mg/Kg bw/week-dose levels, respectively, and for atrophy of the ovaries with the incidence of 6, 15, 28, and 42%. Significant dose responses were also noted for the occurrence of reticulum cell sarcomas (Type A) in the uterus, vagina, bladder, and ovaries. These lesions were absent from the controls, except for 1/182 for RCS in the vagina.

The following incidences were noted for the respective dose levels of 0, 100, 250-275, and 500 mg/Kg bw/week: RCS of the uterus (Type A), 0, 2, 7, and 13%; RCS of the vagina (Type A), 0.5, 1, 5, and 9%; RCS of the bladder (Type A), 0, 2, 3, and 6%; RCS of the ovaries (Type A) 0, 1, 3, and 6%. Adenoma of the Harderian gland was noted in 4, 12, 20, and 16% of the females and in 4, 7, 11, and 10% of the males in the respectively, by 24 months.

Details on results: Lymphoreticular tissue and the liver were the target tissues for tissue morphology. Benign hepatocellular neoplasms (adenomas) were usually small, and they compressed adjacent parenchyma focally. The adenomas were composed of well-differentiated cells in which the cytoplasm was either basophilic, eosinophilic, clear or vacuolated. Little pleomorphism of nuclei was present. The cells were of uniform size and formed either regular cords of not more than two cell layers thick or solid masses.

The hepatocellular carcinomas observed were of the trabecular pattern. Hemorrhage and necrosis occurred in some of the cases. The 4 nonhepatocellular
metastases in the lung were from an undifferentiated sarcoma of muscle and subcutis of the thigh, from a mammary gland tumor, from a Harderian gland tumor, and from a granulosa cell tumor of the right ovary. The other tumor cellular pattern also found was a solid pattern which was composed of either small immature neoplastic hepatocytes or extremely large anaplastic hepatocytes. The cytoplasm was either acidophilic or vacuolated and the nuclei were large with prominent nucleoli. Mitotic figures were not numerous.

The RCS (Type A) of the female genital organs was similar to those described by Dunn (1954). The tumor was composed of sheets of elongated spindled cells with basophilic ovoid nuclei and scanty acidophilic cytoplasm, involving the wall of the vagina, cervix, and uterus.

3. Clinical signs and mortality: Mortality was comparable to or better than that observed in untreated control group

Body weight and weight gain: A less marked reduction in body weight gain was observed in the rats fed the test chemical.

Food consumption and compound intake: No data

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology: No data

Clinical chemistry: No data

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No changes were observed in organ weight as compared to untreated control animals.

Gross pathology: Nodules were observed on the livers only 25% (7/28) of the test rats at the time of final sacrifice. No unusual growth was observed in the control rat livers.

No other outstanding differences were noted in any of the groups upon gross pathologic examination.

Histopathology:

Non neoplastic: Significant liver injury was noted among animals from test groups which was attributed to the test materials. The primary lesion consisted of focal to diffuse hyperplasia of the liver cells. Minor focal lesions of degeneration, necrosis , fatty me tamorphosis , inflammation, bile duct proliferation and cholangiofibrosis were present in the liver of treated animals. The lesions noted upon microscopic examination of the other- tissues from test animal group were attributed to naturally occurring disease and were considered to be normal for albino rats of this age and strain.

Neoplastic: Hepatomas were present in 2/9 male and 1/19 female that survived the length of the investigation. There were no hepatomas observed in any of the treated animals that died prior to the conclusion of the study. However, there were no hepatomas observed in any of the test animals that died prior to the conclusion of the investigation.

Dose descriptor:

NOAEL

Remarks:

1

Effect level:

40 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Changes noted in mortality, body weight, Gross pathology effects

Dose descriptor:

NOAEL

Remarks:

1

Effect level:

30 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Changes noted in mortality, body weight, Gross pathology effects

Dose descriptor:

NOAEL

Remarks:

2

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes noted in mortality and histopathology effects

Dose descriptor:

NOAEL

Remarks:

3

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Male/Female 40 mg/kg/day: Death, Hypertrophy of epithelial cell of intestinal tract, Hypertrophy of centrilobular hepatocyte, Necrosis of centrilobular hepatocyte

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects were noted at the mentioned dose level

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect level (NOAEL) for the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate in male and female rats is considered to be 40 mg/Kg bw.

Executive summary:

Data available for the target chemicals was reviewed to determine the toxic nature of the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate. The studies are as mentioned below:

Combined repeated dose & carcinogenicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using male and female Fischer F344 rats. Male and female weanling animals (F0) were randomly divided into four groups under barrier conditions and administered 0 (control), 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and females: 0, 40, 100 or 200 mg/Kg bw) test chemical in their feed for at least 80 days. All rats had access to feed and drinking-water ad lib. While receiving dosed feed, the females were mated with males (one male/ female) of the same dose level. Brother/sister matings were avoided. Two males and two females were selected randomly from each litter (F1a generation) and allocated three animals per cage as weanlings to the chronic study. Litter mates were not assigned to the same cage. The F1a animals continued on the same dose levels as their respective parents for 12, 18 or 24 months. The animals were observed for changes in body weight, food consumption, mortality and morbundity and the presence of lesions. In females, the body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups. In males, at 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk. Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for the females and 25-30 g food/kg body weight for the males. 253 rats were found to be in the moribund stage throughout the study period. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw. The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels. No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the 160 mg/Kg bw group. The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls. The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered the test chemical in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months. Incidences of leukemia were high in all groups of female rats fed the test chemical for 24 months and statistical analysis showed no significant differences overall or in the dose groups. In the males fed the test chemical for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed the test chemical for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively. No incidence of mononuclear cell leukaemia show a dose response in male rats fed the test chemical for either 18 or 24 months. No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. Innon-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes. Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical in male and female rats is considered to be 30 and 40 mg/Kg bw respectively.

In another study, Combined repeated dose & carcinogenicity study was performed to determine the chronic nature of the test chemical. The study was performed using 720 male and 720 female B6C3F1 mice for 12, 18, and 24 months of continuous dosing. The test chemical was mixed with feed and given by oral gavage route at dose levels of 0, 10, 300 or 600 ppm (0, 75-100, 225-250, 450-475 mg/Kg bw/week for males and 0, 100, 250-275 or 500 mg/Kg bw/week for females). The animals were observed for Clinical signs, body weight changes, food consumption, clinical chemistry and were subjected to gross and histopathology. There was no effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates. Mortality in the controls of both sexes was less than 15% at 24 months, but was approximately 64% in the females and 23% in the males given the high dose of 450-475 mg/Kg bw/week in males and 500 mg/Kg bw/week in females. Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in males at 24 months and in females at 18 and 24 months. Statistical tests for dose-related trends with respect to (1) mortality due to liver neoplasms, (2) prevalence of liver neoplasms, and (3) time to onset of liver neoplasms showed positive trends in both males and females. Histopathological examination revealed several liver neoplasms (hepatocellular carcinoma). A slight dose response at 18 months for the liver neoplasms was noted in females and no other dose responses were noted in the 12- or 18- month sacrifice groups. Essentially, all dose-related lesions were noted in the 24-month sacrifice groups. Malignant liver neoplasms occurred with an incidence of 4, 5, 32, and 77% in the female controls, 100, 250-275, and 500- mg/Kg bw/week-dose groups, respectively, by 24 months. A background incidence of 15% was noted in the control males by 24 months compared to an incidence of 17, 18, and 35% in the 75-100, 225-250, and 450-475 mg/Kg bw/week-dose groups, respectively. The incidence in the females by 18 months was 2, 0, 4, and 13% for the controls, 100, 250-275, and 500 mg/Kg bw/week dose groups, respectively. The incidence of liver neoplasms in males by 18 months did not rise above the background levels of 10% observed in the controls. The males demonstrated a lower susceptibility than females to liver carcinogenicity from gentian violet. While the overall comparisons for both malignant tumors alone and malignant plus benign tumors showed a significant positive trend for mortality, prevalence, and onset in both sexes, the pairwise comparisons of doses to controls showed less positive trends in the males. For malignant liver neoplasms, positive increases from control were noted only in prevalence and onset at 500 mg/Kg in females and 450-475 mg/Kg in males. The results for malignant plus benign lesions were essentially the same as malignant only except positive increases from control were noted for prevalence and onset in the 250-275 mg/Kg in females and 225-250 mg/Kg in males also. Dose responses were noted in females for erythropoiesis in the spleen with 7, 16, 20, and 44% responding for the 0-, 100-, 250-275-, and 500-mg/Kg bw/week-dose levels, respectively, and for atrophy of the ovaries with the incidence of 6, 15, 28, and 42%. Significant dose responses were also noted for the occurrence of reticulum cell sarcomas (Type A) in the uterus, vagina, bladder, and ovaries. These lesions were absent from the controls, except for 1/182 for RCS in the vagina. The following incidences were noted for the respective dose levels of 0, 100, 250-275, and 500 mg/Kg bw/week: RCS of the uterus (Type A), 0, 2, 7, and 13%; RCS of the vagina (Type A), 0.5, 1, 5, and 9%; RCS of the bladder (Type A), 0, 2, 3, and 6%; RCS of the ovaries (Type A) 0, 1, 3, and 6%. Adenoma of the Harderian gland was noted in 4, 12, 20, and 16% of the females and in 4, 7, 11, and 10% of the males in the respectively, by 24 months. Lymphoreticular tissue and the liver were the target tissues for tissue morphology. Benign hepatocellular neoplasms (adenomas) were usually small, and they compressed adjacent parenchyma focally. The adenomas were composed of well-differentiated cells in which the cytoplasm was either basophilic, eosinophilic, clear or vacuolated. Little pleomorphism of nuclei was present. The cells were of uniform size and formed either regular cords of not more than two cell layers thick or solid masses. The hepatocellular carcinomas observed were of the trabecular pattern. Hemorrhage and necrosis occurred in some of the cases. The 4 non hepatocellular metastases in the lung were from an undifferentiated sarcoma of muscle and subcutis of the thigh, from a mammary gland tumor, from a Harderian gland tumor, and from a granulosa cell tumor of the right ovary. The other tumor cellular pattern also found was a solid pattern which was composed of either small immature neoplastic hepatocytes or extremely large anaplastic hepatocytes. The cytoplasm was either acidophilic or vacuolated and the nuclei were large with prominent nucleoli. Mitotic figures were not numerous. The RCS (Type A) of the female genital organs was composed of sheets of elongated spindled cells with basophilic ovoid nuclei and scanty acidophilic cytoplasm, involving the wall of the vagina, cervix, and uterus. Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical is considered to be < 100 mg/L when exposed to male and female B6C3F1 mice.

Combined repeated dose & carcinogenicity was performed to determine the toxic nature of the test chemical upon repeated exposure by oral route of exposure. Male and female albino rats were fed the test chemical at dose levels of 100 mg/Kg bw/day using diet for 20-21 months. Auramine was used as a positive control chemical. The animals were observed for mortality, body weight changes, gross pathology and neoplastic and non-neoplastic histopathology. Survival observed in the test animals was comparable to that noted in untreated control group. A less marked reduction in body weight gain was observed in the treated rats as compared to controls. Nodules were observed on the livers only 25% (7/28) of the test rats at the time of final sacrifice which was less as compared to the nodules observed in positive control 59% (20 /34). No other outstanding differences were noted in any of the groups upon gross pathologic examination. There was significant liver injury among animals from both the positive' control and test groups which was attributed to the positive control and test materials.The incidence of hyperplasia observed was comparable in positive control and treated animals. Minor focal lesions of degeneration, necrosis , fatty metamorphosis , inflammation, bile duct proliferation and cholangiofibrosis were present in the liver of control, positive control and test animals. However the severity of these Iesions was generally greater in the livers of the positive control animals than in the Iivers of control or test animals. The lesions noted in other tissues upon microscopic examination of the from control, positive control and test animals were attributed to naturally occurring disease and were considered to be normal for albino rats of this age and strain. Hepatomas were present in 2/9 male and 1/19 female that survived the length of the investigation. There were no hepatomas observed in any of the treated animals that died prior to the conclusion of the study. On the basis of observations made, the no observed adverse effect level (NOAEL) for the test chemical is considered to be 100 mg/Kg bw/day.

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate in male and female rats is considered to be 40 mg/Kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is from peer reviewed publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Quality of whole database:

Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Data available for the target chemicals was reviewed to determine the toxic nature of the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate. The studies are as mentioned below:

Combined repeated dose & carcinogenicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using male and female Fischer F344 rats. Male and female weanling animals (F0) were randomly divided into four groups under barrier conditions and administered 0 (control), 100, 300 or 600 ppm (Males: 0, 30, 80 or 160 mg/Kg bw and females: 0, 40, 100 or 200 mg/Kg bw) test chemical in their feed for at least 80 days. All rats had access to feed and drinking-water ad lib. While receiving dosed feed, the females were mated with males (one male/ female) of the same dose level. Brother/sister matings were avoided. Two males and two females were selected randomly from each litter (F1a generation) and allocated three animals per cage as weanlings to the chronic study. Litter mates were not assigned to the same cage. The F1a animals continued on the same dose levels as their respective parents for 12, 18 or 24 months. The animals were observed for changes in body weight, food consumption, mortality and morbundity and the presence of lesions. In females, the body weights increased gradually throughout the study in 0, 30, 80 or 160 mg/Kg bw dosed group. However, the rate of increase was lower in the 160 mg/Kg bw group. After about 85 wk, the body weight of animals fed the 80 mg/Kg bw increased at a lower rate than the 30 mg/Kg bw and control groups. In males, at 200 mg/Kg bw dose group, a lower average body weight was noted than those for any other dose group. Body weights of male rats of 40 and 100 mg/Kg bw groups peaked at about 460g at about 70wk and then started a gradual decline at about 85 wk. Food consumption at week 1-20 showed a rapid decrease, then became stable, except for an unexplained increase after 90 wk. Consumption in the controls and test groups was essentially the same and stabilized at about 30-35 g food/kg body weight for the females and 25-30 g food/kg body weight for the males. 253 rats were found to be in the moribund stage throughout the study period. At the end of the dosing period, the mortality rates in the females were 33, 38, 60 and 66% for the controls and 30, 80 and 160 mg/Kg dose groups, respectively. For males, the same respective dose groups had mortality rates after 104 wk of 33, 33, 48 and 39% for 0, 40, 100 or 200 mg/kg bw. The mortality in females was significantly different from the controls at the 0.001 level in the 80 mg/Kg bw (P = 0.00007) and 160 mg/Kg bw groups (P= 0.00005). In males, only the 100 mg/Kg bw (P = 0.0057) had a higher mortality than the control animals at the 0.05 levels. No dose-related pathology was noted in rats necropsied at 12 months. Although statistical analysis of the incidence of hepatocellular adenomas in females showed a significant difference in the 80 mg/Kg bw dose group, the incidence was very low and there was no significant difference in the 160 mg/Kg bw group. The incidence of follicular cell adenocarcinomas of the thyroid gland in female rats at the 24-month necropsy was 1, 1, 5 and 8% in the controls, and 30, 80 and 160 mg/Kg bw groups, respectively. The 80 and 160 mg/kg bw dose groups were significantly different from the controls. The incidence of mononuclear cell leukemia appeared to be a time-related response, that is, the leukemia showed a dose response in female rats administered the test chemical in the diet for 18 months, but these effects were not observed in those rats necropsied at 24 months. Incidences of leukemia were high in all groups of female rats fed the test chemical for 24 months and statistical analysis showed no significant differences overall or in the dose groups. In the males fed the test chemical for 24 months, the only statistically significant differences from the controls for neoplastic lesions were noted in the 100 and 200 mg/Kg bw dosed groups for hepatocellular adenomas and in the high-dose group for follicular cell adenocarcinomas of the thyroid gland. The incidence of follicular cell adenocarcinomas of the thyroid gland in rats fed the test chemical for 24 months was 1, 5, 3 and 6% in the controls and 40, 100 and 200 mg/Kg bw dosed groups, respectively. No incidence of mononuclear cell leukaemia show a dose response in male rats fed the test chemical for either 18 or 24 months. No non neoplastic effects related to administration of the test substance were observed at the 12- and 18-month necropsies in male and female rats. Most non-neoplastic lesions in the female rats that showed a dose response at 24 months were located in the liver. Lesions in the liver included eosinophilic foci, haematopoietic cell proliferation, mixed cell foci, regeneration, centrilobular necrosis and bile duct hyperplasia. Innon-neoplastic lesions noted in the liver of male rats included clear cell foci, eosinophilic foci, mixed cell loci, regeneration and centrilobular necrosis. Lesions in other organs included follicle cyst of the thyroid gland, red pulp hyperplasia of the spleen and hyperplasia of the mesenteric lymph nodes. Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical in male and female rats is considered to be 30 and 40 mg/Kg bw respectively.

In another study, Combined repeated dose & carcinogenicity study was performed to determine the chronic nature of the test chemical. The study was performed using 720 male and 720 female B6C3F1 mice for 12, 18, and 24 months of continuous dosing. The test chemical was mixed with feed and given by oral gavage route at dose levels of 0, 10, 300 or 600 ppm (0, 75-100, 225-250, 450-475 mg/Kg bw/week for males and 0, 100, 250-275 or 500 mg/Kg bw/week for females). The animals were observed for Clinical signs, body weight changes, food consumption, clinical chemistry and were subjected to gross and histopathology. There was no effect on food consumption or body weight gain; however, a dose effect was noted for mortality rates. Mortality in the controls of both sexes was less than 15% at 24 months, but was approximately 64% in the females and 23% in the males given the high dose of 450-475 mg/Kg bw/week in males and 500 mg/Kg bw/week in females. Females appeared to be more susceptible than males. A positive dose response for hepatocellular carcinoma was noted in males at 24 months and in females at 18 and 24 months. Statistical tests for dose-related trends with respect to (1) mortality due to liver neoplasms, (2) prevalence of liver neoplasms, and (3) time to onset of liver neoplasms showed positive trends in both males and females. Histopathological examination revealed several liver neoplasms (hepatocellular carcinoma). A slight dose response at 18 months for the liver neoplasms was noted in females and no other dose responses were noted in the 12- or 18- month sacrifice groups. Essentially, all dose-related lesions were noted in the 24-month sacrifice groups. Malignant liver neoplasms occurred with an incidence of 4, 5, 32, and 77% in the female controls, 100, 250-275, and 500- mg/Kg bw/week-dose groups, respectively, by 24 months. A background incidence of 15% was noted in the control males by 24 months compared to an incidence of 17, 18, and 35% in the 75-100, 225-250, and 450-475 mg/Kg bw/week-dose groups, respectively. The incidence in the females by 18 months was 2, 0, 4, and 13% for the controls, 100, 250-275, and 500 mg/Kg bw/week dose groups, respectively. The incidence of liver neoplasms in males by 18 months did not rise above the background levels of 10% observed in the controls. The males demonstrated a lower susceptibility than females to liver carcinogenicity from gentian violet. While the overall comparisons for both malignant tumors alone and malignant plus benign tumors showed a significant positive trend for mortality, prevalence, and onset in both sexes, the pairwise comparisons of doses to controls showed less positive trends in the males. For malignant liver neoplasms, positive increases from control were noted only in prevalence and onset at 500 mg/Kg in females and 450-475 mg/Kg in males. The results for malignant plus benign lesions were essentially the same as malignant only except positive increases from control were noted for prevalence and onset in the 250-275 mg/Kg in females and 225-250 mg/Kg in males also. Dose responses were noted in females for erythropoiesis in the spleen with 7, 16, 20, and 44% responding for the 0-, 100-, 250-275-, and 500-mg/Kg bw/week-dose levels, respectively, and for atrophy of the ovaries with the incidence of 6, 15, 28, and 42%. Significant dose responses were also noted for the occurrence of reticulum cell sarcomas (Type A) in the uterus, vagina, bladder, and ovaries. These lesions were absent from the controls, except for 1/182 for RCS in the vagina. The following incidences were noted for the respective dose levels of 0, 100, 250-275, and 500 mg/Kg bw/week: RCS of the uterus (Type A), 0, 2, 7, and 13%; RCS of the vagina (Type A), 0.5, 1, 5, and 9%; RCS of the bladder (Type A), 0, 2, 3, and 6%; RCS of the ovaries (Type A) 0, 1, 3, and 6%. Adenoma of the Harderian gland was noted in 4, 12, 20, and 16% of the females and in 4, 7, 11, and 10% of the males in the respectively, by 24 months. Lymphoreticular tissue and the liver were the target tissues for tissue morphology. Benign hepatocellular neoplasms (adenomas) were usually small, and they compressed adjacent parenchyma focally. The adenomas were composed of well-differentiated cells in which the cytoplasm was either basophilic, eosinophilic, clear or vacuolated. Little pleomorphism of nuclei was present. The cells were of uniform size and formed either regular cords of not more than two cell layers thick or solid masses. The hepatocellular carcinomas observed were of the trabecular pattern. Hemorrhage and necrosis occurred in some of the cases. The 4 non hepatocellular metastases in the lung were from an undifferentiated sarcoma of muscle and subcutis of the thigh, from a mammary gland tumor, from a Harderian gland tumor, and from a granulosa cell tumor of the right ovary. The other tumor cellular pattern also found was a solid pattern which was composed of either small immature neoplastic hepatocytes or extremely large anaplastic hepatocytes. The cytoplasm was either acidophilic or vacuolated and the nuclei were large with prominent nucleoli. Mitotic figures were not numerous. The RCS (Type A) of the female genital organs was composed of sheets of elongated spindled cells with basophilic ovoid nuclei and scanty acidophilic cytoplasm, involving the wall of the vagina, cervix, and uterus. Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical is considered to be < 100 mg/L when exposed to male and female B6C3F1 mice.

Combined repeated dose & carcinogenicity was performed to determine the toxic nature of the test chemical upon repeated exposure by oral route of exposure. Male and female albino rats were fed the test chemical at dose levels of 100 mg/Kg bw/day using diet for 20-21 months. Auramine was used as a positive control chemical. The animals were observed for mortality, body weight changes, gross pathology and neoplastic and non-neoplastic histopathology. Survival observed in the test animals was comparable to that noted in untreated control group. A less marked reduction in body weight gain was observed in the treated rats as compared to controls. Nodules were observed on the livers only 25% (7/28) of the test rats at the time of final sacrifice which was less as compared to the nodules observed in positive control 59% (20 /34). No other outstanding differences were noted in any of the groups upon gross pathologic examination. There was significant liver injury among animals from both the positive' control and test groups which was attributed to the positive control and test materials.The incidence of hyperplasia observed was comparable in positive control and treated animals. Minor focal lesions of degeneration, necrosis , fatty metamorphosis , inflammation, bile duct proliferation and cholangiofibrosis were present in the liver of control, positive control and test animals. However the severity of these Iesions was generally greater in the livers of the positive control animals than in the Iivers of control or test animals. The lesions noted in other tissues upon microscopic examination of the from control, positive control and test animals were attributed to naturally occurring disease and were considered to be normal for albino rats of this age and strain. Hepatomas were present in 2/9 male and 1/19 female that survived the length of the investigation. There were no hepatomas observed in any of the treated animals that died prior to the conclusion of the study. On the basis of observations made, the no observed adverse effect level (NOAEL) for the test chemical is considered to be 100 mg/Kg bw/day.

Based on the observations made, the No Observed Adverse Effect level (NOAEL) for the test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate in male and female rats is considered to be non toxic upto a dose level of 40 mg/Kg bw.

Repeated dose toxicity: Inhalation

Reaction mass of Ethanaminium, N-[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl- & acetate has very low vapor pressure (9.77E-014 Pa.), so the potential for the generation of inhalable vapours is very low. Also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point was considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate(as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

Based on the data summarized, it can be concluded that the test chemical Reaction mass of Ethanaminium, N-[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl- & acetate can be considered to be safe in a dose range of 40-100 mg/Kg bw and likely to be toxic at higher doses upon repeated exposure by oral route.

Justification for classification or non-classification

Based on the data summarized, it can be concluded that the test chemical Reaction mass of Ethanaminium, N-[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl- & acetate can be considered to be safe in a dose range upto 40-100 mg/Kg bw and likely to be toxic at higher doses upon repeated exposure by oral route.