[4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL was estimated to be 805 mg/kg bw when Sprague-Dawley male and female rats were orally exposed with N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate. 

Based on the below studies and predictions on N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate and its read across substances, it can be concluded that NOAEL value is less than 805 mg/kg bw. Thus, comparing this effect with the criteria of CLP regulation, N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate can be Not classified for reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

Data is predicted using OECD QSAR toolbox version 3.4 and the supporting QMRF report has been attached

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Prediction is done using QSAR Toolbox version 3.4

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of the test material: (4-((4-(Dimethylamino)phenyl)(4-(methylamino)phenyl)methylene)cyclohexa-2,5-dien-1-ylidene)dimethylammonium acetate
- IUPAC name: N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate
- Molecular formula: C24H28N3.C2H3O2
- Molecular Weight: 417.5499 g/mol
- Substance type: Organic
- Smiles: CC(=O)[O-].CNc1ccc(cc1)C(=C2C=CC(=[N+](C)C)C=C2)c3ccc(cc3)N(C)C

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other:

Remarks:

1% Guar Gum in distilled water

Details on exposure:

not specified

Details on mating procedure:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Daily

Details on study schedule:

not specified

Dose / conc.:

805 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals

Control animals:

yes, concurrent vehicle

Details on study design:

not specified

Positive control:

not specified

Parental animals: Observations and examinations:

not specified

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

not specified

Postmortem examinations (parental animals):

not specified

Postmortem examinations (offspring):

not specified

Statistics:

not specified

Reproductive indices:

not specified

Offspring viability indices:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

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

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

reproductive performance

other: No effect observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

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

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

The prediction was based on dataset comprised from the following descriptors: NOAEL
Estimation method: Takes average value from the 5 nearest neighbours
Domain  logical expression:Result: In Domain

(((("a" or "b" )  and ("c" and ( not "d") )  )  and ("e" and ( not "f") )  )  and ("g" and "h" )  )

Domain logical expression index: "a"

Referential boundary: The target chemical should be classified as SN1 AND SN1 >> Nitrenium Ion formation AND SN1 >> Nitrenium Ion formation >> Secondary aromatic amine AND SN1 >> Nitrenium Ion formation >> Tertiary aromatic amine by DNA binding by OECD

Domain logical expression index: "b"

Referential boundary: The target chemical should be classified as AN2 AND AN2 >> Michael-type addition to quinoid structures  AND AN2 >> Michael-type addition to quinoid structures  >> N-Substituted Aromatic Amines by Protein binding by OASIS v1.4

Domain logical expression index: "c"

Referential boundary: The target chemical should be classified as No alert found by DNA binding by OASIS v.1.4

Domain logical expression index: "d"

Referential boundary: The target chemical should be classified as Non-specific OR Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    OR Non-specific >> Incorporation into DNA/RNA, due to structural analogy with  nucleoside bases    >> Specific Imine and Thione Derivatives OR Radical OR Radical >> Generation of ROS by glutathione depletion (indirect) OR Radical >> Generation of ROS by glutathione depletion (indirect) >> Haloalkanes Containing Heteroatom OR Radical >> Radical mechanism via ROS formation (indirect) OR Radical >> Radical mechanism via ROS formation (indirect) >> Nitroaniline Derivatives OR Radical >> Radical mechanism via ROS formation (indirect) >> Specific Imine and Thione Derivatives OR SN1 OR SN1 >> Nucleophilic attack after reduction and nitrenium ion formation OR SN1 >> Nucleophilic attack after reduction and nitrenium ion formation >> Nitroaniline Derivatives OR SN1 >> Nucleophilic substitution on diazonium ion OR SN1 >> Nucleophilic substitution on diazonium ion >> Specific Imine and Thione Derivatives OR SN2 OR SN2 >> Alkylation, nucleophilic substitution at sp3-carbon atom OR SN2 >> Alkylation, nucleophilic substitution at sp3-carbon atom >> Haloalkanes Containing Heteroatom OR SN2 >> Nucleophilic substitution at sp3 Carbon atom OR SN2 >> Nucleophilic substitution at sp3 Carbon atom >> Haloalkanes Containing Heteroatom by DNA binding by OASIS v.1.4

Domain logical expression index: "e"

Referential boundary: The target chemical should be classified as Acetoxy AND Alkene AND Ammonium salt AND Aromatic amine AND Aryl AND Carboxylic acid by Organic Functional groups

Domain logical expression index: "f"

Referential boundary: The target chemical should be classified as Azo by Organic Functional groups

Domain logical expression index: "g"

Parametric boundary:The target chemical should have a value of log Kow which is >= 0.761

Domain logical expression index: "h"

Parametric boundary:The target chemical should have a value of log Kow which is <= 9.91

Conclusions:

NOAEL was estimated to be 805 mg/kg bw when Sprague-Dawley male and female rats were orally exposed with N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate.

Executive summary:

In a prediction done by SSS (2017) using the OECD QSAR toolbox with log kow as the primary descriptor, the acute oral toxicity was estimated for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate. The NOAEL was estimated to be 805 mg/kg bw when Sprague-Dawley male and female rats were orally exposed with N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate. 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

805 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimish 2 and from OECD QSAR toolbox

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity:

In different studies, N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents, i.e. most commonly in rats for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate along with the study available on structurally similar read across substance 1H-Pyrrole-2,5-dione, 1,1'-(methylenedi-4,1-phenylene)bis (CAS no 13646-54-5),C.I. Basic Violet 1(CAS no 8004-87-3) andGentian violet (CAS no 548-62-9).The predicted data using the OECD QSAR toolbox has also been compared with the experimental studies.

In a prediction done by SSS (2017) using the OECD QSAR toolbox with log kow as the primary descriptor, the acute oral toxicity was estimated for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate. The NOAEL was estimated to be 805 mg/kg bw when Sprague-Dawley male and female rats were orally exposed with N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate. 

In another experimental study conducted by Ministry of Economy, Trade and Industry, Japan (Data sheet (in fiscal 2009): prepared by Hazard-Data Evaluation Committee of National Institute of Technology and Evaluation based on the GLP study report obtained by Japanese Ministry of Economy, Trade and Industry. Shin Nippon Biomedical Laboratories, Ltd)on structurally similar read across substance 1H-Pyrrole-2,5-dione, 1,1'-(methylenedi-4,1-phenylene)bis (CAS no 13646-54-5),Crl:CD (SD)male and female rats treated with1H-Pyrrole-2,5-dione, 1,1'-(methylenedi-4,1-phenylene)bisin the concentration of0, 40, 200 and 1000 mg/kg/day orally by gavage in0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution. No effect on survival, clinical signs, body weight, food consumption, behavior test, hematology, blood chemistry, urinalysis, organ weight and gross pathology were observed in treated male and female rats as compared to control. Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw. The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. Focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rate, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, clinical signs, body weights, development and growth of pups and gross pathological changes were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with1H-Pyrrole-2,5-dione, 1,1'-(methylenedi-4,1-phenylene)bisorally by gavage.

 

In another experimental study conducted by Ministry of Economy, Trade and Industry, Japan (Data sheet (in fiscal 2011): prepared by Hazard-Data Evaluation Committee of National Institute of Technology and Evaluation based on the GLP study report obtained by Japanese Ministry of Economy, Trade and Industry. Shin Nippon Biomedical Laboratories, Ltd)on structurally similar read across substanceC.I. Basic Violet 1(CAS no 8004-87-3),Crl:CD (SD)male and female rats treated withC.I. Basic Violet 1 in the concentration of0, 1.6, 8 and 40 mg/kg/day orally by gavage in0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats. Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at 40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity, incomplete eyelid opening and abnormal gait in died animals and no effects on surviving animals were observed. No effect on ureinanalysis was observed in male rats as compared to control. Similarly, Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated withC.I. Basic Violet 1 orally by gavage.

Again supported by experimental study given by FAO/WHO Expert Committee on Food Additives (JECFA) (FAO/WHO Expert Committee on Food Additives (JECFA),Page no.3-34,2004)on structurally similar read across substanceGentian violet (CAS no 548-62-9), 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group. 90 animals of each sex were used in control group. Gentian violet (548-62-9) was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation. In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2a generation. Same procedure was used to produce F2b generation. After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2 male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues. The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and15 mg/kg bw. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group. Also No dose related effects were observed on gross deformities in each generation. The only significant histopathological changes noted in F3a generation were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female). Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen were observed in male and female rats (p <0.001 for male and p <0.00001 for female). Hence, the NOAEL for reproductive toxicity was considered to be 30mg/kg bw/day. While LOAEL for offspring in F3a generation was considered at 5 mg/kg bw/day when male and female Fischer 344 rats were treated with gentian violet orally in feed for 280-294 days.

 

Thus, based on the below studies and predictions on N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate and its read across substances, it can be concluded that NOAEL value is less than 805 mg/kg bw. Thus, comparing this effect with the criteria of CLP regulation, N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate can be Not classified for reproductive toxicity.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the below studies and predictions on N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate and its read across substances, it can be concluded that NOAEL value is less than 805 mg/kg bw. Thus, comparing this effect with the criteria of CLP regulation, N-(4-{[4-(dimethylamino) phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate can be Not classified for reproductive toxicity.