Registration Dossier

Administrative data

Description of key information

Acute oral Toxicity: 

The acute oral toxicity dose (LD50) for Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate was based on data available for the structurally similar read across chemicals. The LD50 value is between 300-2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified as “Category IV” for acute oral toxicity. 

Acute Inhalation Toxicity: 

Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate has very low vapor pressure (1.08E-010 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.

Acute dermal Toxicity: 

The acute dermal toxicity dose (LD50) for Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate was based on data available for the structurally similar read across chemicals. The LD50 value is greater than 2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate cannot be classified for acute dermal toxicity. 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
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:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on two acute oral toxicity studies as- 1.and 2. Acute Oral toxicity test was carried out to study the effects of the test chemicals on rodents
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
- Name of test material: Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate- Molecular formula: C25H28N2O2- Molecular weight: 388.508 g/mole- Smiles : [N+](=C1/C=CC(\C=C1)=C(\c1ccccc1)c1ccc(cc1)N(C)C)(\C)C.C(C)(=O)[O-]- Inchl: 1S/C23H25N2.C2H4O2/c1-24(2)21-14-10-19(11-15-21)23(18-8-6-5-7-9-18)20-12-16-22(17-13-20)25(3)4;1-2(3)4/h5-17H,1-4H3;1H3,(H,3,4)/q+1;/p-1- Substance type: Organic- Physical state: maroon lustrous liquid
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
1.TEST ANIMALS- Source: National Institute of Biosciences, Pune.- Females nulliparous and non-pregnant: yes- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing.- Weight at study initiation: Body weight range was 196.8 to 204.0 grams.Body weights at the start :FemaleMean : 199.92 g (= 100 %)Minimum : 196.8 g (- 1.56 %)Maximum : 204.0 g (+ 2.04 %)Total No. of animals : 12- Identification: Each female rat was individually identified by the picric acid marking.- Fasting period before study: Approximately 16 hours or more.- Housing: The rats were housed in polycarbonate cages.- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders. - Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.- Acclimation period: 5 days.ENVIRONMENTAL CONDITIONS- Temperature (°C): 20.1 to 22.1 degree centigrade.- Humidity (%): 56.1% to 61.1%- Air changes (per hr): Ten to fifteen air changes per hour.- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.IN-LIFE DATES: 10-07-2017 to 28-07-20172.TEST ANIMALS- Source: National Institute of Biosciences, Pune.- Females nulliparous and non-pregnant: yes- Age at study initiation: Female rats of the age of approximately 8 to 12 weeks old were used at the commencement of its dosing.- Weight at study initiation: Body weight range was 188.0 to 208.2 grams.Body weights at the start:Female Mean : 198.51 g (= 100 %)Minimum : 188.0 g (- 5.29 %)Maximum : 208.2 g (+ 4.88 %)Total No. of animals : 9- Identification: Each female rat was individually identified by the picric acid marking.- Fasting period before study: Approximately 16 hours or more.- Housing: The rats were housed in polycarbonate cages.- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders. - Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.- Acclimation period: 5 days.ENVIRONMENTAL CONDITIONS- Temperature (°C): 20.1 to 22.3 degree centigrade.- Humidity (%): 55.1% to 59.3%- Air changes (per hr): Ten to fifteen air changes per hour.- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.IN-LIFE DATES: 21-06-2017 to 08-07-2017
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Distilled water)
Details on oral exposure:
1.VEHICLE- Concentration in vehicle: 300 mg/kg, 300 mg/kg, 2000 mg/kg and 2000 mg/kg- Amount of vehicle (if gavage): No data- Justification for choice of vehicle: No data- Lot/batch no. (if required): No data- Purity: No dataMAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight.DOSAGE PREPARATION (if unusual): No data availableCLASS METHOD (if applicable)- Rationale for the selection of the starting dose: No data available2.VEHICLE- Concentration in vehicle: 300 mg/kg, 300 mg/kg and 2000 mg/kg- Amount of vehicle (if gavage): No data available- Justification for choice of vehicle: No data available- Lot/batch no. (if required): No data available- Purity: No data availableMAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight.DOSAGE PREPARATION (if unusual): No data availableCLASS METHOD (if applicable) - Rationale for the selection of the starting dose: No data available
Doses:
1.Dose Group I : 300 mg/kgDose Group I : 300 mg/kgDose Group II : 2000 mg/kgDose Group II : 2000 mg/kg2.Dose Group I : 300 mg/kgDose Group I : 300 mg/kgDose Group II : 2000 mg/kg
No. of animals per sex per dose:
1.Three females were used at each step.2.Three females were used at each step.
Control animals:
not specified
Details on study design:
1.- Duration of observation period following administration: 14 days - Frequency of observations and weighing: Twice daily- Necropsy of survivors performed: Yes- Other examinations performed: Clinical Observations and General Appearance:Animals were observed for clinical signs, mortality and morbidity, until sacrifice.Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern. Body weights:Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.Gross Pathology:Necropsy was performed on all animals, found dead and sacrificed at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique. Histopathology:Gross pathological examination revealed stomach, small and large intestine with test item coloured mucosa in animals from 2000 mg/kg dose group, colouration imparted due to the local irritation of the test item and no other systemic abnormality observed hence, no organ collected for histopathology.2.- Duration of observation period following administration: 14 days - Frequency of observations and weighing: Twice daily- Necropsy of survivors performed: Yes- Other examinations performed: Clinical Observations and General Appearance:Animals were observed for clinical signs, mortality and morbidity, until sacrifice.Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at immediately (0 to 5 minutes), 5, 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern. Body weights:Individual animal body weights were recorded, before fasting, prior to administration of the test item (fasting body weights), weekly thereafter and at termination on day 14. Weight changes were calculated and recorded.Gross Pathology:Necropsy was performed on all animals, found dead and sacrificed at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15). Histopathology:Gross pathological examination revealed stomach distended with bluish discoloured mucosa and small and large intestine distended with coloured liquid ingesta and no gross abnormality observed except colouration hence, no organ collected for histopathology.
Statistics:
no data
Preliminary study:
no data
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
500 mg/kg bw
Based on:
test mat.
Remarks on result:
other: 50% mortality was observed
Mortality:
1.Group I Step I :Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days.Group I Step II :Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days.Group II Step I :Animals treated at the dose level of 2000 mg/kg body weight: One animal died at 24 hours after the dosing.Group II Step II :Animals treated at the dose level of 2000 mg/kg body weight: One animal died on day 3, one animal died on day 5 and one animal died on day 10 after the dosing.2.Group I Step I :Animals treated at the dose level of 300 mg/kg body weight: All animals survived through the study period of 14 days.Group I Step II :Animals treated at the dose level of 300 mg/kg body weight: One animal died on day 1 after the dosing. Group II Step I :Animals treated at the dose level of 2000 mg/kg body weight: Three animals died on day 1 after the dosing.
Clinical signs:
1.Group I :Step I :Animals treated at the dose level of 300 mg/kg bw resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 2 hrs after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing. Group I:Step II :Animals treated at the dose level of 300 mg/kg bw resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 1 hour after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity on day 1 after the dosing.Group II :Step I :Animals treated at the dose level of 2000 mg/kg bw resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 6 hours after the dosing. All surviving animals were free of signs of toxicity on day 7 after the dosing.Group II:Step II :Animals treated at the dose level of 2000 mg/kg bw resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 4 hours after the dosing. 2.Group I:Step I :Animals treated at the dose level of 300 mg/kg bw resulted in diarrhoea and test item coloured faeces with onset at 1 to 4 hrs after the dosing. All animals survived through the study period of 14 days and were free of signs of toxicity at on day 2 after the dosing.Group I:Step II :Animals treated at the dose level of 300 mg/kg bw resulted in diarrhoea, test item coloured faeces and reduced locomotor activity with onset at 30 minutes to 1 hour after the dosing. All surviving animals were free of signs of toxicity on day 2 after the dosing.Group II:Step I :Animals treated at the dose level of 2000 mg/kg bw resulted in diarrhoea, test item coloured faeces, reduced locomotor activity and ataxic gait with onset at 1 to 6 hours after the dosing.
Body weight:
1.Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.07% and 18.34% respectively. Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 8.68% and 16.75% respectively. Group II Step I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.77% and 10.96% respectively. Group II Step II (2000 mg/kg) - All animals died, hence, body weight gain could not be calculated. 2.Group I Step I (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.71% and 10.94% respectively. Group I Step II (300 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 6.10% and 14.58% respectively. Group II Step I (2000 mg/kg) - All animals died on day 1, hence, body weight gain could not be calculated.
Gross pathology:
1.Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg dose group.Gross pathological examination revealed stomach, small and large intestine with test item coloured mucosa in animals sacrificed terminally from 2000 mg/kg dose group and stomach distended with test item coloured mucosa and small and large intestine distended with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group. 2.Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg dose group.Gross pathological examination revealed stomach distended with bluish discoloured mucosa and small and large intestine distended with coloured liquid ingesta in found dead animals from 300 mg/kg and 2000 mg/kg dose groups.
Other findings:
no data
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
According to CLP regulation, the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified as “category IV” for acute oral toxicity, as the LD50 value is between 300-2000 mg/kg bw.
Executive summary:

Data available for the structurally similar read across chemicals has been reviewed to determine the acute oral toxicity of the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.The acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 2 hours after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 1 hour after the dosing and no mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 6 hours after the dosing. One animal died at 24 hours after the dosing. As one mortality was observed at 24 hours after the dosing, additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 4 hours after the dosing. One animal died on day 3, one animal died on day 5 and one animal died on day 10 after the dosing. All animals from 300 mg/kg dose group survived through the study period of 14 days and four animals died from 2000 mg/kg dose group after the dosing. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg dose group.Gross pathological examination revealed stomach, small and large intestine with test item coloured mucosa in animals sacrificed terminally from 2000 mg/kg dose group and stomach distended with test item coloured mucosa and small and large intestine distended with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group.Hence, The lethal concentration (LD50-Cut-off value) value for acute oral toxicity test was considered to be 500 mg/kg bw,when female Sprague Dawley rats were treated with test chemical orally via gavage according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method).

2.The acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in diarrhoea and test item coloured faeces with onset at 1 to 4 hours after the dosing and no mortality after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in diarrhoea, test item coloured faeces and reduced locomotor activity with onset at 30 minutes to 1 hour after the dosing. One animal died on day 1 after the dosing. One mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in diarrhoea, test item coloured faeces, reduced locomotor activity and ataxic gait with onset at 1 to 6 hours after the dosing. Three animals died on day 1 after the dosing. All surviving animals from 300 mg/kg dose group exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg dose group. Gross pathological examination revealed stomach distended with bluish discoloured mucosa and small and large intestine distended with coloured liquid ingesta in found dead animals from 300 mg/kg and 2000 mg/kg dose groups.Hence, The lethal concentration (LD50-Cut-off value) value for acute oral toxicity test was considered to be 500 mg/kg bw,when female Sprague Dawley rats were treated with test chemical orally via gavage according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method).

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that LD50 value is between 300 -2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified as “category IV” for acute oral toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
500 mg/kg bw
Quality of whole database:
Data is Klimisch 1 and from study report

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Quality of whole database:
Waiver

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on two acute oral toxicity studies as- 1.and 2. Acute Oral toxicity test was carried out to study the effects of the test chemicals on rodents
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
- Name of test material: Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate- Molecular formula: C25H28N2O2- Molecular weight: 388.508 g/mole- Smiles : [N+](=C1/C=CC(\C=C1)=C(\c1ccccc1)c1ccc(cc1)N(C)C)(\C)C.C(C)(=O)[O-]- Inchl: 1S/C23H25N2.C2H4O2/c1-24(2)21-14-10-19(11-15-21)23(18-8-6-5-7-9-18)20-12-16-22(17-13-20)25(3)4;1-2(3)4/h5-17H,1-4H3;1H3,(H,3,4)/q+1;/p-1- Substance type: Organic- Physical state: maroon lustrous liquid
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
1.TEST ANIMALS- Source: National Institute of Biosciences, Pune.- Females (if applicable) nulliparous and non-pregnant: No data available- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.- Weight at study initiation: The weight range of approximately 218.6 to 246.8 grams at initiation of dosing. Body weights at the start : MaleMean : 244.22 g (= 100 %)Minimum : 241.7 g (- 1.03 %)Maximum : 246.8 g (+ 1.06 %)Total No. of animals : 5FemaleMean : 221.38 g (= 100 %)Minimum : 218.6 g (- 1.26 %)Maximum : 225.6 g (+ 1.91 %)Total No. of animals : 5- Identification: Each rat was individually identified by the cage number.- Fasting period before study: No data available- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding. - Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.- Acclimation period: 5 days.ENVIRONMENTAL CONDITIONS- Temperature (°C): 19.7 to 21.8 degree centigrade.- Humidity (%): 56.2% to 60.1%- Air changes (per hr): Ten to fifteen air changes per hour.- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.IN-LIFE DATES: 13-07-2017 to 28-07-20172.TEST ANIMALS- Source: National Institute of Biosciences, Pune.- Females nulliparous and non-pregnant: No data available- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.- Weight at study initiation: The weight range of approximately 216.9 to 251.9 grams at initiation of dosing. Body weights at the start : MaleMean : 241.18 g (= 100 %)Minimum : 235.8 g (- 2.23 %)Maximum : 251.9 g (+ 4.44 %)Total No. of animals : 5FemaleMean : 221.08 g (= 100 %)Minimum : 216.9 g (- 1.89 %)Maximum : 228.5 g (+ 3.36 %)Total No. of animals : 5- Identification: Each rat was individually identified by the cage number.- Fasting period before study: No data available- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding. - Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.- Acclimation period: 5 days.ENVIRONMENTAL CONDITIONS- Temperature (°C): 19.7 to 21.8 degree centigrade.- Humidity (%): 56.2% to 60.1%- Air changes (per hr): Ten to fifteen air changes per hour.- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.IN-LIFE DATES: 11-07-2017 to 26-07-2017
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
1.TEST SITE - Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area) - % coverage: Approximately 10% of the body surface area. - Type of wrap if used: Porous gauze dressing and non-irritating tape. REMOVAL OF TEST SUBSTANCE - Washing (if done): Distilled water was used to remove residual test item. TEST MATERIAL - Amount(s) applied (volume or weight with unit): 2000 mg/kg bw - Constant volume or concentration used: No data available - For solids, paste formed: No data available VEHICLE - Amount(s) applied (volume or weight with unit): No data available - Concentration (if solution): No data available - Lot/batch no. (if required): No data available - Purity: No data available2.TEST SITE - Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area) - % coverage: Approximately 10% of the body surface area. - Type of wrap if used: Porous gauze dressing and non-irritating tape. REMOVAL OF TEST SUBSTANCE - Washing (if done): Distilled water was used to remove residual test item. TEST MATERIAL - Amount(s) applied (volume or weight with unit): 2000 mg/kg bw - Constant volume or concentration used: No data available - For solids, paste formed: No data available VEHICLE - Amount(s) applied (volume or weight with unit): No data available - Concentration (if solution): No data available - Lot/batch no. (if required): No data available - Purity: No data available
Duration of exposure:
1.24 hours2.24 hours
Doses:
1.A single dose of 2000 mg of the test item per kilogram of body weight was administered to ten rats (five males and five females). 2.A single dose of 2000 mg of the test item per kilogram of body weight was administered to ten rats (five males and five females).
No. of animals per sex per dose:
1.10 (5/sex). 2.10 (5/sex).
Control animals:
not specified
Details on study design:
1.- Duration of observation period following administration: 14 days - Frequency of observations and weighing: Twice daily- Necropsy of survivors performed: Yes- Other examinations performed: Clinical Observations and General Appearance:Animals were observed for clinical signs, mortality, until sacrifice.Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern. Evaluation of Dermal Reaction:Dermal reaction was observed daily for study period of 14 days. Body weights:Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14. Gross Pathology:Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15). Histopathology:No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.2.- Duration of observation period following administration: 14 days - Frequency of observations and weighing: Twice daily- Necropsy of survivors performed: Yes- Other examinations performed: Clinical Observations and General Appearance:Animals were observed for clinical signs, mortality, until sacrifice.Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern. Evaluation of Dermal Reaction:Dermal reaction was observed daily for study period of 14 days. Body weights:Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14. Gross Pathology:Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15). Histopathology:No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed.
Statistics:
no data
Preliminary study:
no data
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality was observed
Mortality:
1.Sex : MaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.Sex : FemaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight: All animals survived through the study period of 14 days.2.Sex : MaleGroup I - All animals survived through the study period of 14 days.Sex : FemaleGroup I - All animals survived through the study period of 14 days.
Clinical signs:
1.Sex : MaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. Sex : FemaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. 2.Sex : MaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. Sex : FemaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days.
Body weight:
1.Sex : MaleGroup I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 19.64% and 29.31% respectively. Sex : FemaleGroup I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 11.09% and 15.97% respectively. 2.Sex : MaleGroup I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.30% and 18.14% respectively. Sex : FemaleGroup I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.60% and 9.06% respectively.
Gross pathology:
1.Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.2.Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.
Other findings:
1.- Other observations:Evaluation of Dermal ReactionSex : MaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days. Sex : FemaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days. 2.- Other observations:Evaluation of Dermal ReactionSex : MaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days. Sex : FemaleGroup I - Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Interpretation of results:
other: not classified
Conclusions:
According to CLP regulation,the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate cannot be classified for acute dermal toxicity, as the LD50 value is >2000 mg/kg bw.
Executive summary:

Data available for the structurally similar read across chemicals has been reviewed to determine the acute dermal toxicity of the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate .The studies are as mentioned below:

1.The acute dermal toxicity profile of test chemical in Sprague Dawley rats.The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.Animals exhibited normal body weight gain through the study period of 14 days.Gross pathological examination did not reveal any abnormalities attributable to the treatment. Hence, The LD50 value was considered to be >2000 mg/kg bw,when male and female Sprague Dawley rats were semiocclusively treated with test chemical  by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

2.The acute dermal toxicity profile of test chemical in Sprague Dawley rats. The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.Animals exhibited normal body weight gain through the study period of 14 days.Gross pathological examination did not reveal any abnormalities attributable to the treatment.Hence, The LD50 value was considered to be >2000 mg/kg bw,when male and female Sprague Dawley rats were semiocclusively treated with test chemical  by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

Thus, based on the above summarised studies,Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that LD50 value is greater than 2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation,Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate cannot be classified for acute dermal toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Data is Klimisch 1 and from study report

Additional information

Acute oral Toxicity: 

Data available for the structurally similar read across chemicals has been reviewed to determine the acute oral toxicity of the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.The acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 2 hours after the dosing and no mortality at 24 hours after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 1 hour after the dosing and no mortality after the dosing. No mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 6 hours after the dosing. One animal died at 24 hours after the dosing. As one mortality was observed at 24 hours after the dosing, additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - II). Administration of the test item at 2000 mg/kg resulted in salivation, distension, diarrhoea, reduced locomotor activity, ataxic gait and test item coloured feces with onset at 30 minutes to 4 hours after the dosing. One animal died on day 3, one animal died on day 5 and one animal died on day 10 after the dosing. All animals from 300 mg/kg dose group survived through the study period of 14 days and four animals died from 2000 mg/kg dose group after the dosing. Gross pathological examination did not reveal any abnormalities in animals from 300 mg/kg dose group.Gross pathological examination revealed stomach, small and large intestine with test item coloured mucosa in animals sacrificed terminally from 2000 mg/kg dose group and stomach distended with test item coloured mucosa and small and large intestine distended with liquid test item coloured ingesta in found dead animals from 2000 mg/kg dose group.Hence, The lethal concentration (LD50-Cut-off value) value for acute oral toxicity test was considered to be 500 mg/kg bw,when female Sprague Dawley rats were treated with test chemical orally via gavage according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method).

2.The acute oral toxicity profile of test chemical in Sprague Dawley rats. Initially, three female animals were treated at the dose level of 300 mg/kg body weight of the test item (Step - I). Administration of the test item at 300 mg/kg resulted in diarrhoea and test item coloured faeces with onset at 1 to 4 hours after the dosing and no mortality after the dosing. As no mortality was observed at 24 hours after the dosing, three female animals were added to the study and treated with the same dose of 300 mg/kg of the test item (Step - II). Administration of the test item at 300 mg/kg resulted in diarrhoea, test item coloured faeces and reduced locomotor activity with onset at 30 minutes to 1 hour after the dosing. One animal died on day 1 after the dosing. One mortality was observed at 300 mg/kg dose group, hence additional three female animals were treated with the higher dose of 2000 mg/kg of the test item (Step - I). Administration of the test item at 2000 mg/kg resulted in diarrhoea, test item coloured faeces, reduced locomotor activity and ataxic gait with onset at 1 to 6 hours after the dosing. Three animals died on day 1 after the dosing. All surviving animals from 300 mg/kg dose group exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities in animals sacrificed terminally from 300 mg/kg dose group. Gross pathological examination revealed stomach distended with bluish discoloured mucosa and small and large intestine distended with coloured liquid ingesta in found dead animals from 300 mg/kg and 2000 mg/kg dose groups.Hence, The lethal concentration (LD50-Cut-off value) value for acute oral toxicity test was considered to be 500 mg/kg bw,when female Sprague Dawley rats were treated with test chemical orally via gavage according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method).

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that LD50 value is between 300 -2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified as “category IV” for acute oral toxicity.

Acute Inhalation Toxicity: 

Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate has very low vapor pressure (1.08E-010 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.

Acute dermal Toxicity: 

Data available for the structurally similar read across chemicals has been reviewed to determine the acute dermal toxicity of the Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate .The studies are as mentioned below:

1.The acute dermal toxicity profile of test chemical in Sprague Dawley rats.The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.Animals exhibited normal body weight gain through the study period of 14 days.Gross pathological examination did not reveal any abnormalities attributable to the treatment. Hence, The LD50 value was considered to be >2000 mg/kg bw,when male and female Sprague Dawley rats were semiocclusively treated with test chemical  by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

2.The acute dermal toxicity profile of test chemical in Sprague Dawley rats. The test item was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days.Animals exhibited normal body weight gain through the study period of 14 days.Gross pathological examination did not reveal any abnormalities attributable to the treatment.Hence, The LD50 value was considered to be >2000 mg/kg bw,when male and female Sprague Dawley rats were semiocclusively treated with test chemical  by dermal application following 14 days of observation period according to OECD Guideline 402 (Acute Dermal Toxicity).

Thus, based on the above summarised studies,Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that LD50 value is greater than 2000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation,Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetatecannot be classified for acute dermal toxicity.

Justification for classification or non-classification

Based on the above experimental studies on Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substances, it can be concluded that LD50 value is between 300-2000 mg/kg bw for acute oral toxicity and >2000 mg/kg bw for acute dermal toxicity.Thus, comparing this value with the criteria of CLP regulation,Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified as “category IV” for acute oral toxicity and cannot be classified for acute dermal toxicity.For Acute inhalation toxicity wavier was added so, not possible to classify.