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

Endpoint summary

Description of key information

Acute oral toxicity: 

Acute oral toxicity dose (LD50) was determined by Sustainability Support Services (Europe) AB and the acute oral LD50 (Cut-off value) was 500 mg/kg bw.It was concluded that LD50 value is between 300 - 2000 mg/kg bw.Thus, acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene] cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Acute inhalation toxicity:

[4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate has very low vapor pressure 5.76E-018Pa, 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:

Acute dermal median lethal dose (LD50) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that [4-[[4-anilino- 1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6) falls into the “Category 5 (>2000)” criteria of CLP.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Principles of method if other than guideline:

The purpose of this study was to assess the Toxicological profile of test item to a single administration via oral route to Sprague Dawley rats. This study was designed to determine the acute toxicity at fixed dose levels by oral route of the test item.

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Test Item: [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) - Source of test material: Sustainability Support Services (Europe) AB, Sweden - Batch No. of test material: KCP/FS//1701/17- Manufacturing Date: April; 2017- Expiration date of the lot/batch: March; 2018- Purity test date: No data available- Consistency: LiquidRADIOLABELLING INFORMATION (not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Test Item and prepared formulation(s) were stored at ambient temperature.- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: The dose of 300 mg/kg of test item was prepared by dilution of the test item in distilled water to obtain 30 mg/ml strength of solution. The formulation was prepared fresh on the day of dosing. The dose of 2000 mg/kg of test item was administered undiluted.- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST (if different from that of starting material): No data availableOTHER SPECIFICS: Safety Precautions: Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

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:

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:

Dose Group I : 300 mg/kgDose Group I : 300 mg/kgDose Group II : 2000 mg/kg

No. of animals per sex per dose:

Three females were used at each step.

Control animals:

not specified

Details on study design:

- 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

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 50% mortality observed

Mortality:

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:

other: Group I Step I :Animals treated at the dose level of 300 mg/kg body weight resulted in diarrhoea and test item coloured faeces with onset at 1 to 4 hours after the dosing. All animals survived through the study period of 14 days and were free of signs of

Gross pathology:

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 available

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Female

Group I :   

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From – to	Mortality
I	300	Diarrhoea	3	1 2 3	1 hr. - Day 1 4 hrs. - Day 1 2 hrs. - Day 1	0/3
		Test item coloured faeces	3	1 2 3	1 hr. - Day 1 4 hrs. - Day 1 2 hrs. - Day 1

 

Group I : 

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From – to	Mortality
II	300	Diarrhoea	3	4,5 6	1 hr. - Day 1 30 min. - 6 hrs.	1/3
		Test item coloured faeces	3	4,5 6	1 hr. - Day 1 30 min. - 6 hrs.
		Reduced locomotor activity	1	6	1 hr. - 6 hrs.

 

Group II :

Step No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days From – to	Mortality
I	2000	Diarrhoea	3	7,8 9	2 hrs. - 6 hrs. 4 hrs. - 6 hrs.	3/3
		Test item coloured faeces	3	7,8 9	2 hrs. - 6 hrs. 4 hrs. - 6 hrs.
		Reduced locomotor activity	3	7,8 9	6 hrs. 1 hr. - 2 hrs.
		Ataxic gait	3	7,8 9	2 hrs. - 6 hrs. 1 hr. - 6 hrs.

 

 

 Table No.II

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Female

Group I :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	300	Mean	202.53	212.03	4.71	224.63	5.95	10.94
		± SD	7.37	6.29	0.72	5.66	0.51	1.28

 

 

Group I :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
II	300	Mean	194.30	202.90	6.10	219.15	8.00	14.58
		± SD	6.20	3.54	0.70	6.43	1.29	0.61

 

 

Group II :

Step No.	Dose (mg/kg body weight)		Before Fasting Body weight	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	2000	Mean	198.70	-	-	-	-	-
		± SD	4.62	-	-	-	-	-

 

 

Table No.III

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Female

Group I :

 

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	300	1, 2, 3	TS	No abnormality detected

 

Group I :

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
II	300	4, 5	TS	No abnormality detected
		6	FD	Stomach distended with bluish discoloured mucosa. Small and large intestine distended with coloured liquid ingesta.

 

Group II :

 

Step No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	2000	7, 8, 9	FD	Stomach distended with bluish discoloured mucosa. Small and large intestine distended with coloured liquid ingesta.

FD = Found dead

TS = Terminal Sacrifice

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Under the condition of the study, the acute oral LD50 (Cut-off value) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) was 500 mg/kg body weight.  Thus, it was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Executive summary:

The study now reported was designed and conducted to determine the acute oral toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) 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. 

The acute oral LD50 (Cut-off value) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) was 500 mg/kg body weight.  

Thus, it was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethyl ammonium acetate (CAS No. 83803-79-6), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

Data is Klimisch 2

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

This study was designed to determine the dermal LD50 of the test item (up to 2000 mg/kg) or to establish a non-lethal dose level of 2000 milligram of test item per kilogram of body weight.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Test Item: [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) - Source of test material: Sustainability Support Services (Europe) AB- Batch No.of test material: KCP/FS//1701/17- Manufacturing Date: April; 2017- Expiration date of the lot/batch: March; 2018- Purity test date: No data available- Consistency: LiquidRADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Ambient Temperature- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: The test item was used undiluted.- Preliminary purification step (if any):No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST:No data availableOTHER SPECIFICS:Safety Precautions : Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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:

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:

24 hours

Doses:

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:

10 (5/sex).

Control animals:

not specified

Details on study design:

- 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

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality observed

Mortality:

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:

other: 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 a

Gross pathology:

Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.

Other findings:

- 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.

Table No. I

Summary of Clinical Signs of Toxicity and Mortality

Test System : Sprague Dawley Rat

Sex : Male

Group  No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No clinical signs observed	5	1 - 5	Day 0 - Day 14	0/5

 

 

Sex : Female

Group  No.	Dose mg/kg	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No clinical signs observed	5	6 - 10	Day 0 - Day 14	0/5

 

  

Table No. II

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group  No.	Dose mg/kg	Dermal Reaction	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No dermal reaction observed	5	1 - 5	Day 0 - Day 14	0/5

 

Sex : Female

Group  No.	Dose mg/kg	Dermal Reaction	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No dermal reaction observed	5	6 - 10	Day 0 - Day 14	0/5

 

 

 

Table No.III

Mean Body Weight and Percent Body Weight Gain (g)

Test System : Sprague Dawley Rat

Sex : Male

Group No.	Dose (mg/kg body weight)		Body weight Day 0	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	2000	Mean	241.18	263.58	9.30	284.84	8.09	18.14
		± SD	6.54	6.02	1.15	3.97	1.25	1.58

 

Sex : Female

Group No.	Dose (mg/kg body weight)		Body weight Day 0	Body weight Day 7	% body weight gain day 0-7	Body weight Day 14	% body weight gain day 7- 14	% body weight gain day 0- 14
I	2000	Mean	221.08	231.26	4.60	241.14	4.27	9.06
		± SD	4.43	5.79	0.78	6.90	0.57	1.14

 

  

 Table No.IV

Summary of Gross Pathological Findings

Test System : Sprague Dawley Rat

Sex : Male

Group No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	2000	1 - 5	TS	No abnormality detected

 

Sex : Female

Group No.	Dose mg/kg	Animal Numbers	Animal Fate	Gross Pathological Findings
I	2000	6 - 10	TS	No abnormality detected

   

 TS = Terminal Sacrifice

 

                        

Interpretation of results:

other: Not Classified

Conclusions:

It was concluded that the acute dermal median lethal dose (LD50) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2 ,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that [4-[[4-anilino- 1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6) does not classify as an acute dermal toxicant.  CLP Classification: “Unclassified”.

Executive summary:

The study now reported was designed and conducted to determine the acute dermal toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6)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. 

It was concluded that the acute dermal median lethal dose (LD50) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6)does not classify as an acute dermal toxicant.  

CLP Classification: “Unclassified”.

Endpoint conclusion

Endpoint conclusion:

no study available

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 2

Additional information

Acute oral toxicity: 

In an experimental study conducted by Sustainability Support Services (Europe) AB (study no.19154, 2017) was designed and conducted to determine the acute oral toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) 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. The acute oral LD50 (Cut-off value) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) was 500 mg/kg body weight.  Thus, it was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethyl ammonium acetate (CAS No. 83803-79-6), when administered via oral route in Sprague Dawley rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

In another study conducted by Sustainability Support Services (Europe) AB has letter of access from BASF SE, (XXIV/56, 1976) was designed and conducted to determine the acute oral toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) in 50 male/female rats by using the methods described in OED Guideline 401. The test material was diluted in water and administered via oral gavage route at the concentration of 464, 681, 1000, 4640 and 6810 mg/kg bw. Animals were observed for clinical signs, mortality, body weight, pathology for 7 days. At 6810, 4640 and 1000 mg/kg bw: 10/10 animals died; At 681 mg/kg bw: 7/10 animals died; and At 464 mg/kg bw: 2/10 animals died. Animals showed clinical signs such as, dyspnea, apathy, blue feces, acute dilatation in heart and diarrhea.Weight loss was observed in animals.Hence, the LD50 was considered to be 588 mg/kg bw. It was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethyl ammonium acetate (CAS No. 83803-79-6), when administered via oral route in rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

This study is supported by Sustainability Support Services (Europe) AB has letter of access from BASF AG, Department of Toxicology, Ludwigshafen, Germany (78/512, 1983) was designed and conducted to determine the acute oral toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethyl ammonium acetate (CAS No. 83803-79-6) in 30 Sprague-Dawley male/female rat. The test material was diluted in water and administered via oral gavage route at the concentration of 681, 1000 and 1470 mg/kg bw. Animals were observed for clinical signs, mortality, body weight, pathology for 14 days. At 1470 mg/kg bw: 9/10 animals died; At 1000 mg/kg bw: 6/10 animals died; and At 681 mg/kg bw: 1/10 animals died after 14 days. Animals showed clinical signs such as,dyspnea, stertor, apathy, abnomal position, staggering, blue faeces, blue urine, exsiccosis, exopthalamus, paresis, and poor general state. Dead animals showed: acute right diluatation in heart, acute congestive hyperemia; peripheral obule marking in liver; In stomach/intestine: mucosa stained by the test substance; intestine; atonic; pale grey muscle were observed. In sacrificed animals: nothing abnormal detected in organs (n.a.d.). Hence, the LD50 was considered to be 965 mg/kg bw.It was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene] dimethyl ammonium acetate (CAS No. 83803-79-6), when administered via oral route in rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

In another study conducted bySustainability Support Services (Europe) AB has letter of access from INBIFO, Institut für biologische Forschung, Köln (77/461, 1978), for the structurally similar read across substance [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride (2580-56-5) in Sprague-Dawley male/female rat. The test substance at the concentrations used: 10, 31.6, 100, 178 mg/l was diluted in water at application volume: 31.6 ml/kg and administered via oral gavage route at the dose of 316, 1000, 3160, and 5620 mg/kg bw. Animals were observed for clinical signs, mortality, body weight, pathology for 14 days.Body weight was determined shortly before and 7 and 8 days after administration. Clinical symptoms and mortality was recorded as < 15 min, 15 min, 30 min, 1 h, 2 h, 4 h, 5 h and 24 h after administration, then at least daily until day 14. At 316 mg/kg: no deaths; At 1000 mg/kg: 6/10; At 3160 mg/kg: 9/10; At 5620 mg/kg: 5/5 after 14 days of observation. Clinical signs such as, apathy, tight palpebral fissures, decreased and shapeless faeces, squeaking and stertorous respiratory sounds. Among the animals of the highest dose group gasping, muscular hypotonia, and a reduced reaction to pain were also observed. 2 days after administration, the majority of surviving animals were without findings. Body weight gain was observed. Animals that died: In some cases the adrenal glands and kidneys were discoloured and bluish; in 2 animals the liver was blackish; in 2 animals the spleen was considerably diminished; in another rat spleen and thymus were considerably diminished, and the adipose tissue extremely reduced; in one animal the adipose tissue was considerably reduced. Animals sacrificed at study termination: In one animal the spleen was considerably enlarged. All other animals were without findings. Therefore, the LD50 was considered to be 1037.0 mg/kg bw.It was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride (2580-56-5), when administered via oral route in rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

The above study is further supported by Sustainability Support Services (Europe) AB has letter of access from BASF AG, Department of Toxicology, Ludwigshafen (XVII/198, 1967), for the structurally similar read across substance [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride (2580-56-5) in male/female "US" rats. The test substance was diluted in water at concentrations used were 10, 16 and 30% (G/V) and administered via oral gavage route at the dose of 1000, 1600 and 3200 mg/kg bw. Animals were observed for clinical signs, mortality, body weight, pathology for 7 days.At1000 mg/kg: 1/10 animals died; at 1600 mg/kg: 5/10 animals died; and at 3200 mg/kg: 9/10 animals died after 7 days of observation.Clinical signs was observed after 4 hours long-legged gait, intermittently, partly accelerated breathing, restlessness, partly diarrhea. Female animals showed dark-green coloured feces and urine. After one day all animals showed a dark-green colouration of feces and urine, accelerated breathing, partly crouched position. One animal of the 1000 mg/kg dose group showed dyspnea. After 4 - 6 days all surviving animals were without symptoms.Animals that died showed blue colouration of the gastro-intestinal tract, slight blue-smeared ani and snouts. Sacrificed animals showed: slight blue up to greenish colouration parts on the tails. Hence, the LD50 was considered to be 1600 mg/kg bw.It was concluded that the acute toxicity study of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride (2580-56-5), when administered via oral route in rats falls into the “Category 4 (300 – ≤ 2000)” criteria of CLP.

Acute inhalation toxicity:

[4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene] cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate has very low vapor pressure 5.76E-018Pa, 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:

In a experimental study conducted by Sustainability Support Services (Europe) AB (report no.19155, 2017) was designed and conducted to determine the acute dermal toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) 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. It was concluded that the acute dermal median lethal dose (LD50) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered to male and female Sprague Dawley rats was found to be greater than 2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6) falls into the “Category 5 (>2000)” criteria of CLP.

In another experimental study conducted by Sustainability Support Services (Europe) AB has letter of access from BASF AG, Department of Toxiclolgy, Ludwigshafen, Germany (Report no. XXIV/56, 1976) was designed and conducted to determine the acute dermal toxicity profile of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) in Sprague Dawley rats. The test item was applied to the 50 cm² area of skin of 5 male/female animals at 5000 mg/kg body weight. Administration of the test item at 5000 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.No abnormalities were noted at necropsy.All animals obtained local staining (dark blue) from residual substance. It was concluded that the acute dermal median lethal dose (LD50) of [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6), when administered to male and female Sprague Dawley rats was found to be 5000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene] dimethylammonium acetate (CAS No. 83803-79-6) falls into the “Category 5 (>2000)” criteria of CLP.

Justification for classification or non-classification

Thus, based on the above study on [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate (CAS No. 83803-79-6) and its read across substance, it can be concluded that LD50 value is between 300 - 2000 mg/kg bw for acute oral toxicity and is greater 2000 mg/kg bw for acute dermal toxicity. Thus, comparing this value with the criteria of CLP regulation, [4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl] methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate can be classified as category 4 for acute oral and category 5 dermal toxicity. For Acute inhalation toxicity wavier were added so, not possible to classify.