4,11-diamino-2-(3-methoxypropyl)-1H-naphth[2,3-f]isoindol-1,3,5,10(2H)-tetrone

Endpoint summary

Description of key information

FAT 36152/M is considered to have low toxicity by oral route. 

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

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
Female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the body weight of the initially dosed animal.

The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.


Justification
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

For the purpose of the study the test item was freshly prepared, as required, as a dispersion in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

Following a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were tested.

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose. In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated. A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.

Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily.

Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.

At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

No data

Preliminary study:

No data

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period. Black staining of the feces was noted in all animals from Day 1 and up to 5 days after dosing.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

No data

Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Female	0	0	0	0	0F	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-3 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

F =   Black staining of the feces

Individual Body Weights and Body Weight Changes

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
2000	1-0 Female	166	192	203	26	11
	2-0 Female	183	196	203	13	7
	2-1 Female	168	195	196	27	1
	2-2 Female	169	196	207	27	11
	2-3 Female	179	189	209	10	20

 

Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Female	Killed Day 14	No abnormalities detected
	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Killed Day 14	No abnormalities detected
	2-2 Female	Killed Day 14	No abnormalities detected
	2-3 Female	Killed Day 14	No abnormalities detected

Interpretation of results:

not classified

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Unclassified).

Executive summary:

A key study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat according to OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method) and EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure).

  

After a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were given a single oral dose of test item, as adispersioninarachis oil BP, at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

 There were no deaths. Black staining of the feces was noted in all animals on Day 1 and for up to 5 days after dosing. There were no signs of systemic toxicity noted in the additional four treated animals. All animals showed expected gains in body weight. No abnormalities were noted at necropsy.

 

 

Therefore, the acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System-Unclassified).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

The acute oral toxicity of the target chemical (FAT 36152/M) was assessed in the Wistar strain rat according to OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method) and EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure).There were no deaths. Black staining of the feces was noted in all animals on day 1 and for up to 5 days after dosing. There were no signs of systemic toxicity noted in the additional four treated animals. All animals showed expected gains in body weight. No abnormalities were noted at necropsy.  According to the data, the LD50 for FAT 36152/M in rats is >2000 mg/kg.

FAT 36152/E, FAT 36152/B and FAT 36152/A were the mixtures of Disperse Blue 60 long amine and Disperse Blue 60 short amine (target chemical) with the mixture constituting 67.4, 26.6 and 43.5% respectively. FAT 36152/E and FAT 36152/B were found to have LD50 >2000 mg/kg bw. However, FAT 36152/A (mixture purity 43.5%) was reported to have a LD50 of 715 mg/kg bw in females. However, in the most recently conducted studies (2015), FAT 36152/N (Disperse Blue 60 long amine with purity >90%) was found to have a LD50 >2000 mg/kg bw. Similarly FAT 36152/M (Disperse Blue 60 short amine or the target chemical with purity >90%) was also found to have low toxicity with LD50 >2000 mg/kg bw. These results indicate that both chemicals have low acute toxicity poptential when administered orally. In case of FAT 36152/A (as well as FAT 36152/E and FAT 36152/B), the purity of either the Disperse Blue 60 long amine or Disperse Blue 60 short amine is not clear (purity of mixture of Disperse Blue 60 long amine and Disperse Blue 60 short amine is however indicated). No clear inference about the individual toxicity potential of either Disperse Blue 60 long amine and Disperse Blue 60 short amine could be made from the studies with mixtures. Hence, the studies with mixtures were not considered for hazard assessment or classification and most recently conducted study with FAT 36152/M was taken as key study. Based on the results of the key study, target chemical was considered to have a LD50 >2000 mg/kg bw and no classification was considered necessary.

Inhalation:
The test substance has very low vapour pressure (above the lower detection limits) and a melting point of 194 ºC, therefore the potential for the generation of inhalable forms is low. Also all uses considered in the CSR for this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Furthermore, during the acute oral toxicity study in rats, the test substance administration does not exacerbate systemic toxicity effects which suggest a low toxicity potential. Results from the toxicokinetics studies confirmed that upon oral administration the substance is rapidly and completely excreted without potential for bioaccumulation. Owing to the physical chemical properties of the test substance, oral ingestion and subsequent gastro-intestinal absorption is considered to be the most relevant route of exposure. Since this route does not result in systemic toxicity, inhalation exposure is considered to be negligible for systemic toxicity. Therefore the study will be waived and the intrinsic property/toxicity potential can be extrapolated from the acute oral route administration.

Exposure considerations:
Production and spray drying is performed in closed process without isolation of reaction products. Isolated products consist dust free granules (non-dusty solid) or liquid formulations only. In addition the test substance and its related products are marketed for industrial use and professional digital printing applications without inhalation exposure only. Therefore established risk management measures are considered appropriate to enable safe handling of the substance. In summary, also in the light of animal welfare further testing is not considered.

Dermal:
The physicochemical (molecular weight 379.4 g/mol, water solubility <1 mg/L and n-octanol/water partition coefficient >4) and toxicological properties suggest a low potential for significant rate of absorption through the skin. Furthermore, results of laboratory animal studies performed to assess skin irritation and skin sensitization potential displayed no acute dermal toxicity. In addition, the target chemical was found to have low acute toxicity when administered orally with LD50 >2000 mg/kg bw. Results from the toxicokinetics studies confirmed that upon oral administration the substance is rapidly and completely excreted without potential for bioaccumulation. Owing to the physical-chemical properties oral ingestion and subsequent gastro-intestinal absorption is considered to be the most relevant route of exposure. Since this route does not result in systemic toxicity, dermal exposure is considered to be negligible for systemic toxicity. Therefore the study will be waived and the intrinsic property/toxicity potential can be extrapolated from the acute oral route administration.

Exposure considerations: Production and spray drying is performed in closed process without isolation of reaction products. The substance and its related products are only marketed for industrial use and for professional digital printing applications without dermal exposure only. Therefore established risk management measures are considered appropriate to enable safe handling of the substance. In summary also in the light of animal welfare further in vivo testing is not considered appropriate.

Justification for classification or non-classification

Based on the above stated assessment of the acute oral toxicity the substance does not need to be classified for acute oral toxicity according to GLP (Regulation (EC) No. 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.