1,4-diamino-2,3-diphenoxyanthraquinone

Endpoint summary

Description of key information

Several valid acute oral toxicity studies according or similar to OECD guideline 401 are available. The LD50 value in all studies was greater than 5000 mg/kg bw (discriminating dose).

In a valid acute inhalation study according to OECD guideline 436 a LC50 greater than 5.04 mg/l (discriminating dose) was determined.

No study for acute dermal toxicity is available: According to Commission Regulation (EU) 2016/863 of May 2016 acute toxicity testing by the dermal route (Annex VII, point 8.5.3., column 2) ‘does not need to be conducted if the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure’. The registered substance conforms with the requirements given above. Therefore, it can be concluded for acute dermal toxicity that the available information is conclusive for non-classification.

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:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

Five male and five female rats received a single dose of 5000 mg/kg bw Resolin Rotviolett FBL fl., 3. Umstellung by gavage. After the application the animals were observed for mortality, clinical signs and body weight for 14 days. A necropsy was performed on the surviving animals at the end of the study.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The acute toxicity experiment was carried out with SPF-bred Wistar rats (strain Bor: WISW (SPF Cpb). At the start of study the male rats were about 9 weeks old and females 14 weeks; the mean initial weight of males was 183 and that of females 175 g.
The rats were housed in groups of five animals each under conventional conditions in Makrolon Type-III cages on dust-free wood granules; they were exposed to a room temperature of 22 ± 2° C, a 12-hour light/dark cycle (artificial light from 6 a.m. to 6 p.m. CET), and relative humidity of about 50 ± 10 %.
During the study period the animals received tap water ad libitum. Feed was withdrawn from approx. 16 hours prior to until approx. 4 hours after application. The other time it was available ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test substance was prepared immediately before the application.

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 male and 5 female rats/dose

Control animals:

no

Details on study design:

The animals were inspected several times on the day of administration, and twice daily during the following 14-day observation period (once on Weekends and bank holidays). During inspections, the type, onset, duration, and intensity of clinical signs were recorded and dead animals removed if necessary. The animals were individually weighed at application, after one week and at the end of the 14-day observation period. All animals which died intercurrent or were sacrificed at the end of study were necropsied.

Key result

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

5 000 mg/kg bw

Based on:

test mat.

Mortality:

none of the rats died.

Clinical signs:

other: No signs of poisoning were observed.

Gross pathology:

No adverse findings were observed.

Interpretation of results:

GHS criteria not met

Conclusions:

LD50 was greater than 5000 mg/kg bw (discriminating dose).

Executive summary:

A single dose of 5000 mg/kg bw of the test substance was applied to 5 male and 5 female Wistar rats per gavage. The animals were inspected several times on the day of administration, and twice daily during the following 14-day observation period. The dose of 5000 mg/kg body weight was tolerated without adverse effects by all male and female animals.

No adverse gross pathological findings were observed. The LD50 is greater than 5000 mg/kg bw (discriminating dose).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Reliability 1 (reliable without restriction)

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 09 May 2016 Experimental completion date: 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Identification: Macrolex Rotviolett R
Physical state/appearance: Violet solid
CAS Number: 6408-72-6
Expiry: 28 July 2017
Storage/Usage Conditions: Stored in darkness at room temperature; may be used/formulated in light

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.
Animal Care and Husbandry
The animals were housed in groups of up to three by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, 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 and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
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.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.48 µm

Geometric standard deviation (GSD):

3.12

Remark on MMAD/GSD:

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm was calculated.
The resulting values were converted to probits and plotted against Log10 cut point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

Details on inhalation exposure:

Atmosphere Generation
A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.

Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410.

The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.

Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied in an attempt to achieve the required atmospheric conditions.

Exposure Procedure
One day prior to the day of exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
Following an appropriate equilibration period a single group of six rats (three males and three females) was exposed to an atmosphere of the test item for a period of 4 hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 101 % of target and no deaths occurred, no further levels were required.

Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4 Hour exposure period.

Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
The actual chamber concentration was measured at regular intervals during each exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.

Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.

The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.

The nominal concentration was 242 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric method

Duration of exposure:

4 h

Concentrations:

5.00 mg/L

No. of animals per sex per dose:

3 males at 5.00 mg/L
3 females at 5.00 mg/L

Control animals:

no

Details on study design:

Serial Observations
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.
Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
Terminal Investigations
Necropsy
At the end of the 14 day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.04 mg/L air

Based on:

test mat.

Mortality:

There was no mortality at Mean Achieved Atmosphere Concentration 5.04 (mg/L)

Clinical signs:

other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. Generalized fur stai

Body weight:

All animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period. In contrast, two female animals showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.

Gross pathology:

No macroscopic abnormalities were detected amongst animals at necropsy.

Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were as follows:

The mean values obtained were as follows:

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.04	0.12	12.2

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T₉₉) was 3 minutes*(Silver, 1946).

 Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone was as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
5.04	3.48	54.9	3.12

*= Test atmospheres were generated for a total of 17 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

Interpretation of results:

GHS criteria not met

Conclusions:

No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.04 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of Macrolex Rotviolett R in the Wistar strain rat was greater than 5.04 mg/L (Globally Harmonized Classification System – Un-classified).

Executive summary:

Summary

Introduction

A study was performed to assess the acute inhalation toxicity of Macrolex Rotviolett R. The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals (2009) No. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method” and Method B.52. Acute Inhalation Toxicity – Acute Toxic Class Method, 2014, of Commission Regulation (EC) No. 440/2008.

Methods

A group of six RccHan^TM: WIST strain rats (three males and three females) was exposed to a dust atmosphere of the test item. The animals were exposed for 4 hours using a nose only exposure system, followed by a fourteen day observation period.

Results

The mean achieved atmosphere concentration was as follows:

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.04	0.12	12.2

The characteristics of the achieved atmosphere were as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
5.04	3.48	54.9	3.12

The mortality data were summarized as follows:

Mean Achieved Atmosphere Concentration (mg/L)	Deaths
	Male	Female	Total
5.04	0/3	0/3	0/6

Clinical Observations. Common abnormalities noted during the study included decreased respiratory rate, hunched posture, pilo-erection, generalized purple fur staining caused by the test item and wet fur. An isolated occurrence of noisy respiration was also noted. All animals recovered such that no significant observations were apparent on Day 2 post-exposure.

Body Weight. All animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted for all male animals during the remainder of the recovery period.  In contrast, two female animals showed no body weight gain from Days 3 to 7 post-exposure. All female animals exhibited body weight gains during the final week of the recovery period.

 

Necropsy. No macroscopic abnormalities were detected amongst animals at necropsy.

 

Conclusion

No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.04 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC₅₀) of Macrolex Rotviolett R in the Wistar strain rat was greater than5.04 mg/L (Globally Harmonized Classification System – Un-classified).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Reliability 1 (reliable without restriction)

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the acute oral toxicity studies the LD50 is greater than 5000 mg/kg bw (discriminating dose) and the acute inhalation LC50 value is greater 5.04 mg/L (discriminating dose).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.

No study for acute dermal toxicity is available: According to Commission Regulation (EU) 2016/863 of May 2016 acute toxicity testing by the dermal route (Annex VII, point 8.5.3., column 2) ‘does not need to be conducted if the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure’. The registered substance conforms with the requirements given above. Therefore, it can be concluded for acute dermal toxicity that the available information is conclusive for non-classification.