Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

Endpoint summary

Description of key information

In an acute oral toxicity study in rats the acute oral LD50 was determined to be > 2000 mg dye/kg bw (corresponding to > 2160 mg test item/kg bw).

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:

11 May 2017 - 05 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI rats

Sex:

female

Details on test animals or test system and environmental conditions:

Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: The Wistar rat as a rodent is one of the standard species of acute toxicity studies
Number of animals: 3 animals/group
Sex: Female, nulliparous and non pregnant animals
Age of animals: Young adult rat, 8 weeks old in the first and second step
Body weight range at starting (first step): 180 - 183 g
Body weight range at starting (second step): 198 - 203 g
Acclimatization time: 6 days in the first step and 7 days in the second step

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the study director.
Room: 13/2
Housing: Group caging (3 animals/cage)
Cage type: Type III. polypropylene/polycarbonate.
Bedding: Laboratory bedding.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: above 10 air exchanges/hour by central air-condition system.
The temperature and relative humidity were recorded daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from municipal supply, as for human consumption from bottle ad libitum. The diet and drinking water are periodically analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Helianthi annui oleum raffinatum

Details on oral exposure:

All doses were formulated in the vehicle. Concentration of formulations was adjusted to maintain a treatment volume of 10 mL/kg bw. The test item was applied in a concentration of 200 mg/mL. Correction of concentrations for active component content was made (correction factor: 1.08). Formulations were prepared just before the administration and were stirred continuously during the treatment.

Vehicle
Name: Helianthi annui oleum raffinatum
Batch number: 1607-4569
Date of expiration: 30.11.2017
Produced by: Parma Produkt Kft.

Doses:

2000 mg dyestuff/kg bw (corresponding to 2160 mg product/kg bw)

No. of animals per sex per dose:

6 female animals per dose (3 female animals/step)

Control animals:

no

Details on study design:

A single oral administration - followed by a fourteen-day observation period - was performed by gavage. The day before treatment the animals were fasted. The food but not water was withheld overnight. Animals were weighed before the application and the food was given back 3 hours after the treatment.

6 days in the first step and 7 days in the second step of acclimatization, day of treatment, 14 days post-treatment observation period and necropsy on Day 15.

Animals were observed individually after dosing at least once during the first 30 minutes, then 1 h, 2 h, 3 h, 4 h after the treatment and twice each day for 14 days thereafter.
Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

The body weights were recorded on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g.

At the end of the observation period all rats were sacrificed under isofluran anaesthesia. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size.

Statistics:

The method used is not intended to allow the calculation of a precise LD50 value.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Mortality:

No lethality was noted at a single oral dose of 2000 mg/kg bw.

Clinical signs:

other: In the first ans second step, dark faeces were observed in all animals on Day 1, being related to the physical property of test item. No systemic clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the gen

Gross pathology:

All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes.

Other findings:

The method used is not intended to allow the calculation of a precise LD50 value.

Summary of Lethality: Post-treatment observation period (14 days) 

Groups	Treatment		Lethality
	Test Item	Dose (mg/kg bw)	Females
1	Step 1	2000	0/3
2	Step 2	2000	0/3

Summary of Clinical Symptoms

Groups	Treatment		Symptoms	Incidence
	Test Item	Dose (mg/kg bw)
1	Step 1	2000	Dark faeces	3/57
			Normal	54/57
2	Step 2	2000	Dark faeces	3/57
			Normal	54/57

 Remark: Incidence = Number of symptoms/Summarized number of observations inside the group

Summarized number of observations inside the group =(number of observations of first animal) + (number of observations of second animal) + (number of observations of third animal)

Summary of Body Weights (g)

FEMALES		Day 0	Day 7	Day 15
Group 1:
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		181.7	202.0	216.7
SD:		1.53	4.36	5.13
FEMALES		Day 0	Day 7	Day 15
Group 2:
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		201.3	233.3	249.7
SD:		2.89	10.21	19.86

Summary of Body Weight Gains (g)

FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 1:
2000 mg/kg bw, Step 1
Group size:		3	3	3
Mean: (g)		20.3	14.7	35.0
SD:		5.69	1.15	6.56
FEMALES		Day 0-7	Day 7-15	Day 0-15
Group 2:
2000 mg/kg bw, Step 2
Group size:		3	3	3
Mean: (g)		32.0	16.3	48.3
SD:		9.54	9.71	19.22

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 was determined to be > 2000 mg dye/kg bw (corresponding to > 2160 mg test item/kg bw).

Executive summary:

An acute oral toxicity study was performed according to OECD guideline 423. The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg dyestuff/kg bw (corrected concentration, correction factor: 1.08, corresponding to 2160 mg product/kg bw) as the starting dose in three female rats. No animal died in the first step at 2000 mg dyestuff/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. No animal died in the second step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on the 15th day after the treatment.

No lethality was noted at a single oral dose of 2000 mg dyestuff/kg bw. In the first and second step, dark faeces were observed in all animals on Day 1, being related to the physical property of test item. No systemic clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of the experimental animals were normal. The body weight development was not affected in all animals. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes. The method used is not intended to allow the calculation of a precise LD50 value. The oral LD50 was determined to be > 2000 mg dyestuff/kg bw (corrsponding to 2160 mg product/kg bw).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

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

Acute oral toxicity:

An acute oral toxicity study was performed according to OECD guideline 423. The acute toxic class method was carried out involving a stepwise procedure with the use of 2000 mg dyestuff/kg bw (corrected concentration, correction factor: 1.08, corresponding to 2160 mg product/kg bw) as the starting dose in three female rats. No animal died in the first step at 2000 mg dyestuff/kg bw dose level, so treatment with 2000 mg/kg bw was repeated on further three female rats. No animal died in the second step, too, so the test was finished, the stopping criteria of Annex 2d of OECD Guideline No. 423 (presented in Appendix VII) were met. Animals were weighed, observed for lethality and toxic symptoms for 14 days after the treatment. Gross pathological examination was carried out on the 15th day after the treatment.

No lethality was noted at a single oral dose of 2000 mg dyestuff/kg bw. In the first and second step, dark faeces were observed in all animals on Day 1, being related to the physical property of test item. No systemic clinical symptoms were observed on the day of the treatment and during the 14-day observation period, the general state and behaviour of the experimental animals were normal. The body weight development was not affected in all animals. All organs of the animals treated with 2000 mg/kg bw proved to be free of treatment related gross pathological changes. The method used is not intended to allow the calculation of a precise LD50 value. The oral LD50 was determined to be > 2000 mg dyestuff/kg bw (corrsponding to 2160 mg product/kg bw).

Acute inhalation toxicity:

The study does not need to be conducted as exposure of humans via inhalation is not the appropriate route of exposure. Furthermore, physicochemical and toxicological properties do not suggest a potential for a significant rate of respiratory absorption.

Acute dermal toxicity:

According to REACH Annex VIII point 8.5.3 the study does not need to be conducted because 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 (e.g. skin irritation, skin sensitisation). Due to the absence of acute oral toxicity and the fact that dermal absorption is not expected to occur, a dermal LD50 above 2000 mg/kg bw is assumed.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on acute toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.