4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Endpoint summary

Description of key information

Two key studies are available for acute toxicity, an oral and a dermal study.

Oral route:

A single oral application of the registered substance to rats at a dose of 2000 mg/kg body weight was not associated with signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days was > 2000 mg/kg bw.

Dermal route:

A single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of dermal irritation. The dermal LD50 was determined to be > 2000 mg/kg body weight.

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:

2016-2017

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:

adopted 17 December 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA 712-C-02-190, December 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Procedures and facilities comply with the requirements of Directive 2010/63/EU and the national legislation defined in the animal protection law concerning the protection of animals used for experimental and other scientific procedures

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: WISTAR Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks old
- Weight at day of admnistration: step 1: 165 – 198 g; step 2: 156 – 173 g
- Fasting period before study: Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test item was administered. Food was provided again approximately 4 hours post dosing.
- Housing: The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: Acclimatisation Period:
Step 1, animals no. 1 - 3: 17 November 2016 to 29 November 2016
Step 2, animals no. 4 - 6: 29 November 2016 to 05 December 2016

To: 20 December 2016

Route of administration:

oral: gavage

Vehicle:

other: aqua ad injectionem

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 2 g of the test item was dissolved in the vehicle to gain a final volume of 10 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight.
- Justification for choice of vehicle: In order to get the test item in a solution or suspension, which is applicable to the animals, aqua ad injectionem was evaluated as vehicle and was considered to be adequate. This vehicle was chosen due to its non-toxic characteristics.
- Lot/batch no. (if required): AlleMan Pharma, lot no. 605070, expiry date: 04/2019

MAXIMUM DOSE VOLUME APPLIED: The test item was administered at a dose volume of 10 mL/kg body weight.

DOSAGE PREPARATION (if unusual): The test item was weighed out into a tared plastic vial on a precision balance.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before each dose administration.
For all animals of the first and second step, 2 g of the test item was dissolved in the vehicle to gain a final volume of 10 mL and to achieve a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight.
The dose formulations were made shortly before each dosing occasion.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The starting dose was selected to be 2000 mg/kg body weight. According to OECD 423 guideline, when available information suggests that mortality is unlikely at highest starting dose level (2000 mg/kg), then a limit test should be conducted. The provided safety data sheet of the sponsor indicated no toxicity at the highest starting dose level.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 per step / 2 steps performed

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
All animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality. A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weighgross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Based on:

test mat.

Remarks on result:

other: no mortality at limit dose of 2000 mg/kg bw

Mortality:

The test item showed no mortality after a single dose administration.

Clinical signs:

other: The test item showed no mortality and no other acute oral toxicity characteristics after a single dose administration.

Gross pathology:

With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Interpretation of results:

other: No classification required according to regulaation (EC) No. 1272/2008

Conclusions:

Under the conditions of the present study, a single oral application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no signs of toxicity or mortality.
The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): ∞
According to Annex I of Regulation (EC) 1272/2008 the test item has no obligatory labelling requirement for toxicity and is not classified.

Executive summary:

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight (limit test). The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study without showing any test-item related signs of toxicity.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

On the basis of the test results given below and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008, the substance should not be classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Reliable

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

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

Study period:

28.03-12.04.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

24 Feb, 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

30 May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3°C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period. The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

Duration of exposure:

The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed using aqua ad injectionem

Doses:

The test item was applied at a single dose of 2000 mg/kg body weight to each animal.

No. of animals per sex per dose:

5 male and 5 female animals

Control animals:

no

Details on study design:

Preparation of the Animals
The animals were marked for individual identification by tail painting.
Approximately 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper. Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals. Only healthy animals were used

Observation Period
All animals were observed for 14 days after dosing.

Primary Skin Irritation
Signs of erythema and oedema were assessed using the scoring system laid down in OECD Guideline 404.

Weight Assessment
The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

Clinical Examination
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology
At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw.
All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.

Evaluation of Results
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed.

Clinical signs:

other: Signs of systemic toxicity related to dose level used, time of onset and duration: No treatment-related effects were observed.

Gross pathology:

Effect on organs (related to dose level):
No treatment-related effects were observed.

Other findings:

Signs of irritation:
No erythema or oedema nor any other signs of irritation were observed.

Interpretation of results:

other: No classification required according to Regulation No. 1272/2008

Conclusions:

According to Annex I of Regulation (EC) 1272/2008 the test item Reactive Yellow 42 has no obligatory labelling requirement for percutaneous toxicity and is unclassified.

Executive summary:

Under the conditions of the present study, a single dermal application of Reactive Yellow 42 to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of dermal irritation.

The effects on weight loss are a common observation for female animals within this study type and might be secondary to the dressing, and toxicological relevance of this finding cannot clearly be concluded.

The dermal LD50 was determined to be > 2000 mg/kg body weight.

According to Annex I of Regulation (EC) 1272/2008 the test item Reactive Yellow 42 has no obligatory labelling requirement for percutaneous toxicity and is unclassified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Reliable

Additional information

Acute oral toxicity

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight (limit test). The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study without showing any test-item related signs of toxicity.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

Acute dermal toxicity

Under the conditions of the present study, a single dermal application of Reactive Yellow 42 to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of dermal irritation.

The effects on weight loss are a common observation for female animals within this study type and might be secondary to the dressing, and toxicological relevance of this finding cannot clearly be concluded.

Justification for classification or non-classification

Acute oral toxicity

Two groups, each of three female Wistar rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight (limit test). All animals survived until the end of the study without showing any test-item related signs of toxicity.

According to the EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) the substance is not considered to be classified as acute toxic because the LD50 is > 2000 mg/kg bw (Table 3.1.1).

Acute dermal toxicity

A single dermal application of Reactive Yellow 42 to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of dermal irritation. The dermal LD50 was determined to be > 2000 mg/kg body weight.

According to Annex I of Regulation (EC) 1272/2008 the test item Reactive Yellow 42 has no obligatory labelling requirement for percutaneous toxicity and is unclassified.