Reaction mass of trisodium [m[7-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-8-hydroxynaphthalene-1,6-disulphonato(7-)]]dicuprate(3-) and trisodium [m[4-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-3-hydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

Endpoint summary

Description of key information

Oral LD50 (females) > 5000 mg/kg bw

Dermal LD50 (males and females) > 2000 mg/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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

not specified

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:

TEST ANIMALS
- Source: animals were purchased from KFM, CH-4414 Füllinsdorf, Switzerland.
- Weight at study initiation: weight ranged between 160 - 190 g.
- Fasting period before study: access of food only was prevented approximately 18 hours prior and four hours after the dosing.
- Housing: rats were housed individually in Macrolon cages.
- Diet: standard laboratory pelleted diet (KLIBA no. 24-343-4 fro Klingentalmfihle AG., Basle), ad libitum. The batch of diet used for the study was analysed for chemical and microbiological contaminants.
- Water: ad libitum. The water bottles were with-drawn two hours prior and four hours after dosing.
- Acclimation period: about 5 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: the room temperature was maintained within a median of 23 ± 2 °C recorded by a maximum-minimum thermometer.
- Humidity: 30 - 70 %, measured by a hygrometer.
- Air changes: the rate of air exchange was maintained at approximately 15 changes/hour.
- Photoperiod: the light/dark cycle was 12 hours.

Route of administration:

oral: gavage

Vehicle:

other: the test item was diluted with WEM (33.3 %)

Details on oral exposure:

Dosed volumee: 15 ml per kg body weight.
The sample was prepared immediately before dosing.

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 male and 5 female rats

Details on study design:

PRELIMINARY STUDY
A preliminary study was carried out to establish a dosing regimen, using groups of two males and two females at one dosage, using 15 ml/kg body weight.
- Duration of observation period following administration: 14 days

MAIN STUDY
- Duration of observation period following administration: 14 days
- Frequency of weighing: individual body weights on day 1, 7 and 14.
- Necropsy of survivors performed: surviving animals were killed after two weeks. All animals which died during the study and those killed after two weeks were subjected to a macroscopic postmortem examination. The macroscopic appearance of the abnormal organs was recorded.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No deaths occurred during both the preliminary and main experiments.

Clinical signs:

The animals were flaccid, weak and showed a rough coat.

Gross pathology:

No special findings.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

LD50 (male and female) > 5000 mg/kg bw

Executive summary:

Young adult male and female rats were used to evaluated the oral acute toxicity potential of the test item. A group of 5 males and 5 females rats were dosed at 5000 mg/kg bw. The substance was administered at the dose volume of 15 ml/kg bw, by gavage. During the observation period of 14 days, all signs of toxicity were recorded. All rats were sacrificed terminally and examined macroscopically in an attempt to identify any target organs.

No deaths occurred during both the preliminary and main experiments; the animals were flaccid, weak and showed a rough coat. Gross examination did not revealed any special finding.

Conclusion

LD50 (male and female) > 5000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From October 18th to November 01st, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details for read across approach are included into the IUCLID section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted February 24, 1987

Qualifier:

according to guideline

Guideline:

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

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:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland.
- Age at study initiation: approx. 11 weeks.
- Weight at study initiation: males 309 to 322 g and females 202 to 240 g
- Housing: individually housed in polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by Broekman Institute, Someren, The Netherlands).
- Diet: free access to standard pelleted laboratory animal diet (Kliba 343 from Klingentalmdhle AG, Kaiseraugst, Switzerland).
- Water: free access to tap-water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Humidity: 55 %:
- Air changes: 15 air changes per hour.
- Photoperiod: lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: one day before exposure an area of approximately 5x7 cm on the back of the animal was clipped. The formulation was applied to an area of approximately 25 cm2 (5x5 cm) for males and 18 cm2 (3.5x5 cm) for females by application on a gauze patch.
- Type of wrap if used: gauze patch was fixed to aluminium foil end flexible bandage, with drops of petrolatum.

REMOVAL OF TEST SUBSTANCE
- Washing: whereafter residual test substance was removed with tissue moistened with tap-water.
- Time after start of exposure: 24 hours.

TEST SUBSTANCE PREPARATION
The formulations were prepared immediately prior to dosing. The test substance was weighed into a glass flask on an analytical balance and the vehicle (wlw) was added. Following stirring the test substance preparation formed a solution.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females

Details on study design:

- Duration of observation period following administration: 14 days
- Mortality / Viability: at periodic intervals on the day of dosing (day 1) and twice dai1y thereafter for 14 days.
- Body Weights: days 1 (pre-administration), 8 and 15.
- Symptoms: at periodic intervals on the day of dosing (day 1) and once daily thereafter For 14 days. All signs of reaction to treatment were recorded with particular attention paid to changes in the skin (treated skin), fur, eyes and mucous membranes, as well as to behaviour pattern, tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- Necropsy of survivors performed: all animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide asphyxiation and subjected to necropsy. All animals assigned to the study were subjected to necropsy. The necropsies were performed by experienced prosectors and descriptions of all macroscopic abnormalities recorded.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No mortality occurred during the study period.

Clinical signs:

No clinical signs of ill health or behavioural changes were seen during the study.
No skin irritation was observed on the treated area during the study period. All animals showed blue staining of the treated skin area by the test substance during the study period.

Body weight:

Slight body weight gain was noted among all animals over the first week of observation. All animals showed improved body weight gain over the second week of the study.

Gross pathology:

Macroscopic post mortem examination of the surviving animals at termination did not reveal any abnormalities.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

LD50 (males and females) > 2000 mg/kg bw

Executive summary:

The purpose of the study was to assess the toxicity of the test article when administered to rats as a single dermal dose. The study was carried out in accordance with OECO Guideline No. 402, “Acute Dermal Toxicity‘ and EEC Directive 84/449/EEC, Part B.3, "Acute Toxicity ~ Dermal". Test item was administered by dermal application, to five rats of each sex, at 2000 mg/kg body weight. Macroscopic examination was performed at the end of the experimental period.

No animals died during the study. No clinical signs of ill health or behavioural changes were seen during the observation period. Slight body weight gain was noted over the first week with recovery of body weight gain over the second week of observation.

No signs of skin irritation were observed on the treated area during the study period. All animals showed blue staining of the treated skin by the test substance.

Macroscopic post mortem examination of the surviving animals at termination did not reveal any abnormalities.

Conclusion

LD50 (males and females) > 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

ORAL ROUTE

The oral acute toxicity potential of the Direct blue 251 was evaluated in young adult male and female rats. A group of 5 males and 5 females rats were dosed at 5000 mg/kg bw. The substance was administered diluted with WEM (33.3 %) at the dose volume of 15 ml/kg bw, by gavage. No deaths occurred during both the preliminary and main experiments; the animals were flaccid, weak and showed a rough coat. Gross examination did not revealed any special finding.

The study outcomes are confirmed by those obtained in a test performed on the structural analogous Similar Substance 01; the read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13). The study was performed with the Sprague-Dawley CD strain rat, following the method and procedures outlined into the OECD Guidelines 423. A group of three fasted females was treated with the test material at a dose level of 2000 mg/kg bodyweight. There were no deaths and there were no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy. The acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD strain rat was estimated to be greater than 2000 mg/kg bodyweight.

INHALATION ACUTE TOXICITY

No acute toxicity studies by inhalation route are available on Direct Blue 251. Nevertheless, because of the physical state and the trade forms of the substance under registration, inhalation is not an appropriate route of exposure. The substance is manufactured and handled with suitable risk management measures and with the suitable personal protective equipments; in addition, the commercial powder form contains small amount of oil to reduce the dusts and allow to handle it more easily.

DERMAL ACUTE TOXICITY

The inhalation and the skin contact of Direct Blue 251 are unlikely. According to the Commission Regulation (EU) 2016/863, testing by the dermal route does not need to be conducted if no systemic effects have been observed in in vivo studies with dermal exposure. Furthermore, it is explained that recent scientific analysis of available data from in vivo acute toxicity studies have shown that substances that are not toxic via the oral route may be expected with high certainty to be also non-toxic via the dermal route. Therefore, testing those substances via the dermal route does not provide essential information for their safety assessment.

The amendment is the consequence of studies and scientific debates. In particular, the 15th Meeting of Competent Authorities for REACH and CLP (CARACAL, 2014) concluded that an adaptation of point 8.5.3 of Annex VIII to REACH was justified in order to not require information on acute dermal toxicity for substances that have shown no toxicity in acute oral toxicity test up to the limit dose of 2000 mg/kg bw.

In the oral acute toxicity test, no signs of systemic toxicity were recorded up to 2000 mg/kg bw.

The excpectation is confirmed by data available on the structural analogous Similar Substance 02; the read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

The purpose of the study was to assess the toxicity of the Similar Substance 02 when administered to rats as a single dermal dose. The study was carried out in accordance with OECO Guideline No. 402 and EU Method B.3. Test item was administered by dermal application to five rats of each sex, at 2000 mg/kg body weight. No animals died during the study. No clinical signs of ill health or behavioural changes were seen during the observation period. Slight body weight gain was noted over the first week with recovery of body weight gain over the second week of observation. No signs of skin irritation were observed on the treated area during the study period. All animals showed blue staining of the treated skin by the test substance. Macroscopic post mortem examination of the surviving animals at termination did not reveal any abnormalities.

REFERRENCE

CARACAL, 2014. 15th Meeting of Competent Authorities for REACH and CLP (CARACAL), 8 – 9 July 2014. Charlemagne building, Brussels, Belgium. Brussels, 26 July 2014. Doc. CA/61/2014. Stakeholder proposal to modify REACH standard information requirements for acute toxicity (REACH Annex VIII, point 8.5)

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.1 Acute toxicity section, substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

The oral LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute oral toxicity (oral acute toxicity category 4: 300 < ATE ≤ 2000 mg/kg bw).

The dermal LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute dermal toxicity (dermal acute toxicity category 4: 1000 < ATE ≤ 2000 mg/kg bw).

Inhalation exposure is unlikely, thus no acute toxicity value is available and no further investigation is required.

In conclusion, the test substance does non meet the criteria to be classified for either oral, neither dermal acute toxicity, according to the CLP Regulation (EC) No 1272/2008.