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Description of key information

To assess the acute toxicity by oral route of the substance, an adequate study in the rat following oral administration has been conducted on a similar substance (read-across). Additionnally one supporting study done with a preparation of the substance (at a max. 35%) is available. Although the data with the preparation is not representative to assess the oral toxicity of the pure dye, it supports that the substance is not toxic by oral route.

The dermal acute toxicity of the substance has been determined in an adequate study in the rat following dermal administration.

No studies are available for inhalation.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance Direct Red 254 TEA salt (CAS No 64683-40-5 / EC 265-016-4) is defined as a mono-constituent substance with cations triethanolammonium.
The available toxicological data on this substance are insufficient to fulfil the data requirements for a REACH Annex VIII dossier.
In order to prevent unnecessary animal testing, the occurring data gaps on toxicity studies might be filled by applying read-across from the similar substance Direct Red 254 sodium salt (CAS No 6300-50-1 / EC 228-589-1). Both salts of Direct Red 254 are synthetized using the same raw materials and following the same manufacturing process.
The only difference between the query structure Direct Red 254 TEA salt (CAS 64683-40-5) and Direct Red 254 sodium salt (CAS 6300-50-1) is the counter ion. CAS 6300-50-1 is the result of a neutralization with NaOH, whilst the alkaline agent used in CAS No. 64683-40-5 is triethanolamine.
Both substances are identical in relation to the anionic components.

The read-across is based on the hypothesis that source and target substances have similar toxicological properties because both molecules have the following similarities: a) Identical raw materials; b) Identical manufacturing process; c) Identical anionic structure composition; d) Identical degradation products by reductive cleavage; e) Both have affinity to the same type of substrates/molecules. The substances are able to be adsorbed on the same type of substance, e.g. polysaccharides (cellulose), polyphenols (lignin) and proteins; f) Both substances have similar physicochemical properties

In summary, it is considered that both substances have the same mode of action with regard to the following endpoints:Acute Oral toxicity, Skin irritation, Eye irritation, Skin sensitisation, Mutagenicity and Repeated dose and reproduction / developmental (screening).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. For both, impurities are comparable.
The composition and impurities of the target and source substances are shown in table 1 of the attached document to this record.

3. ANALOGUE APPROACH JUSTIFICATION

The target chemical Direct Red 254 TEA salt (CAS No 64683-40-5) is a mono-constituent substance, with cations triethanolammonium.
Direct Red 254 Sodium salt (CAS No 6300-50-1) is assumed as source chemical since it is identical to the target chemical Direct Red 254 TEA salt (CAS No 64683-40-5) in respect of the different chemical anionic component but varies in the cation. The physicochemical properties of both substances are nearly identical. No experimental data on absorption, distribution and excretion is available for the source and target substances and their hydrolysis products. The toxicokinetics assessment is based on the physicochemical properties and the available toxicological data of the substances.
The source chemical CAS No 6300-50-1 is ionisable and is assumed that will be dissociated in aqueous media or in biological fluids to the anionic component and free Na+ cation. Sodium ion is a naturally occurring cation in the body with a blood plasma concentration of 140 mmol/L. It is excreted with the urine and does not cause any toxic effects when administered in low concentrations.
In analogy to the source chemical also CAS No 64683-40-5 (target chemical) is expected to be dissociated shortly after absorption and the cation TEA+ is also assumed to be readily available in the body. The TEA+ cation can be assimilated to triethanolamine (CAS No 102-71-6).
Base on that the only difference between the target structure (CAS No 64683-40-5) and the source structure (CAS 6300-50-1) is the counter ion, and the influence to the human health toxicity, irritation and / or sensitisation effects due to the presence of TEA+ in the target chemical CAS No 64683-40-5 is not expected. Consequently, read-across to the source chemical CAS No 6300-50-1 is regarded as feasible.
A broad and more detailed explanation is included in the attached document in section 13 of this dossier.


4. DATA MATRIX
Two data matrix are included in the attached document in section 13 of this dossier: Matrix 1 (Toxicity data on the source and target substance) and Matrix 2 (Main potential metabolites data)
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
assessment report
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
2 516 mg/kg bw
Based on:
act. ingr.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 516 mg/kg bw
Quality of whole database:
The study is not GLP compliant but the Klimisch score is 1

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
There isn’t any test available for acute inhalation toxicity. According to the column 2 of annex VIII, the choice for the second route will depend on the nature of the substance and the likely route of human exposure. Information on dermal toxicity is provided, as considered more relevant for this substance.

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:
May to October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Species:
rat
Strain:
Wistar
Remarks:
RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: no
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: at least 200 g
- Fasting period before study:
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24-Hour exposure period and in groups of four, by sex, for the remainder of the study.
- Diet (e.g. ad libitum): free acces to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): free access to drinking water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area).
- Type of wrap if used: A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the 24-Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- For solids, paste formed: yes (powder moistened)

VEHICLE
- Amount(s) applied (volume or weight with unit): the substance was moistened with distilled water.
Duration of exposure:
24h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 males / 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration:
On the day before treatment the back and flanks of each animal were clipped free of hair. In the absence of data suggesting the test item was toxic, one male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg.
The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage. The animals were caged individually throughout the study. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24-Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item.
As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. The animals were caged individually for the 24-Hour exposure period. After the 24-Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. These animals were returned to group housing for the remainder of the test period.

- Frequency of observations and weighing:
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1,2 and 4 hours after dosing and subsequently once daily for 14 days.
After removal ofthe dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored based on the erythema and eschar formation and edema formation. Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.

- Necropsy of survivors performed: At the end of the study 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.

- Other examinations performed: Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioral and clinical
observations, gross lesions, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute dermal median lethal dose (LD50) of the test item was made.
Statistics:
The following computerized system was used in the study: Delta Controls - ORCA view
Preliminary study:
In the absence of data suggesting the test item was toxic, one male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg. As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths.
Clinical signs:
There were no signs of systemic toxicity.
Body weight:
One female showed body weight loss during the first and second week. Two other females showed body weight loss or no gain in body weight during the first week with expected gain in body weight during the second week. One further female showed expected gain in body weight during the first week but no gain in body weight during the second week. The remaining animals showed expected gains in body weight over the study period.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
Dermal Irritation. There were no signs of dermal irritation noted.
Interpretation of results:
GHS criteria not met
Conclusions:
The acute dermal median lethal dose (LDso) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The study is a GLP compliant and has Klimisch score 1

Additional information

Justification for classification or non-classification

Acute oral toxicity

Based on the results of acute oral toxicity testing, the substance has not to be classified for this hazard class following the criteria of the EU Regulation 1272/2008 (CLP Regulation). The criteria in CLP Regulation stablishes the limit for classification as Acute Tox Oral Category 4 in 2000 mg/kg, and the value obtained is >2000 mg/kg bw.

 

Acute inhalation toxicity

There isn’t any test available for acute inhalation toxicity. According to the column 2 of annex VIII, the choice for the second route will depend on the nature of the substance and the likely route of human exposure. Information on dermal toxicity is provided, as considered more relevant for this substance.

 

Acute dermal toxicity

Based on the results of acute dermal toxicity testing, the substance has not to be classified for this hazard class following the criteria of the EU Regulation 1272/2008 (CLP Regulation).The criteria in CLP Regulation stablishes the limit for classification as Acute Tox Dermal Category 4 in 2000 mg/kg, and the value obtained is >2000 mg/kg bw.