Reaction products of 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, coupled twice with diazotized 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate, sodium salts

Endpoint summary

Description of key information

Based on data from 28-day´s and 90-days studies performed with similar substance according to OECD guideline 407 and 408 in accordance with GLP, NOAEL (No Observed Adverse Effect Level) for males and females was established at 1000 mg/kg body weight/day. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May to 14 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Compliant to OECD 408 and GLP Guideline. Read-across from other vinyl-sulfone dye: common metabolites: The chlorinated triazine group is likely to be metabolized to cyanuric acid based groups following exposure with eventual degradation to chloride salts, ammonia, CO2 and water.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP Ltd. 1032 Budapest, Cserkesz u. 91
- Age at study initiation: 6 weeks old
- Weight at study initiation: Male: 170 -194 g; Female: 120 -152 g
- Fasting period before study: NA
- Housing: Five animals/cage
- Diet: ssniff SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: tap ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): 8 to 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 May To: 14 Sep 2006

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in concentrations of 6.25 mg/mL, 25 mg/mL and 100 mg/mL prepared with distilled water. Formulations were prepared daily during working days and beforehand for weekends by Central Dispensary Unit of LAB International Hungary Ltd. The read across substance B was stable at the applied concentrations at room temperature for 24 hours and in refrigerator (5 ± 3 °C) for 4 days.

VEHICLE
- Concentration in vehicle: 6.25 mg/mL, 25 mg/mL and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of concentration of dosing formulations was conducted on first and last weeks of the treatment. All the measured concentrations varied in the range of 92 % and 109 % of the nominal concentrations.

Duration of treatment / exposure:

90 days for male animals
91 days for female animals

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
6.25 mg/mL, 25 mg/mL and 100 mg/mL
Basis:
actual ingested

No. of animals per sex per dose:

15 animals/sex in the control and high dose groups (including 5 animals/sex for recovery groups)
10 animals/sex in the low and mid dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of the results of the acute oral toxicity study with the test item.
- Rationale for selecting satellite groups: reversibility of findings
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

not investigated

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality & morbidity: twice daily
general clinical signs: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior first treatment - weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups: prior to treatment - all animals
week 13 - control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study and end of recovery period
- Anaesthetic used for blood collection: Yes - Euthanyl
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [3.5.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study and end of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [3.5.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: for 16 hours aft termination of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [3.5.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 89
- Dose groups that were examined: all animals
- Battery of functions tested:
- sensory activity
- grip strength
- motor activity
- modified Irwin

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Dead animal was subjected to gross pathology just after he was found dead. Terminally and at the end of the recovery period, a gross necropsy was performed on each animal just after the blood harvesting for clinical pathology examinations. One day after the last treatment, animals were sacrificed by exsanguination under pentobarbital (Euthanyl) 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. Any abnonnality was recorded with details of the location, colour, shape and size.
- Organ Weight Measurement: The following organs were weighed recorded (paired organs were weighed together):
With precision of 0.01 g: Liver, kidneys, testes, epididymides, uterus, heart, thymus, spleen, brain.
With precision of 0.001 g: Adrenals Ovaries

HISTOPATHOLOGY: Yes
- The following organs and tissues were preserved in 10% formaldehyde solution:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), larynx, femur + bone marrow, spinal cord (cervical, lumbar, thoraeie level), pituitary, thymus, trachea, lungs (with main stern bronchi), heart, thyroid + parathyroid, oesophagus, stomaeh, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, Harderian glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve, aorta.
- Full histological examinations were performed on the organs or tissues of the animals ofthe control and high dose groups. In groups 2 and 3, liver, kidneys and organs with macroscopic alteration were also processed histologically. The listed organs or their specimens were embedded into paraffin after dehydration. Slides were stained with haematoxylin eosin and examined by a light microscope.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data: body weight, food consumption, hematology, clinical chemistry, urinalysis, organ weight
- The heterogeneity of variance between the groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a oneway analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance ofthe inter-group differences. Where significant heterogeneity was found, the normal distribution of data by Kolmogorov-Smimov test was used. In case of not-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied.
- If positive results were obtained, the inter-group comparisons were performed using Mann-Whitney U-test. Frequency of clinical symptoms, necropsy and histopathological findings were calculated. At the end of recovery period, the homogeneity of variance between groups was determined by F-test. Depending on the result Pooled or Separate variance estimate of the Two-Sample t-test was performed. For data, which was not normally distributed, the data were compared between groups according to Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

males at 1000 mg/kg

Mortality:

mortality observed, treatment-related

Description (incidence):

males at 1000 mg/kg

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

males at 1000 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

males at 1000 mg/kg

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

at 1000 mg/kg

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: There was no test item related mortality. One male rat of 1000 mg/kg bw/day dose was found dead without preceding signs on day 66. Cause of death was para-gastric treatment resulting in peripheral circulatory and breathing disturbance, which is in line with the histological observation: congestion and alveolar edema causing death of animal.
- Clinical observations: 1000 mg/kg bw/day dose of read across substance B caused polyuria in male and female animals from day 2 up to the end of the treatment, which recovered 10 days after the cessation of test item administration. There were no test item related effects on animal's behavior or reaction to different type of stimuli at the functional observation battery.

BODY WEIGHT AND WEIGHT GAIN
The lower body weight gain of male animals dosed at 1000 mg/kg bw/day resulted in statistically significant body weight differences when compared to the control. The recovery groups showed increased weight gain after cessation of treatment, which is a typical response to non-specific toxicity. The body weight change in males at 1000 mg/kg was considered to be an effect of treatment, although all weights were within the normal range.

FOOD CONSUMPTION
There were no differences in the mean food consumption between any dose levels except weeks 9 and 10 in male 1000 mg/kg bw/day dose, where statistically significantly less food intake was measured.

WATER CONSUMPTION
Water consumption was not measured. But the routine control of animals showed a hiher water consumption in high-dose animals. The bottle size was therefore increased to 1000 mL bottles for this group.

OPHTHALMOSCOPIC EXAMINATION
No effects observed

HAEMATOLOGY
There were no toxicologically significant test item induced alterations in the examined haematological parameters at termination of the treatment.

CLINICAL CHEMISTRY
There were no test item re1ated differences in the examined biochemical parameters.

URINALYSIS
A test item influence on the urine excretion was observed in all groups, which manifested in an elevated volume of urine at 62.5 mg/kg bw/day (no statistical significance in male group), 250 mg/kg bw/day, and 1000 mg/kg bw/day and in a decreased specific gravity at 250 mg/kg bw/day male and at 1000 mg/kg bw/day (male and female animals). Although the effect was related to treatment, it was considered to be a physiological effect, related to the high salt level in the test item, and not an adverse effect of treatment.

NEUROBEHAVIOUR
There were no differences related to the test item in the examined parameters.

ORGAN WEIGHTS
No test item related differences were noted in the weighed organs of test groups.

GROSS PATHOLOGY
No test item related macroscopic findings were detected at the necropsy.
Dead animal : Dark red lungs, blood filled thoracic cavity and yellow colour of the organs were noted for the rat which died from misgavage.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test item related lesions in the organs of animals subjected to histopathological examination.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 1000 mg/kg bw/day: - increased water consumption and urine excretion due to a high salt concentration (lithium and sodium) in the test substance - minimally reduced body weight gain and food consumption in males

Critical effects observed:

not specified

Conclusions:

The read across substance B caused an increased water consumption and urine excretion in the course of 90-day consecutive oral administration at 1000 mg/kg bw/day dose level to CRL:(WI) BR rats, which recovered within 10 days after cessation of the treatment. This effect was ascribed to high levels of salts in the test item (lithium and sodium) and is considered to be a physiological response and not an adverse toxic effect.
The body weight and food consumption was slightly depressed in male group of 1000 mg/kg bw/day. This reversed in the recovery phase.
Urinalysis revealed a higher volume of urine at 62.5 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day after 16 hours collection at termination of the treatment. The increased volume of urine indicates an increased physiological function without any related pathologie (clinical or histopathology) alterations.
The changes observed at 1000 mg/kg were minor in nature and were reversible. There were no signs of serious irreversible toxicity at 1000 mg/kg bw/day.
The no observed adverse effect level (NOAEL) of the read across substance B was 250 mg/kg bw/day.

Executive summary:

In a GLP-compliant study performed according to OECD guideline 408, a 90-day repeated dose oral (gavage) toxicity study was performed in CRL:(WI) BR rats (n= 10 animals/sex/group and further 5 animals/sex in the control and Group 4 for recovery) on the read-across substance with dose levels of 0 (vehicle only), 62.5, 250 and 1000 mg/kg bw/day. Animals were treated by daily oral gavage with test item or vehic1e (distilled water) at a constant dosing volume of 10 mL/kg body weight. Recovery animals were retained for further 28-day observation without treatment.

The stability of test item in this vehicle was found to be adequate (at least 24 hours at room temperature and at least 4 days at 5 ± 3 °C.) General clinical observations were made daily and detailed clinical observations were performed weekly. A functional observation battery was conducted on week 13. Body weight and food consumption were measured weekly. Ophthalmoscopy was made on all animals before starting of the treatment and in the control and high dose groups on week 13. Urinalysis was conducted on week 12, clinical pathology and gross pathology one day after the last treatment. Selected organs were weighed. A full histological examination was performed on the preserved organs and tissues of the animals of the control group and Group 4 (1000 mg/kg bw/day). The liver, kidneys and organs with macroscopic findings were evaluated histologically in Group 2 and Group 3. Animals of recovery groups were processed in the same manner at termination of the recovery period.

Results

Mortality:

There was no test item related mortality. One male rat of 1000 mg/kg bw/day dose was found dead without preceding signs on day 66. Cause of death was para-gastric treatment resulting in peripheral circulatory and breathing disturbance, which is in line with the histological observation: congestion and alveolar edema causing death of animal.

Clinical observations:

1000 mg/kg bw/day dose of read-across substance caused polyuria in male and female animals from day 2 up to the end of the treatment, which recovered 10 days after the cessation of test item administration. There were no test item related effects on animal's behavior or reaction to different type of stimuli at the functional observation battery.

Body weight:

The mean body weight gain of male animals at 1000 mg/kg bw/day was less than the control value during the treatment period resulting in significant difference in the body weight, which was not reversible at the end of the recovery period.

Food consumption:

There were no differences in the mean food consumption between any dose levels except weeks 9 and 10 in males of the 1000 mg/kg bw/day dose, where significantly less food intake was measured.

Clinical pathology:

There were no test item related adverse effects of treatment in the examined hematological or clinical chemistry parameters. A higher volume of the urine was noted for 62.5 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day doses at the terminal urinalysis.

Gross pathology:

No test item related macroscopic finding were found at the necropsy.

Organ weight:

There were no relevant test item organ weight alterations (male and female).

Histopathology:

There were no histopathological changes related to the test item effect detected.

 

Conclusions

The read-across substance caused an increased water consumption and urine excretion in the course of 90 -day consecutive oral administration at 1000 mg/kg bw/day dose level to CRL:(WI) BR rats, which recovered within 10 days after cessation of the treatment. This effect was ascribed to high levels of salts in the test item (lithium and sodium) and is considered to be a physiological response and not an adverse toxic effect.

The body weight and food consumption was slightly depressed in male group of 1000 mg/kg bw/day. This reversed in the recovery phase.

Urinalysis revealed a higher volume of urine at 62.5 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day after 16 hours collection at termination of the treatment. The increased volume of urine indicates an increased physiological function without any related pathologie (clinical or histopathology) alterations.

The changes observed at 1000 mg/kg were minor in nature and were reversible. There were no signs of serious irreversible toxicity at 1000 mg/kg bw/day.

The no observed adverse effect level (NOAEL) of the read-across substance was 250 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Key study with reliability rating 1 performed according to OECD guideline 408 and in accordance with GLP.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 21-day study with 14 applications with a single dose of 500 mg/kg bw/day was performed without revealing any adverse effects. The only effects seen were substance stained urine and feces.

From radio-kinetic studies with the test substance it is well known that the major metabolite is sulfuric acid mono-[2-(4-amino-benzenesulfonyl)-ethyl] ester. The other main component of the test substance, 3,5-Diamino-4-hydroxy-naphthalene-2,7-disulfonic acid was not absorbed and only found as not recoverable substance in the faeces.

Therefore, a 28-day and a 90-day study with structurally similar reactive dyes were used for cross-reading. The read-across substance A was used for cross-reading of the 28-day study. Besides the chloro-triazin fragment, the dye has almost the same structure as the test substance. The only effects observed during treatment with the test substance were reddish discolorations of urine, feces, and several organs. Microscopically, renal re-absorption of the dye was observed in two high-dose females without resulting in any adverse effect. Consequently, the NOAEL after 28 days of oral treatment with the read across substance A in rats was set to 1000 mg/kg bw/day.

The read-across substance B covering the major metabolite, sulfuric acid mono-[2-(4-amino-benzenesulfonyl)-ethyl] ester metabolite was used for cross-reading of the 90-day study. Besides an increased urine excretion and water consumption at 1000 mg/kg bw/day and a minimally decreased food consumption and body weight gain in males there were no treatment related effects observed. Polyuria and polydipsia are well-known side effects for lithium. As the content of lithium is rather high in this test substance, these effects were most likely related to the lithium salt content. Due to the salt-effects observed at the highest dose level, the NOAEL for the reactive substance B was set to 250 mg/kg bw/day. Due to the fact that no lithium is contained in the test substance no comparable effects are expected for the test substance up to the limit dose of 1000 mg/kg body weight.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was conducted according to OECD guideline 408 and in accordance with GLP

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Waiving argument:
The test substance has very low vapour pressure (above the lower detection limits) and a melting point of >180 ºC, so the potential for the generation of inhalable forms is low, also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Furthermore, in the 28-days and 90 -days repeated dose study via oral gavage administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby, there is low toxicity potential. This intrinsic property/toxicity potential can be extrapolated to repeated inhalation route administration.

Exposure considerations: Production and spray drying is performed in closed processes without isolation of reaction products. Isolated product is consisting of dust free granules (non-dusty solid).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Dermal penetration is very unlikely. The physicochemical and toxicological properties suggest low potential for significant rate of absorption through the skin. Furthermore, the results of laboratory animal studies show low acute toxicity. In the 28 -days and 90-days repeated dose study via oral gavage administration does not exacerbate systemic toxicity effects which suggest bioavailability is low, thereby, there is low toxicity potential. Due to the good water solubility, the toxicologically most relevant route of exposure is the oral one. This intrinsic property/toxicity potential can be extrapolated to repeated dermal route administration.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the test substance does meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and therefore no classification is required.