reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

Endpoint summary

Description of key information

The No Observed Effect Level (NOEL) of is 200 mg/kg body weight/day. As the effects seen in this study are either singular findings or of no toxicological relevance for humans, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg body weight/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 1998 to Feb. 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

"Repeated dose (28 days) toxicity (oral)", September 30, 1996.

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes (incl. QA statement)

Remarks:

Switzerland GLP

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR 50
- Expiration date of the lot/batch: September 1, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (ca. 20°C) away from direct sunlight.
- Stability under test conditions: up to seven days

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf / Switzerland
- Age at delivery : 6 weeks
- Weight at acclimatization : Males: 129 - 176 grams (mean 156 grams) ; Females: 122 - 147 grams (mean 138 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet : Pelleted standard Provimi Kliba 3433 (batch no. 28/98) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum.
- Water : tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h fluorescent light/ 12 h dark (light period between 06.00 and 18.00)

Route of administration:

oral: gavage

Details on route of administration:

Rational for oral by gavage: Accidental oral ingestion is a possible route of human exposure.

Vehicle:

water

Remarks:

bidistilled water

Details on oral exposure:

Dose volume : 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 h and 7 days) of the dose formulations were determined in samples taken during acclimatization. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Ltd, Environmental Chemistry & Pharmanalytics Division, according to a method supplied by the Sponsor (summarised in Analytical Report to: FAT 40'571/A: 28-Day Oral Toxicity (Gavage) Study in the Wistar Rat) applying a calibrated photometric quantification at 500 nm wavelength.

Duration of treatment / exposure:

Test duration: 28 days
duration of recovery : 14 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose.
Rational: Based upon the results of a non-GLP 5-day dose-range finding study (RCC Project 706408) in which Reactive Brown 49 was administered by gavage to 2 rats per group and sex. Animals showed no signs of toxicity up to and including 1000 mg/kg body weight/ day.

No. of animals per sex per dose:

5.
However, an additional 5 rats per sex and group were used at 0 and 1000 mg/kg as a recovery group animals.

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

MORTALITY/VIABILITY
Observations for mortality/viability were recorded twice daily.
CLINICAL LABORATORY INVESTIGATIONS
Blood samples for hematology and clinical biochemistry + Urinalysis : after 4 and 6 weeks.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once weekly (weeks 1-3) thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 5/sex/dose and additional 5/sex for 0 and 1000 mg/kg dose group as recovery animal

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 5/sex/dose and additional 5/sex for 0 and 1000 mg/kg dose group as recovery animal

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks and after 6 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: after 4 weeks
- Dose groups that were examined: all dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (after 4 and 6 weeks)

HISTOPATHOLOGY: Yes (after 4 and 6 weeks from the animals of control and high-dose groups)

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to macroscopical findings

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dark feces were noted from treatment days 7-28 in males and females treated with 200 mg/kg. Black feces were seen from treatment days 4-28 in all animals treated with 1000 mg/kg. The latter finding was also noted in all remaining animals during days 29-32 of the recovery period. Discoloration of the feces was considered to be a common passive effect caused by oral ingestion of a dyestuff, rather than a specific toxic effect of the test article.
Additionally, kinked tail was noted in one male (no. 13) of group 2 during weeks 2 and 3. Dyspnea was noted in one female (no. 55) of group 4 during week 3. These findings were considered to be incidental. All other animals were without findings.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test article-treated animals compared favorably with those of the control animals. Evaluation of the relative food consumption did not indicate test article-related differences between groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slightly elevated absolute reticulocyte count was noted in males and females treated with 1000 mg/kg after four weeks' treatment when compared with the control animals. The difference from the control value attained statistical significance (p<0.05) in males only. Elevated reticulocyte counts persisted after 2 weeks' recovery in males only, and were therefore considered to be incidental. Slightly elevated proportions of high-fluorescence reticulocytes were noted in the females treated at 50 and 1000 mg/kg. In the absence of a dose-response relationship, these findings were considered to be incidental.
The plasma concentrations of methemoglobin was slightly higher in males treated with 1000 mg/kg (p<0.05) for four weeks when compared with the controls. This finding was attributed to a low control value as the post-recovery plasma methemoglobin concentration of the test article-treated group was almost unchanged, whereas the control group was more than twice that of its post-treatment value. The plasma methemoglobin concentration of test articletreated females compared favorably with that of the control females.
The slightly higher absolute white blood cell concentration (p<0.05) noted in the males treated with 1000 mg/kg after 4 weeks' treatment was attributed chiefly to the marginal increase in the absolute lymphocyte count (p<0.05). This finding was considered to be incidental, as the relative lymphocyte counts of the test article-treated and control males compared favorably. After two weeks' recovery, the absolute lymphocyte count of the test article-treated males was lower (p<0.05) than that of the controls. This finding was considered to be incidental as the relative lymphocyte count of the test article-treated males did not significantly differ from that of the control groups.
The statistically significant differences noted in females treated with 50 mg/kg (erythrocyte, platelet and absolute segmented neutrophil counts, p<0.05) and 200 mg/kg (absolute segmented neutrophil counts, p<0.05) were not noted in the females treated with 1000 mg/kg and therefore considered to be incidental.
All other parameters were similar to the control values.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher levels of albumin were noted in females treated with 200 mg/kg and 1000 mg/kg (p<0.05 and 0.01, respectively). Total protein was also significantly increased (p<0.05) in females treated with 1000 mg/kg. These changes were considered to be incidental as similar values were recorded after two weeks' recovery in females treated with 1000 mg/kg and in the absence of any effect upon these parameters in the males.
All remaining clinical biochemistry parameters of test article-treated females were considered to be similar to those of the control females.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The urine osmolality of the males treated with 1000 mg/kg was significantly higher (p<0.05) than that of the control males. This value was considered to be incidental and related to a low control value as the osmolality of the urine of males treated with 1000 mg/kg was similar with that of the historical control data.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

After 4 Weeks:
A small number of statistically significant, differences to the control values were noted in males.
These comprised reduced absolute liver weights (p<0.01 at 50 mg/kg and p<0.05 at 1000 mg/kg) and reduced absolute thymus weights (p<0.01 at 50 mg/kg). In the absence of a doseresponse relationship or microscopical abnormalities, these and all differences noted between the relative organ weights of control and test article-treated males, including those which attained statistical significance, were considered to be incidental. In females, there were no statistically significant differences of absolute or relative organ weights of the test article-treated females when compared with the control females.

After 6 Weeks:
The absolute brain weight of males treated with 1000 mg/kg was increased with statistical significance (p<0.05). This finding was considered to be fortuitous as the brain to body weight ratio of these animals was identical to that of the control males. All other organ weights and ratios compared favorably with those of the control males.
The organ weights of the females treated with 1000 mg/kg were similar to those of the control females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the four-week treatment period, dilation of the renal pelvis was noted in two males of the control group, two males treated with 200 mg/kg and one male treated with 1000 mg/kg. After the two-week recovery period, this finding was noted in one male of the control group, and one male and one female treated with 1000 mg/kg. Dilation of the uterine horns was seen after four weeks' treatment in one female treated with 1000 mg/kg. Discoloration of the seminal vesicles was noted in one control male and thickening of the thyroid gland was noted after four weeks' treatment in one male treated with 50 mg/kg.
All macroscopical abnormalities were considered to be common findings in rats of this strain and age and unrelated to the test article.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following histological abnormalities were noted at 1000 mg/kg bw/d :
- Kidneys: A slightly increased incidence and markedly increased severity of hyaline droplets were noted in males (which is only seen in male rats, as the male rat is prone to hyaline droplet formation in renal proximal tubular cells)
- Stomach: Moderate focal erosion of the nonglandular stomach associated with moderate inflammation, slight squamous cell hyperplasia and moderate hyperkeratosis of the nonglandular stomach was noted in one female rat.
- Lung: Minimal to slight interstitial fibrosis associated with slight, multifocal alveolitis were noted in two females. In the female rat with slight fibrosis, slight aggregations of brownish, pigment-laden alveolar macrophages were seen.
The incidence, severity and morphologic appearance of findings noted in the other organs and tissues examined at the end of the treatment and recovery periods did not distinguish test article-treated rats from controls.
All findings noted at the end of the treatment period were fully reversible within the 14-day recovery period.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

FUNCTIONAL OBSERVATIONAL BATTERY:
No qualitative or quantitative differences between the behavioral parameters of test article treated or control animals were noted during the fourth treatment week.
Grip Strength :
No differences in the grip strength of the fore and hind limbs was observed in the test article-treated animals compared to the control animals.
Locomotor Activity : No effect related to the treatment at 50, 200 and 1000
mg/kg.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

The No Observed Effect Level (NOEL) of is 200 mg/kg body weight/day. As the effects seen in this study are either singular findings or of no toxicological relevance for humans, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.

Executive summary:

A sub-acute toxicity study was carried out with Reactive Brown 49 according to OECD 407 and EU B.7 guideline. The test substance was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated concurrently with the vehicle (bidistilled water) only.

There was no mortality found. There were no treatment related effects seen for clinical signs, body weight, food consumption, haematology, clinical chemistry, urine analysis, organ weights and gross pathology. A number of microscopic findings were considered to distinguish rats treated with the test article at 1000 mg/kg bw/day from those of the control group at the end of the treatment period. These comprised of an increased incidence and severity of hyaline droplets in the kidneys of males treated with 1000 mg/kg bw/day; erosions in the non-glandular stomach, associated with inflammation, squamous cell hyperplasia and moderate hyperkeratosis in one female treated with 1000 mg/kg bw/day; interstitial fibrosis in the lungs of two female rats treated with 1000 mg/kg bw/day, associated in one rat with pigment-laden alveolar macrophages. All other microscopic findings noted a termination of treatment were considered to be incidental findings which occur commonly in rats of this strain and age. All test article-related findings noted at the end of the treatment period were fully reversible during the 14-day recovery period.

Based on the results of this study, 200 mg/kg body weight/day (group 3) of test substance was established as the no-observed effect- level (NOEL). As the above mentioned findings are either singular findings or of no toxicological relevance for humans, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study according to OECD TG 407

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

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:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The male rat has a predisposition of to develop hyaline droplet nephropathy that relates to (1) the large amounts of the low-molecular-weight protein α2U globulin in the glomerular filtrate, (2) the resistance of the globulin to proteolysis, and (3) the low protease activity in the proximal tubule lysosomes (Read, N.G. The role of lysosomes in hyaline droplet nephropathy induced by a variety of pharmacological agents in the male rat. Histochem J 23, 436–443 (1991)). Female rats, and all other species excrete only small amounts of α2U globulin or similar proteins, which are more easily hydrolysed. Hence, this type of hyaline droplet nephropathy is unique to the male rat and of little relevance to man.

Additional information

Sub-acute oral toxicity:

A sub-acute toxicity study was carried out with Reactive Brown 49 according to OECD 407 and EU B.7 guideline. The test substance was administered daily by gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated concurrently with the vehicle (bidistilled water) only.

There was no mortality found. There were no treatment related effects seen for clinical signs, body weight, food consumption, haematology, clinical chemistry, urine analysis, organ weights and gross pathology. A number of microscopic findings were considered to distinguish rats treated with the test article at 1000 mg/kg bw/day from those of the control group at the end of the treatment period. These comprised of an increased incidence and severity of hyaline droplets in the kidneys of males treated with 1000 mg/kg bw/day; erosions in the non-glandular stomach, associated with inflammation, squamous cell hyperplasia and moderate hyperkeratosis in one female treated with 1000 mg/kg bw/day; interstitial fibrosis in the lungs of two female rats treated with 1000 mg/kg bw/day, associated in one rat with pigment-laden alveolar macrophages. The male rat has a predisposition of to develop hyaline droplet nephropathy that relates to (1) the large amounts of the low-molecular-weight protein α2U globulin in the glomerular filtrate, (2) the resistance of the globulin to proteolysis, and (3) the low protease activity in the proximal tubule lysosomes (Read, N.G. The role of lysosomes in hyaline droplet nephropathy induced by a variety of pharmacological agents in the male rat. Histochem J 23, 436–443 (1991)). Female rats, and all other species excrete only small amounts of α2U globulin or similar proteins, which are more easily hydrolysed. Hence, this type of hyaline droplet nephropathy is unique to the male rat and of little relevance to man. All other microscopic findings noted a termination of treatment were considered to be incidental findings which occur commonly in rats of this strain and age. All test article-related findings noted at the end of the treatment period were fully reversible during the 14-day recovery period.

Based on the results of this study, 200 mg/kg body weight/day (group 3) of test substance was established as the no-observed effect- level (NOEL). As the above mentioned findings are either singular findings or of no toxicological relevance for humans, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.

 

Repeat dose inhalation toxicity:

Currently no study to assess the repeated dose inhalation toxicity potential of Reactive Brown 49 is available. However, the vapour pressure for the substance can be considered low (1.22 x 10^-23Pa) owing to the high melting point (>400 °C). Hence the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, 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. Further the chemical is found to have water solubility of 277 g/L and n-octanol/water partition coefficient (log P) of -4.5, indicating it being too hydrophilic to cross the lipid rich environment of the stratum corneum, hence in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption.No systemic toxicity was observed when Reactive Brown 49 was administered to Wistar rats upto 200 mg/kg bw/day via gavage in a 28 day repeated dose oral toxicity study. Similarly, absence of systemic toxicity in skin irritation as well as sensitization studies, further supports the conclusion that low toxicity is expected for the chemical via the inhalation route. Further experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon inhalation exposure and not after systemic application.Taking into consideration the above arguments, low toxicity potential is expected on repeated exposure of Reactive Brown 49 via inhalation route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via inhalation route for Reactive Brown 49 is considered to be scientifically not necessary.

 

Repeat dose inhalation toxicity:

 

Currently no study to assess the repeated dose dermal toxicity of Reactive Brown 49 is available. However, the molecular weight of the chemical is 823 g/mol, indicating it being too large for dermal absorption. It has water solubility of 277 g/L and n-octanol/water partition coefficient (log P) of -4.5, indicating it being too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance will be low. No systemic toxicity was observed when Reactive Brown 49 was administered to Wistar rats upto 200 mg/kg bw/day via gavage in a 28 day repeated dose oral toxicity study. Similarly, absence of systemic toxicity in skin irritation as well as sensitization studies, further supports the conclusion that low toxicity is expected for the chemical via the dermal route. Further experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon dermal exposure and not after systemic application. Taking into consideration the above arguments, low toxicity potential is expected on repeated exposure via dermal route and safety for human health can be estimated using the principles of route to route extrapolation. Hence, the conduct of repeated dose toxicity study via dermal route for Reactive Brown 49 is considered to be scientifically not necessary.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, the Reactive Brown 49 does not considered to be classified for specific target organ toxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.