2-Naphthalenesulfonic acid, 6-amino-5-[2-[4-[[4-[bis(2-hydroxyethyl)amino]-6-[(2-sulfoethyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:3)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 1996 to 29 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed to valid guidelines and the study was conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese guideline Notification No's; No. 700 Environment Agency, No. 1039 Ministry of Health and Welfare and No. 1014 Ministry of International Trade and Industry (1986)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

419-720-5

EC Name:

-

Cas Number:

182061-89-8

Molecular formula:

C25H26N9O12S3 . 3 Na

IUPAC Name:

trisodium 6-amino-5-{2-[4-({4-[bis(2-hydroxyethyl)amino]-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazen-1-yl}-4-hydroxynaphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): M-377
- Physical state: solid
- Appearance: dark purple powder
- Storage condition of test material: room temperature and protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl CD (SD) BR
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: Mean male body weight 202 g (193 - 217 g); Mean female body weight 169 g (157 - 182 g)
- Housing: individually in suspended wire mesh cages
- Diet: pelleted maintenance diet, ad libitum
- Water: filtered tap water (0.22 µm), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 8 February 1996 to 29 March 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(reverse osmosis purified)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was administered as a solution in the vehicle. The test material was dissolved in order to achieve concentrations of 30, 90 and 270 mg/mL and then homogenised with a magnetic stirrer. The quantities of test material used were calculated in order to take account of its purity (91.6%). The test material preparations were made for up to 9 days and kept stored at 4 °C and protected from light, until administration.

STABILITY:
The stability of the solutions was checked. Samples of the 30 mg/mL and 270 mg/mL solutions were taken in duplicate 5 hours after preparation and again after 4 and 9 days. Samples were stored at 4 °C and protected from light. These samples were then stored frozen at -20°C pending the last sampling occasion (day 9) when all samples were assayed together according to the method outlined below.

ADMINISTRATION:
The quantity of test material was adjusted according to the most recently recorded bodyweight.
A constant dosage volume of 5 mL/kg/day was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each group, including the control, the preparations intended for use in weeks 1 and 4 were sampled for analyses.
The solution was stirred with a magnetic stirrer. 1 mL was diluted tenfold with water. After vortex mixing, serial dilutions were carried out with water ranging from 0 – 50 μg/mL. An aliquot of the test material solutions was analysed by UV spectrometry and a calibration curve was obtained by linear regression analysis of UV absorbance at 518 nm versus concentration of standard solutions for concentrations ranging from 0 – 50 μg/mL.
The regression analysis of the calibration data gave an equation as follows:
Y = a X + b
Where
Y = absorbance of test material
X = concentration of test material (μg/mL)
a = slope
b = intercept
The concentrations of test material were calculated using this equation.

Experimental conditions:
- Instrumentation: Shimadzu UV-260 (Roucaire) double beam
- Cell: quartz cell
- Path length: 1 cm
- Blank: water
- Wavelength: 518 nm

Validation:
- Specificity: Demonstrated as no absorbance measured in water.
- Linearity: Satisfactory linearity was obtained in the range of 0 to 50 µg/mL for the test material (4 levels in duplicate; 5, 10, 20 and 50 µg/mL) The coefficient of determination was 0.9994.
- Repeatability: Replicate analysis (n = 10) of a solution containing 20 µg/mL of the test material was satisfactory since the coefficient of variation obtained was 0.3%.

Duration of treatment / exposure:

28 days, with an additional 2 week reversibility period (animals not treated during this time)

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 450, 1350 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females (0 and 1350 mg/kg/day)
5 males and 5 females (150 and 450 mg/kg/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were selected on the basis of a 7 day preliminary range finding study during which none of the animals died and no clinical signs were observed. Animals received the same doses during the preliminary study.
- Rationale for selection of route of exposure: The oral route was selected since it is one of the route of exposure in humans.
- Rationale for animal assignment: During the acclimatisation period , the required number of animals were selected according to body weight and clinical conditions, and allocated, by sex to the groups, according to computerized randomisation.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for mortality and morbidity at least twice a day during the treatment period and at least once a day during the reversibility period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day before allocation into groups, on the first day of treatment and once a week until the end of the reversibility period.

FOOD CONSUMPTION
- Food consumption for each animal was determined once a week, until the end of the study (including the reversibility period). Food intake per animal and per day was calculated using the amount of food given and left in each feeder.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 27
- Anaesthetic used for blood collection: Yes (light ether anaesthetic)
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: 5 males and 5 females per treatment level and control
- Parameters checked included: erythrocytes, haemoglobin, mean cell volume, packed cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, thrombocytes, leucocytes, differential white cell count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 27
- Animals fasted: Yes (overnight period of at least 14 hours)
- How many animals: 5 males and 5 females of per treatment level and control
- Parameters checked included: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase

URINALYSIS: Yes
- Time schedule for collection of urine: day 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight period of at least 14 hours)
- Parameters checked included: volume, pH, specific gravity, proteins, glucose, ketones, blood, appearance, and colour

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: Five animals per sex per dose were sacrificed on completion of treatment after at least a 14 hour fasting period. On completion of the reversibility period the remaining 5 animals per sex in the control and high dose group were sacrificed.

ORGAN WEIGHTS
For all animals killed at the end of the treatment or reversibility period, the body weight was recorded before necropsy and the organs, were weighed wet as soon as possible after dissection. Paired organs were weighed separately (except for thyroids with parathyroids).
Organs weighed; adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and the thyroids with parathyroids.

GROSS PATHOLOGY: Yes
- A complete macroscopic examination was performed on all animals.

TISSUE PRESERVATION
For all animals, the macroscopic lesions and the tissues specified below were preserved in 10 % buffered formalin (except for the eyes which were fixed in Davidson’s fixative.
- Tissues examined included: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, eyes with Harderian glands, heart, ileium,, Jejenum, kidneys, liver, lungs with bronchi, lymph nodes (mandibular, mesenteric), mammary glands, oesophagus, optic nerve, ovaries, pancreas, pituitary gland, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus (horns and cervix), vagina.

HISTOPATHOLOGY: Yes
Microscopic examination was performed on:
- All macroscopic lesions and tissues listed below for animals of the control and high dose groups (1350 mg/kg/day) killed at the end of the treatment period.
- All macroscopic lesions and organs identified as showing effects in the low and intermediate dose groups (150 and 450 mg/kg/day).
- All macroscopic lesions and organs identified as showing effects in the control and high dose groups (1350 mg/kg/day) killed at the end of the reversibility period.
- Tissues examined included: macroscopic lesions, adrenals, epididymides, heart, kidneys, liver, ovaries, spleen and testes.
- All tissues were embedded in paraffin wax, sectioned at 4 microns and stained with hematoxylin-eosin.

Statistics:

Kolmogorov-Lilliefore's test for normality was performed. Bartlett's test or Fischer's test was performed where there was a normal distribution. Dunnett's test was then performed on homogenous data and Dunn's test, or Mann-Whitney's test, was performed on heterogeneous data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
None of the animals died during the study and no clinical signs were noted.
The following observations were noted during the study, however they were considered to be related to elimination of the test material or its metabolites and not of toxicological significance:
- Pink coloured faeces in all treated animals (from day 2).
-Pink coloured urine in 3/5 males given 450 mg/kg/day, and 4/10 males and 1/10 females given 1350 mg/kg/day. At the time of urine collection during week 4, pale to dark pink urine colouration was noted in 2/5 males given 150 mg/kg/day, in 4/5 males and 2/5 females given 450 mg/kg/day and in all animals given 1350 mg/kg/day.
- Pink coloured extremities in 6/10 males and 7/10 females given 1350 mg/kg/day (from weeks 3 or 4).
- Pink coloured tail in 3/10 males and females given 1350 mg/kg/day (from week 3 or 4).
During the reversibility period, the coloured faeces and urine were no longer noted in the animals of the high dose group, and the only sign observed was pink coloured tails in 4/5 and d5/5 treated males and female, respectively.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was comparable in control and treated animals

FOOD CONSUMPTION
Food consumption was considered to be similar in control and treated animals

HAEMATOLOGY
No test material-related changes were noted at the end of the treatment period. The only differences from controls (eosinophil count in females) were slight and not dose related.

CLINICAL CHEMISTRY
No test material-related changes were noted at the end of the treatment period. The only differences from controls (increase in aspartate aminotransferase activity in males, albumin/globulin ratio in females) were only noted in a few animals and were within or close to historical values. Therefore, they were not considered to be toxicologically significant.

URINALYSIS
No test material-related changes were noted at the end of the treatment period other than the change in colour.

ORGAN WEIGHTS
The following differences in organ weights were noted:
- Statistically significant higher mean absolute and relative kidney weights in females at 450 mg/kg/day (absolute: 11%, relative: 20%) and at 1350 mg/kg/day (absolute: 25%, relative: 29%).
- Statistically significant higher mean absolute and relative liver weights in females at 1350 mg/kg/day (absolute: 19%, relative: 23%).
- Higher mean absolute and relative adrenal weights in males at 450 mg/kg/day (absolute: 8%, relative: 20%) and at 1350 mg/kg/day (absolute: 21, relative: 20%).
- Higher mean absolute and relative thymus weights in males at 1350 mg/kg/day (absolute: 20%, relative: 19%).
- Higher mean absolute and relative thyroid weights in males and females given 1350 mg/kg/day (absolute: 9% and 12% and absolute: 17% and 14% respectively).

The higher adrenals weight was considered to be treatment related and was well correlated with cortical cell hypertrophy found in these animals.
After the 2 week observation period following treatment, higher absolute and relative adrenal weight was noted in the males (20% and 19%) and females (25% and 17%) of the same group indicating partial reversibility. The differences in kidneys, liver, thyroids and thymus were considered to be without relationship to treatment with the test material as they were not associated with relevant histopathological findings.

GROSS PATHOLOGY
No test material-related changes were noted at gross pathology apart from observations related to colouration of the mucosa and other parts of the gastrointestinal tract. These changes were considered to be related to the physical properties of the test material.
- Pink colouration of the mucosa of the different parts of the gastro-intestinal tract was noted among the males and females given 450 mg or 1350 mg/kg/day.
- Pink colouration of the extremities and/or hair was found in 1/5 females given 450 mg/kg/day and in 1/5 males and 2/5 females given 1350 mg/kg/day.
- Pink colouration of the tail was noted.in 1/5 females given 150 mg/kg/day, in 2/5 males given 450 mg/kg/day and in 5/5 males and 4/5 females given 1350 mg/kg/day.
- Partial reversibility was found after the 2-week reversibility period, as pink colouration of the extremities was still observed among the animals of both sexes (3/5 males and 4/5 females) given 1350 mg/kg/day.

The few other macroscopic findings noted were those which are commonly recorded spontaneously in the untreated laboratory rat of this strain and age and none was considered to be of toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Slight to moderate cortical cell hypertrophy in the zona fasciculata and the zona reticularis of the adrenal glands was seen in all animals dosed at 1350 mg/kg/day at the end of the treatment period. This was considered to be treatment-related. This finding was not observed on the animals given 150 or 450 mg/kg/day.
Following the 2 week observation period, this finding was detected to a lesser degree in 2/5 males and 4/5 females dosed at 1350 mg/kg/day, indicating partial reversibility of this change.
The other microscopic findings were all spontaneously recorded in the rat of this strain and age and considered to be of no toxicological importance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Higher adrenal weight correlated with cortical cell hypertrophy was observed (a partial reversibility of this finding was noted after 2 weeks).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean Organ Weightsat Necropsy

			Weight (mean in g)
Sex			Males				Females
Organ	Dose group		1	2	3	4	1	2	3	4
	No. of animals		5	5	5	5	5	5	5	5
Final body weight		Absolute	361.6	340.2	337.5	365.9	227.3	214.4	210.7	221.0
Heart		Absolute	1.40	1.35	1.31	1.38	0.898	0.870	0.938	0.975
		Relative	0.388	0.397	0.389	0.377	0.396	0.407	0.445	0.441
Liver		Absolute	11.87	10.50	10.56	12.08	7.21	7.19	6.22	8.60*
		Relative	3.2	3.09	3.13	3.31	3.18	3.35	2.95	3.91**
Adrenal glands		Absolute	0.073	0.065	0.079	0.088	0.072	0.079	0.070	0.075
		Relative	0.020	0.019	0.024	0.024	0.032	0.037	0.033	0.034
Kidneys		Absolute	3.07	2.90	3.10	3.25	1.70	1.77	1.89	2.12**
		Relative	0.847	0.852	0.918	0.889	0.750	0.825	0.897#	0.965##
Spleen		Absolute	0.732	0.690	0.614	0.692	0.481	0.483	0.467	0.441
		Relative	0.202	0.205	0.182	0.190	0.213	0.226	0.221	0.199
Testes/ ovaries		Absolute	3.23	3.34	3.25	3.19	0.142	0.126	0.116	0.153
		Relative	0.892	0.983	0.967	0.873	0.063	0.059	0.055	0.069
Epididymides		Absolute	0.986	0.984	0.970	1.03	Not applicable
		Relative	0.272	0.289	0.288	0.281	Not applicable
Brain		Absolute	2.04	2.03	2.02	2.07	0.19	1.88	1.92	1.88
		Relative	0.563	0.599	0.600	0.568	0.838	0.877	0.913	0.857
Thymus		Absolute	0.505	0.473	0.452	0.605	0.528	0.389	0.415	0.461
		Relative	0.139	0.138	0.134	0.166	0.233	0.181*	0.196	0.207
R & L thyroid glands		Absolute	0.023	0.021	0.023	0.025	0.017	0.017	0.017	0.019
		Relative	0.006	0.006	0.007	0.007	0.007	0.008	0.008	0.008

Relative organ weight: mean % of body weight.

Dunnett’s test based on pooled variances; * (p = 0.05), ** (p = 0.01).

Dunn’s test; # (p = 0.05), ## (p = 0.01).

 

Table 2: Mean Organ WeightsAfter the Recovery Period

			Weight (mean in g)
Sex			Male		Female
Organ	Dose group		1	4	1	4
	No. of animals		5	5	5	5
Final body weight		Absolute	419.9	410.4	236.1	240.1
Heart		Absolute	1.50	1.38	0.854	0.886
		Relative	0.356	0.336	0.363	0.370
Liver		Absolute	13.04	12.40	6.89	7.59
		Relative	3.10	3.01	2.91	3.15
Adrenal glands		Absolute	0.066	0.079*	0.064	0.080**
		Relative	0.016	0.019*	0.028	0.033*
Kidneys		Absolute	3.34	3.23	1.78	2.19**
		Relative	0.797	0.785	0.759	0.917*
Spleen		Absolute	0.702	0.781	0.460	0.567*
		Relative	0.167	0.190*	0.195	0.237*
Testes/ ovaries		Absolute	3.43	3.42	0.114	0.121
		Relative	0.818	0.835	0.149	0.051
Epididymides		Absolute	1.17	1.20	Not applicable
		Relative	0.281	0.293	Not applicable
Brain		Absolute	2.09	2.10	1.94	1.93
		Relative	0.500	0.512	0.829	0.804
Thymus		Absolute	0.474	0.452	0.355	0.433
		Relative	0.114	0.110	0.150	0.179
R & L thyroid glands		Absolute	0.023	0.024	0.017	0.017
		Relative	0.006	0.006	0.007	0.007

Relative organ weight: mean % of body weight.

Student’s Test; * (p = 0.05), ** (p = 0.01).

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level of the test material was determined to be 450 mg/kg/day, based on higher adrenal weight correlated with cortical cell hypertrophy observed at 1350 mg/kg/day. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The oral repeat dose toxicity of the test material was determined in accordance with standardised guidelines OECD 407 and EU Method B.7. Two groups of five male and five female Sprague-Dawley rats received the test material, daily by gavage at 150 and 450 mg/kg/day, and one group of ten males and ten females was given 1350 mg/kg/day for 4 weeks. An additional group of ten males and ten females received the vehicle alone (purified water) under the same conditions, and acted as a control group. On completion of the 4-week treatment period, the first five animals of each sex in the control and high dose-level group (1350 mg/kg/day) were kept for a 2-week reversibility period. Clinical observations, bodyweights and food consumption were measured throughout the study. At the end of the scheduled period, the animals were killed and subjected to an examination post mortem (day 27). Blood and urine samples were taken for analysis, selected organs were weighed and specified tissues were taken for subsequent histopathology examination. Dosing preparations were analysed for achieved concentration, homogeneity and stability and were considered to be satisfactory.

During the study none of the animals died and no clinical signs were recorded. Body weight gain, and food consumption of treated animals was comparable to the controls. No test material related changes were noted at the end of the treatment period following haematology, blood biochemistry and urinanalysis investigations. No abnormalities were noted at gross pathology other than colouration effects caused by the physical properties of the test material.

The higher absolute and relative adrenal weights noted at the end of the treatment period in males dosed at 450 and 1350 mg/kg/day and at the end of the reversibility period in males dosed at 1350 mg/kg/day were considered to be treatment related. This weight increase was well correlated with cortical hypertrophy found in these animals. This finding was noted with a lower incidence and severity after the 2 week observation period. These observations were not observed in animals dosed at 150 or 450 mg/kg/day.

Under the conditions of the study the No Observed Adverse Effect Level of the test material was determined to be 450 mg/kg/day.