2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:4)

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Data is from publication.

Data source

Reference

Reference Type:

publication

Title:

Long-term toxicity study of black PN in mice

Author:

J.J.-P. Drake, K.R. Butterworth, I.F. Gaunt, P. Grasso

Year:

1977

Bibliographic source:

Food and Cosmetics Toxicology Volume 15, Issue 6, December 1977, Pages 503-508

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Evaluation on the testicular toxicity and esterogenic activity of test chemical in male and female mice.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

EC Number:

219-746-5

EC Name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

Cas Number:

2519-30-4

Molecular formula:

C28H21N5O14S4.4Na

IUPAC Name:

tetrasodium 4-acetamido-5-hydroxy-6-({7-sulfonato-4-[(4-sulfonatophenyl)diazenyl]-1-naphthyl}diazenyl)naphthalene-1,7-disulfonate

Details on test material:

- Name of test material (as cited in study report):Black PN- Molecular formula :C28H21N5O14S4.4Na- Molecular weight :867.6873 g/mol- Substance type:Organic

Test animals

Species:

mouse

Strain:

other: CFW

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: No data available- Age at study initiation: No data available- Weight at study initiation: yes - Fasting period before study: No data available- Housing: They were caged in groups of 15 in a room .- Diet (e.g. ad libitum): Oxoid pasteurized Breeding diet - Water (e.g. ad libitum): water available- Acclimation period: No data availableENVIRONMENTAL CONDITIONS- Temperature (°C): No data- Humidity (%):50-60%- Air changes (per hr): No data- Photoperiod (hrs dark / hrs light): 21 ± 1°CIN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

other: Oxoid pasteurized Breeding diet

Details on exposure:

DIET PREPARATION- Rate of preparation of diet (frequency):- Mixing appropriate amounts with (Type of food): Oxoid pasteurized Breeding diet- Storage temperature of food:VEHICLE- Justification for use and choice of vehicle (if other than water):- Concentration in vehicle: 0.1, 0.25,0.5 or 1.0%- Amount of vehicle (if gavage):- Lot/batch no. (if required):- Purity:

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Daily

Details on study schedule:

Groups of 30 male and 30 female mice were given diets containing 0.1, 0.25, 0.5 or 1.0% test chemical for 80 wk, with groups of 60 mice of each sex as controls. Effects on mortality ,body-weight gain, hematology, organ weights and histopathology was observed.

Doses / concentrations

Remarks:

Doses / Concentrations:0, 0.1, 0.25,0.5 and 1.0%Basis:nominal in diet

No. of animals per sex per dose:

0%-60 male and 60 female0.1%-30 male and 30 female0.25% -30 male and 30 female0.5-30 male and 30 female1%-30 male and 30 female

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

The general condition and behavior of the animals were observed frequently and any mouse that showed signs of ill-health was isolated, to be returned to its cage on recovery or to be killed If its condition deteriorated. Body weight-The mice were weighed at the start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment. Hematology-Blood samples were taken at wk 28 and 55 from the caudal vein of ten males and ten females from the control group and from the groups given the 0.5 and 1% dietary levels. At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy. The samples were examined for haemoglobin concentration and packed cell volume, as well as for counts of erythrocytes and leucocytes. In addition, the methaemoglobin concentrations were determined in the samples collected at 80 wk. Preparations for counting the reticuiocytes and the different types of leucocytes were made but, in the absence of consistent effects on the other measurements, these counts were not carried out.Urine analysis-During wk 28, urine samples were collected over a 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels (0.5 and 1%) of Black PN. These samples were examined for protein, reducing substances, bile salts and blood as well as for colour, pH and microscopic constituents. Organ weight was also measured.

Oestrous cyclicity (parental animals):

No data available.

Sperm parameters (parental animals):

No data available.

Litter observations:

No data available.

Postmortem examinations (parental animals):

The animals were killed by exsanguinations from the aorta under sodium pentobarbitone anesthesia following an overnight period without food. At autopsy, macroscopic abnormalities were recorded and the brain, heart, liver, spleen, kidneys, adrenal glands and gonads were weighed. Samples of these organs and of salivary glands, pituitary, thyroid, thymus, various lymph nodes, pancreas, urinary bladder, lungs, stomach, duodenum, ileum, colon, caecum,rectum, striped muscle (hind limb), spinal cord, uterus, aortic arch and any other tissue that appeared abnormal were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin. All tissues from the control mice and from those fed diet containing 1% Black PN were examined histologically. At the lower dose levels, the examination was confined to the liver, kidneyand any tissues seen to be abnormal at autopsy

Postmortem examinations (offspring):

No data available

Statistics:

Statistics was observed by different method for different parameters

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) Mortality- No statistically significant differences between the number of deaths in the control mice and those given Black PN was observed.Clinical sign-No significant change were observed in treated group compare to control group at any dose level (0, 0.1, 0.25,0.5 and 1.0%).BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)No significant change was observed in treated group compare to control group at any dose level (0, 0.1, 0.25, 0.5 and 1.0%).TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)No data availableORGAN WEIGHTS (PARENTAL ANIMALS)There were slight difference in mean organ weights between treated and control animals.A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1% Black PN. This difference, which did not occur in the males, was only marginally significantand there was no significant difference when the weights were expressed relative to body weight By contrast, the relative brain weight of females fed 0.25% Black PN was higher than the control.Liver weights of female but not of male mice fed 0.25% Black PN were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5%level, at which a higher value was recorded for the treated mice. The only other signifi cant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25% Black PN. The changes in organ weight showed no doserelated effects. Moreover the isolated changes seen at the lower levels were not found in both sexes and were not evident when expressed relative to body weight. It is considered that these random findings were not associated with Black PN treatment.HISTOPATHOLOGY (PARENTAL ANIMALS)No significant change was observed in treated group compare to control group at any dose level (0, 0.1, 0.25, 0.5 and 1.0%).OTHER FINDINGS (PARENTAL ANIMALS)Hematology - The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing 0.5 and 1-0% Black PN than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% Black PN in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the colouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1% Black PN were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80. This suggests that some degree of haemoconcentration in this group may have led to artificially high control values. It is suggested, therefore, that the observed differences may have been related to technical problems rather than to an effect of treatment.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effect were observed on mortality ,body-weight gain, hematology, organ weights and histopathology

Remarks on result:

other: No toxic effect were observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

other: Not specified

Based on:

not specified

Sex:

not specified

Remarks on result:

other: Not secified

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Relative organ weights of mice fed diets containin# 0-1% Black PN for 80 wk

Sex /dietery level %	No. Of rats examined	Brain	Heart	Liver	Spleen	Kidney	Adrenals	Gonades	Terminal body weight
0	40	1.07	0.64	5.47	0.38	1.67	27.5	0.45	35
0.1	20	1.21	0.59	5.84	0.45	1.50	27.8	0.41	36
0.25	20	1.15	0.63	5.88	0.44	1.62	27.1	0.45	33
0.5	12	1.32	0.71*	5.80	0.51	1.77*	29.6	0.49	32
1	23	1.28	0.63	5.29	0.39	1.60	28.5	0.48	35
Female
0	32	1.53	0.57	5.80	0.51	1.46	40.6	84.2	29
0.1	11	1.53	0.61	5.87	0.56	1.56	37.8	84.8	28
0.25	17	1.72	0.64*	5.29	0.41	1.51	45.9	100.8	26
0.5	15	1.55	0.56	5.43	0.54	1.41	42.8	101.2	27
1	18	1.53	0.53	5.27	0.47	1.40	42.3	87.2	30
Weights are expressed in mg/100g body weight. Weights of female gonads only are expressed in mg/100g body weight. Values are means for the numbers of mice shown and those marked with an asterisk differ significantly (Student's t test) from those of controls: *P < 0.05.

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1300 mg/kg/day for test chemical in male and femle CFW mice for 80 weeks by oral feed.

Executive summary:

Reproductive study was conducted for test chemical in male and female CFW mice for 80 weeks by oral feed. When Groups of 30 male and 30 female CFW mice were given diets containing 0.1, 0.25, 0.5 and 1.0% test chemical for 80 wk, with groups of 60 mice of each sex as controls.There were no dose-related effects on body-weight gain, haematology and organ weights. The incidence of histopathological findings was not altered by the feeding of test chemical. Therefore it is considered that test chemical to the diet of male and female CFW mice at levels up to 1% (equivalent to approximately 1300 mg/kg/day) for 80 wk does not induce any reprotoxic effect.