6-[(2,4-diaminophenyl)azo]-3-[[4-[[4-[[7-[(2,4-diaminophenyl)azo]-1-hydroxy-3-sulpho-2-naphthyl]azo]phenyl]amino]-3-sulphophenyl]azo]-4-hydroxynaphthalene-2-sulphonic acid

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviwed juornal

Data source

Reference

Reference Type:

publication

Title:

Acute (Mouse and Rat) and Short-term (Rat) Toxicity Studies on Black PN

Author:

I.F. Gaunt, Madge Farmer, P. Grasso, S.D. Gangolli

Year:

1967

Bibliographic source:

Food and Cosmetics Toxicology Volume 5, 1967, Pages 171-177

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 of Black PN in rats .

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
- Physical state: no data
- Analytical purity:83.6%
- Impurities (identity and concentrations): Dye content (70 % min .); chloride and sulphate as sodium salts plus moisture (30% max); subsidiary dyes (15 % max); intermediates (1% max); water-insoluble material (0.2% max); arsenic (5 ppm max); lead (20 ppm max); antimony, copper, chromium, zinc, barium sulphate (100 ppm max taken separately or 200 ppm max taken together).

Specific details on test material used for the study:

- 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:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data available.
- Age at study initiation: (P) x wks; (F1) x wks- No data available.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g- No data available.
- Fasting period before study: No data available.
- Housing: Rats were housed four /cage.
- Diet (e.g. ad libitum): Spillers Small Laboratory Animal Diet ad libitum.
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available.
- Humidity (%):No data available.
- Air changes (per hr): No data available.
- Photoperiod (hrs dark / hrs light): No data available.

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Spillers Small Laboratory Animal Diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): Spillers Small Laboratory Animal Diet
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 0.3, 1.0 and 3.0 %
- 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:

90 days

Frequency of treatment:

Daily

Details on study schedule:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

3 000 mg/kg bw/day

No. of animals per sex per dose:

Total no. of animals-128
0 mg/kg/day- 16 male and 16 female
300 mg/kg/day-16 male and 16 female
1000 mg/kg/day-16 male and 16 female
3000 mg/kg/day-16 male and 16 female

Control animals:

yes, concurrent vehicle

Details on study design:

not specified

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

Parental animal: observation and examination-
Body weight and food intake were observed weekly.

Hematology-Terminal haematological investigations involved the determination of haemoglobin and methaemoglobin levels, haematocrits, erythrocyte, reticulocyte, and total and differential leukocyte counts. Erythrocytes were examined for the presence of Heinz bodies.

Clinical chemistry- Liver and kidney function tests were carried out terminally. Levels of serum glutamic-oxaloacetic and glutamic-pyruvic transaminase and of blood urea nitrogen were determined.

Urine analysis- The urine was examined at wk 6 and 12 for colour, pH, microscopic constituents and content of protein, reducing substances, bile salts and blood, and activity of glutamicoxaloacetic Transaminase. A concentration test conducted terminally, involved measurements of volume and specific gravity of the urine excreted during a 6-hr period of water deprivation and during a 4-hr period beginning 16 hr after a water load of 25 ml/kg.

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

not specified

Postmortem examinations (parental animals):

At autopsy, the gross appearance of the organs and the weights of the liver, kidneys, brain, spleen, heart, adrenals and gonads were noted.
Histopathology- Paraffin wax sections of these organs together with a wide range of other organs were stained with haematoxylin and eosin

Postmortem examinations (offspring):

not specified

Statistics:

Statistics was observed by Litchfield, J. T, Jr. & Wilcoxon, F. (1949) method.

Reproductive indices:

not specified

Offspring viability indices:

not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant changes were observed at any dose level in both treated male and female rats compare to control. Except for two females, one was from control group and other was from 300 mg/kg/day showed unaccountable tremors and ataxia. They were killed at day 17. The nervous disorder observed in two female rats at an early stage of the experiment cannot be attributed to Black PN since the two animals were in the control and lowest level groups, respectively. Moreover, no comparable effect was seen after more prolonged treatment or at 3000 mg/kg bw.

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant growth retardation was observed in males at the 3000 mg/kg/day level and it associated with a reduced food intake.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food intake were observed in treated rat at 3000 mg/kg bw

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No Hematological changes were observed in treated rats.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No clinical chemistry changes were observed in treated rats.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No urine analysis changes were observed in treated rats.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Significant change was observed in the foci of inflammatory cell infiltration of the myocardium in treated males at 3000 mg/kg/day. The foci of inflammatory cell infiltration of the myocardium which occurred occasionally in males of the 3000 mg/kg/day group were of spontaneous origin. Similar Historical control data was found in the Wilens & Sproul (1938) study.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

Remarks on result:

other: No significant increase in the relative weights of the testes.

2

Dose descriptor:

NOEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: No significant change in the gonades

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

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 specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Body weight and food consumption:

Mean values of body weight in rats fed Black PN at 0-3% (1000mg/kg/day ) of the diet for 90 days

Dietary level	Body weight (g) at wk
	0‡	4	8	12
	Males
0.0	102	276	352	387
0.3	108	289	364	405
1.0	107	275	345	374
3.0	105	267	327	357٭*
	Female
0.0	101	194	226	235
0.3	99	190	220	233
1.0	99	188	215	235
3.0	100	193	222	225

"†'Calculated from data on body weight and food consumption.

"‡'Day 1 of feeding.

Values of body weight are the means for groups of 16 animals.

Value marked with asterisk differs significantly from that of the control:**P <0 01.

Food consumption-

Food consumption in rats fed Black PN at 0-3% of the diet for 90 days

Dietary level	Food consumption (g/rat/day) at wk
	0‡	4	8	12
	Males
0.0	14.4	18.0	19.7	19.2
0.3	14.3	16.2	19.0	19.5
1.0	15.1	17.7	19.0	17.1
3.0	14.4	19.7	16.1	15.9
	Female
0.0	12.7	13.7	11.4	10.1
0.3

	289	364	405
1.0	107	275	345	374
3.0	105	267	327	357٭*
	Female
0.0	101	194	226	235
0.3	99	190	220	233
1.0	99	188	215	235
3.0	100	193	222	225

"†'Calculated from data on body weight and food consumption.

"‡'Day 1 of feeding.

Values of body weight are the means for groups of 16 animals.

Value marked with asterisk differs significantly from that of the control:**P <0 01.

Food consumption-

Food consumption in rats fed Black PN at 0-3% of the diet for 90 days

Dietary level	Food consumption (g/rat/day) at wk
	0‡	4	8	12
	Males
0.0	14.4	18.0	19.7	19.2
0.3	14.3	16.2	19.0	19.5
1.0	15.1	17.7	19.0	17.1
3.0	14.4	19.7	16.1	15.9
	Female
0.0	12.7	13.7	11.4	10.1
0.3	12.8	9.9	12.8	10.8
1.0	13.7	11.3	12.7	10.5
3.0	13.5	12.6	12.7	10.2

Intake of colouring in rats fed Black PN at 0-3% of the diet for 90 days.

 

Dietary level	Intake of colouring (g/kg/day) t at wk
	0‡	4	8	12
	Males
0.0	-	-	-	-
0.3	0.40	0.17	0.16	0.14
1.0	1.41	0.64	0.55	0.46
3.0	4.11	2.20	1.47	1.34
	Female
0.0	-	-	-	-
0.3	0.39	0.16	0.17	0.14
1.0	1.38	0.60	0.60	0.43
3.0	4.05	1.95	1.71	1.36

 

"†'Calculated from data on body weight and food consumption.

"‡'Day 1 of feeding.

Values of body weight are the means for groups of 16 animals. Values of food consumption are the means for four cages of four animals. Although growth and food consumption were recorded ‘weekly, values at monthly intervals are included in the Table.

Value marked with asterisk differs significantly from that of the control:**P <0.01.

Relative organ weight
Sex/Dietary level(%)	Brain	Heart	Liver	Spleen	Kidney Left Right		Adrenal		Gonades
Male
0.0	0.55	0.30	3.14	0.15	0.27	0.28		0.017	0.45
0.3	0.51	0.30	2.97	0.17	0.27	0.28		0.017	0.43
1	0.55	0.30	3.10	0.16	0.29	0.29		0.018	0.49
3	0.57	0.30	3.12	0.16	0.30**	0.31***		0.019	0.50*
Female
0.0	0.80	0.34	3.29	0.19	0.30	0.32		0.036	0.048
0.3	0.82	0.36	3.04*	0.20	0.30	0.32		0.037	0.052
1	0.81	0.35	3.11	0.21	0.32	0.33		0.037	0.052
3	0.81	0.33	2.95*	0.18	0.31	0.32		0.035	0.046
Values are the means of groups of 16 ammals and those marked with asterisks differ significantly from those of controls: *P <0"05; **P <0.01 ; ***P <0 001. Decreased liver weight in these instances arose from the fact that autopsles were performed late in the day, so that the animals were not fasted overmght, in accordance with the standard procedure but were only fasted during the day.

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1000 mg/kg bw for male and 3000 mg/kg bw for female rats when treated with Black PN orally in feed for 90 days.

Executive summary:

In sub-chronic toxicity study, male and female rat were treated with Black PN in the concentration of 0,300, 1000 and 3000 mg/kg bw orally in feed for 90 days. No significant changes were observed at any dose level in both treated male and female rats compare to control. Except for two females, one was from control group and other was from 300 mg/kg/day showed unaccountable tremors and ataxia. They were killed at day 17. The nervous disorder observed in two female rats at an early stage of the experiment cannot be attributed to Black PN since the two animals were in the control and lowest level groups, respectively. Moreover, no comparable effect was seen after more prolonged treatment or at 3000 mg/kg bw. Significant growth retardation was observed in males at the 3000 mg/kg/day level and it associated with a reduced food intake. No effect on Hematology, clinical chemistry and urine analysis of treated male and female rats were observed as compared to control. In addition, there were significant increase in the relative weights of the testes and kidneys at the dose level of 3000 mg/kg/day in treated male was observed as compare to control. The elevated relative kidney weights were not accompanied by changes in the kidney function tests or in organ pathology. Decrease liver weight was also observed in 1000 and 3000 mg/kg/day in treated female rat as compare to control. Decreased liver weight in these instances arose from the fact that autopsles were performed late in the day, so that the animals were not fasted overnight, in accordance with the standard procedure but were only fasted during the day. No significant change in the female gonads weight was observed. Significant change was observed in the foci of inflammatory cell infiltration of the myocardium in treated males at 3000 mg/kg/day. The foci of inflammatory cell infiltration of the myocardium which occurred occasionally in males of the 3000 mg/kg/day group were of spontaneous origin. Similar Historical control data was found in the Wilens & Sproul (1938) study. Therefore, NOAEL was considered to be 1000 mg/kg bw for male and 3000 mg/kg bw for female rats when treated with Black PN orally in feed for 90 days.