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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
Chronic toxicity
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 reviewed journal

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:
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

Reference
Name:
Unnamed
Type:
Constituent
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/mole
- Substance type:Organic

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals 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 % (0, 150, 500, 1000 mg/kg/day)
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
not specified
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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 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
150 mg/kg/day-16 male and 16 female
500 mg/kg/day-16 male and 16 female
1000 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 150 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 higher levels of dosage.
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 1000 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 was observed in males at the 1000 mg/kg/day.
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 significant change was observed in Hematology of treated male female group compare to control.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant change was observed in clinical chemistry of treated male female group compare to control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant change was observed in urine analysis of treated male female group compare to control.
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 1000 mg/kg/day . The foci of inflammatory cell infiltration of the myocardium which occurred occasionally in males of the 1000 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:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: No significant increase in the relative weights of the testes.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
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 500 mg/kg/day in male rats and 1000 mg/kg/day in female rats respectively when male and female rats were treated with Black PN for 90 days.
Executive summary:

In a Chronic toxicity study, male and female rats were treated with Black PN in the concentration of 0, 150, 500, 1000 mg/kg/day oral in diet for 90 days. No significant changes were observed at any dose level in both treated male and female rats compare to control. Significant growth retardation was observed in males at the 1000mg/kg/day level and it associated with a reduced food intake. There were significant increase in the relative weights of the testes and kidneys at the dose level of 1000 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 at 150 and 1000 mg/kg/day in treated female as compare to control. Significant change was observed in the foci of inflammatory cell infiltration of the myocardium in treated males at 1000 mg/kg/day .The foci of inflammatory cell infiltration of the myocardium which occurred occasionally in males of the 1000 mg/kg/day group were of spontaneous origin. Therefore, NOAEL was considered to be 500 mg/kg/day in male rats and 1000 mg/kg/day in female rats respectively when male and female rats were treated with Black PN for 90 days.