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Toxicological information

Toxicity to reproduction

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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 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
Justification for study design:
No data available

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/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).

Test animals

Species:
rat
Strain:
not specified
Details on species / strain selection:
No data available
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
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:
No data available
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.3, 1.0 and 3.0 % (0, 150, 500, 1000 mg/kg/day)
Basis:
nominal in diet
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:
No data available
Positive control:
No data available.

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):
No data available.
Sperm parameters (parental animals):
No data available.
Litter observations:
No data available.
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):
No data available
Statistics:
Statistics was observed by Litchfield, J. T, Jr. & Wilcoxon, F. (1949) method.
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
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
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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant change were observed at any dose level in both treated male and female rats compare to control.Except for two femals ,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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight- Significant growth retardation was observed in males at the 1000mg/kg/day level and it associated with a reduced food intake

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 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 were also observed in 150 and 1000 mg/kg/day in treated female 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 overmght, in accordance with the standard procedure but were only fasted during the day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Significant change was observed in thefoci 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.

OTHER FINDINGS (PARENTAL ANIMALS)
No significant change were observed in Hematology,clinical chemistry and urine analysis in treated male female group compare to control.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Remarks on result:
other: No significant increase in the relative weights of the testes.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
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 examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Remarks on result:
not measured/tested

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:
NOEL for Black PN was considered to be 500 mg/kg/day in male rats and 1000 mg/kg/day in female rats respectivley ,for 90 days reprotox study.
Executive summary:

Reproductive toxicity study for Black PN was conducted on male and female rats for 90 days by administrating in diet. When they were exposed at dose concentration of 0, 150, 500, 1000 mg/kg/day by oral diet for 90 days. Clinical sign, body weight, food consumption, colour intake, Haematology, clinical chemistry, urine analysis, organ weight and histopathology was observed. No significant change was 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 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 150 and 1000 mg/kg/day in treated female 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.  On the basis of results NOEL was considered to be 500 mg/kg/day in male rats and 1000 mg/kg/day in female rats respectively, for 90 days reproductive toxicity study.