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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: meets generally acceptable scientific standards, well documented and accepted for assessment
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1971

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
Principles of method if other than guideline:
2-generation study in rats as part of a combined Reproduction and Teratology Studies
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium nitrilotriacetate
EC Number:
225-768-6
EC Name:
Trisodium nitrilotriacetate
Cas Number:
5064-31-3
Molecular formula:
C6H9NO6.3Na
IUPAC Name:
trisodium nitrilotriacetate
Details on test material:
- Name of test material (as cited in study report): Trisodium nitrilotriacetate
- Substance type: pure substance
- purity: not further specified

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Rat study
- Charles River CD rats
- Housing: caged individually
- Diet (e.g. ad libitum): ground Purina Chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 weeks (after weaning)

ENVIRONMENTAL CONDITIONS
Carefully controlled environment

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): ground Purina Chow


Details on mating procedure:
Parent rats (F0) were bred three times to bear the generations F1a, F1b and F1c.
The F1a generation was discarded at weaning.
The F1c generation was used for teratological studies (see chapter 7.8.2 Developmental toxicity / teratogenicity).
The F1b generation were bred twice to bear the generations F2a and F2b.
The F2a generation was discarded at weaning.
The F2b generation was used for teratological studies (see chapter 7.8.2 Developmental toxicity / teratogenicity).

No further specification of the mating procedure.
Day of conception (day 0) was determined by vaginal smears.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
NTA had been mixed to ground Purina Chow to yield the target concentrations
For each generation, records of individual weekly feed consumption and body-weight gain were kept during the first 8 wk from weaning. Afterwards, the parent rats were weighed at the beginning of each mating phase and no records of feed consumption were kept.
Duration of treatment / exposure:
Continuously throughout two generations.
Frequency of treatment:
Continously feeding
Details on study schedule:
The original parent rats (F0) were bred three times to bear the generations F1a, F1b and F1c.
The F1a generation was discarded at weaning.
The F1c generation was used for teratological studies (see chapter 7.8.2 Developmental toxicity / teratogenicity).
The F1b generation were bred twice to bear the generations F2a and F2b.
The F2a generation was discarded at weaning.
The F2b generation was used for teratological studies (see chapter 7.8.2 Developmental toxicity / teratogenicity).

No detailed description of mating and age of rats.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.1 and 0.5 %
Basis:
nominal conc.
approximately 90 and 450 mg/kg bw/day for adult female rats
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels had been chosen from previous subacute and long-term studies, from which the lower level had been reported to be without any effect, whereas the 0.5% level had been reported to produce some mild toxicity (not further specified).
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly feed consumption and body weight gain during the first 8 weeks from weaning

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food and feed efficiency (%): Yes
Feed efficiency = body-weight gain/feed consumed × 10
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):
not determined
Sperm parameters (parental animals):
not determined
Litter observations:
STANDARDISATION OF LITTERS
- All live-born litters were counted at birth and again 4 days later, when the pups were weighed and their sex determined.
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded on day 4.

PARAMETERS EXAMINED
The following parameters were examined in [F1a / F1b / F2a] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weight gain, lactation index, average weaning weight of pups per sex

GROSS EXAMINATION OF DEAD PUPS:
no; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- No post mortem examinations of parental animals

GROSS NECROPSY
- No gross necropsy of parental animals

HISTOPATHOLOGY / ORGAN WEIGTHS
No microscopic examination of parental animals
Postmortem examinations (offspring):
SACRIFICE
- No post mortem examinations of animals (exept for teratogenicity study, see 7.8.2)

GROSS NECROPSY
- No gross necropsy

HISTOPATHOLOGY / ORGAN WEIGTHS
No microscopic examination (exept for teratogenicity study, see 7.8.2)
Statistics:
Significance test, analysis of variance, not further specified
Reproductive indices:
Conception in %
Offspring viability indices:
Average no. born alive / litter
Average no. alive 4 days post partum
Average no. weaned / litter
lactation index = no. of pups weaned/no. alive at 4 days after litters reduced × 100
average weaning weight of pups per sex

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in body weight gain was observed in high-dose females only, fed continuously, as compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food consumption was observed in high-dose males only, fed continuously, as compared to controls.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food efficacy was observed in mid-dose males only, fed continuously, as compared only to groups fed during gestation days 6 - 15. It is believed that this effect iwas probably due mostly to a reduced palatability of the feed.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
no significant differences in the conception rate during the specific phases of the study and no significant differences in the measures of fertility

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean total food consumption (determined by week 8) at the high dietary level (0.5% Na3NTA) was somewhat less than in the control and in the low dietary level (0.1% Na3NTA) group in both sexes but statistically significantly different only for the males. Mean body weight gain in male and female rats was slightly reduced for the high dietary level (0.5 %) in comparison to either controls or to the 0.1% group, statistically significantly different, however only for females. A slight, but statistically significant, decrease in food efficacy was observed in mid-dose males only, fed continuously, as compared only to groups fed during gestation days 6 - 15. It is believed that this effect was probably due mostly to a reduced palatability of the feed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences in the conception rate during the specific phases of the study and no significant differences in the measures of fertility.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effect
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: depressed body weight gain

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in body weight gain was observed only in males exposed in-utero from the highest dose group fed continuously as compared to controls and as compared to the high dose group fed during gestation days 6 thru 15.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food consumption was observed only in males exposed in-utero from the low dose group fed during gestation days 6 thru 15 as compared to controls and the high dose group fed during gestation days 6 thru 15. The lack of dose-response suggests this is probably not treatment related.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food efficacy was observed only in females exposed in-utero from the highest dose group fed continuously as compared to the groups fed only during gestation days 6 - 15.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P1)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A slight, but statistically significant, decrease in body weight gain was observed only in males exposed in-utero from the highest dose group fed continuously as compared to controls and as compared to the high dose group fed during gestation days 6 thru 15. A slight, but statistically significant, decrease in food consumption was observed only in males exposed in-utero from the low dose group fed during gestation days 6 thru 15 as compared to controls and the high dose group fed during gestation days 6 thru 15. The lack of dose-response suggests this is probably not treatment related. A slight, but statistically significant, decrease in food efficacy was observed only in females exposed in-utero from the highest dose group fed continuously as compared to the groups fed only during gestation days 6 - 15.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences in the conception rate during the specific phases of the study and no significant differences in the measures of fertility.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: depressed body weight gain

Target system / organ toxicity (P1)

Critical effects observed:
no

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:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in body weight gain was observed only in males exposed in-utero from the highest dose group fed continuously as compared to controls and as compared to the high dose group fed during gestation days 6 thru 15.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food consumption was observed only in males exposed in-utero from the low dose group fed during gestation days 6 thru 15 as compared to controls and the high dose group fed during gestation days 6 thru 15. The lack of dose-response suggests this is probably not treatment related.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
A slight, but statistically significant, decrease in food efficacy was observed only in females exposed in-utero from the highest dose group fed continuously as compared to the groups fed only during gestation days 6 - 15.
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
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
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

Details on results (F1)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A slight, but statistically significant, decrease in body weight gain was observed only in males exposed in-utero from the highest dose group fed continuously as compared to controls and as compared to the high dose group fed during gestation days 6 thru 15. A slight, but statistically significant, decrease in food consumption was observed only in males exposed in-utero from the low dose group fed during gestation days 6 thru 15 as compared to controls and the high dose group fed during gestation days 6 thru 15. The lack of dose-response suggests this is probably not treatment related. A slight, but statistically significant, decrease in food efficacy was observed only in females exposed in-utero from the highest dose group fed continuously as compared to the groups fed only during gestation days 6 - 15.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences in the conception rate during the specific phases of the study and no significant differences in the measures of fertility.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1b
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Generation:
F1b
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Generation:
F1b
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
exposed in-utero
Sex:
male/female
Basis for effect level:
other: depressed body weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Group

Treatment

1

F0, F2a, Control Diet

2

F0, F2a, Control Diet + 0.1% Na3NTA Continuously

3

F0, F2a, Control Diet + 0.5% Na3NTA Continuously

4

F0, F2a, Control Diet + 0.1% Na3NTA GD 6 - 15

5

F0, F2a, Control Diet + 0.5% Na3NTA GD 6 – 15

6

F1b, F2b, Control Diet

7

F1b, F2b, Control Diet + 0.1% Na3NTA Continuously

8

F1b, F2b, Control Diet + 0.5% Na3NTA Continuously

9

F1b, F2b, Control Diet + 0.1% Na3NTA GD 6 - 15

10

F1b, F2b, Control Diet + 0.5% Na3NTA GD 6 – 15

11

F1c, Control Diet

12

F1c, Control Diet + 0.1% Na3NTA Continuously

13

F1c, Control Diet + 0.5% Na3NTA Continuously

14

F1c, Control Diet + 0.1% Na3NTA GD 6 - 15

15

F1c, Control Diet + 0.5% Na3NTA GD 6 – 15

8-wk F0 Body Weight Gain

Group

Body Weight Gain (g) - Males

Body Weight Gain (g) - Females

F0, 1

359

205

F0, 2

356

200

F0, 3

335

1883

F0, 4

352

200

F0, 5

358

206

3  Significantly different (P ≤ 0.05) from Group 1 and Group 5.

 

8-wk F0 Food Consumption

Group

Food Consumption (g) - Males

Food Consumption (g) - Females

F0, 1

1261

943

F0, 2

1285

926

F0, 3

11914

891

F0, 4

1210

925

F0, 5

1227

942

4  Significantly different (P ≤ 0.05) from Group 2.

 

8-wk F0 Feeding Efficiency1

Group

Feeding Efficiency (g) - Males

Feeding Efficiency (g) - Females

F0, 1

28.5

21.7

F0, 2

27.72

21.5

F0, 3

28.2

21.2

F0, 4

29.1

21.6

F0, 5

29.2

21.9

1  Body weight gained/Food consumed X 100.

2  Significantly different (P ≤ 0.05) from Group 4 and Group 5.

8-wk F1b Body Weight Gain (in-utero Exposure Only)

Group

Body Weight Gain (g) - Males

Body Weight Gain (g) - Females

F1b, 6

322.3

137.2

F1b, 7

311.1

141.1

F1b, 8

284.32

132.8

F1b, 9

301.1

143.7

F1b, 10

320.6

149.9

2  Significantly different (P ≤ 0.05) from Group 6 and Group 10.

 

8-wk F1b Food Consumption (in-utero Exposure Only)

Group

Food Consumption (g) - Males

Food Consumption (g) - Females

F1b, 6

1462.5

955.6

F1b, 7

1405.1

1002.1

F1b, 8

1397.9

1018.2

F1b, 9

1362.43

955.6

F1b, 10

1462.3

1009.0

3  Significantly different (P ≤ 0.05) from Group 6 and Group 10

 

8-wk F1b Feeding Efficiency1(in-utero Exposure Only)

Group

Feeding Efficiency (g) - Males

Feeding Efficiency (g) - Females

F1b, 6

22.3

14.5

F1b, 7

22.3

14.2

F1b, 8

20.7

13.44

F1b, 9

22.4

15.0

F1b, 10

22.1

14.7

1  Body weight gained/Food consumed X 100.

4  Significantly different (P ≤ 0.05) from Group 9 and Group 10.

F0-Generation to produce F1a

 

Group 1

Group 2

Group 3

Group 4

Group 5

Females paired

25

25

24

25

25

Females pregnant

24

24

24

21

24

Percent pregnant1

96

96

100

84

95

1  Fertility Index

 

F0-Generation to produce F1b

 

Group 6

Group 7

Group 8

Group 9

Group 10

Females paired

20

20

20

20

20

Females pregnant

19

19

19

16

18

Percent pregnant1

95

95

95

80

90

1  Fertility Index

F0-Generation to produce F1c

F1c Group:

11

12

13

14

15

Days:

13

21

13

21

13

21

13

21

13

21

Females paired

10

10

10

10

10

10

10

10

10

10

Females pregnant

7

6

6

5

9

8

8

5

8

7

Percent pregnant1

70

60

60

50

90

80

80

50

80

70

F1b-Generation to produce F2a

 

F2a Group 1

F2a Group 2

F2a Group 3

F2a Group 4

F2a Group 5

Females paired

24

25

22

23

24

Females pregnant

22

23

19

23

22

Percent pregnant1

92

92

86

100

92

1  Fertility Index

F1b-Generation to produce F2b

F2b Group:

6

7

8

9

10

Days:

13

21

13

21

13

21

13

21

13

21

Females paired

11

11

11

11

10

10

10

10

10

12

Females pregnant

9

10

10

9

7

10

10

9

7

8

Percent pregnant1

82

91

91

82

70

100

100

90

70

67

1  Fertility Index

Applicant's summary and conclusion

Conclusions:
No significant effects on reproduction at 450 mg/kg/d.
Executive summary:

In a two-generation reproduction study Na3NTA was administered to 20 Sprague Dawley rats/sex/dose in dietary concentrations of 0, 0.1% and 0.5% (resp. daily intake for adult female rats approximately 90 and 450 mg/kg/d).

There were no significant differences in food efficiency. With respect to reproductive performance there were no significant differences in the conception rate during the specific phases of the study and no significant differences in the measures of fertility and lactation in terms of average numbers of live-borns per litter, live pups on p.n. day 4, average number of weaned pups per culled litter and in the lactation index. Body weights of the new-borns were not reported. Offspring weaning weights were reduced at the 0.5% level in both sexes, an effect observed in litters of the first breeding trials and statistically significant for the F1 generation only, but not consistent across successive breedings and/or across generations.

No examination of effects on reproductive parameters, testes weights or morphology, epididymal sperm counts, motility, or morphology, daily sperm production, efficiency of daily sperm production, or prostate or dorsal prostate weights or histopathology was performed.

The only effect of Na3NTA on rats in this study was some growth depression in both adult and weanling animals fed the 0 .5% level continuously. This effect was probably due mostly to a reduced palatability of the feed, since no such depression was seen in the weights of foetuses removed by Caesarean-section or in the weights of 4-day-old liveborn animals.

The study shows that Na3NTA, even at highly exaggerated levels, causes no deleterious effects on reproduction in rats.

This study meets generally acceptable scientific standards, is sufficiently documented and accepted for assessment.

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