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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:
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no significant differences in food efficiency (body weight gain per food intake)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no significant differences in food efficiency (body weight gain per food intake)
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-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:
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 in the F0 generation and in the F1 males, statistically significantly different, however only for the F0 males.
Mean body weight gain in male and female rats of both generations 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 F0 females and for F1 males.
No significant differences in food efficiency (body weight gain per food intake)

For detailed data see table 1.

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)
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: F0, F1 (migrated information)
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
90 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effect
Remarks on result:
other: Generation: all (migrated information)
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: depressed body weight gain
Remarks on result:
other: Generation: F0, F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weights of new born not reported, offspring weaning weights were reduced at 0,5% in both sexes.
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
No significant differences in the following measures: average number of live-borns per litter, live pups on day 4, average number of weaned pups per culled litter, lactation index.
For details see table 2.

BODY WEIGHT (OFFSPRING)
not reported for new born.
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.
For some detailed data see table 1.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Effect of feeding 0.1 and 0.5% dietary Na3NTA on the growth, feed consumption and feed efficiency of first- and second-generation rats

Gain in body weight (g) by wk 8  Total feed consumed (g) by wk 8  Feed efficiency (%)
Generation
male female male female male female
Treatment  F0 F1b   F0 F1b   F0 F1b   F0 F1b   F0 F1b   F0 F1b  
Control  359 322 205 137 1261 1463 943 956 28,5 22,3 21,7 14,5
0.1 % NTA 356 311 200 141 1285 1405 926 1003 27,7 22,3 21,5 14,2
0.5 % NTA  335  284* 188* 133 1191 t 1398 891 1018 28,2 20,7 21,2 13,4

Values are means of groups of 20 rats. Those marked with an asterisk differ significantly (Analysis of Variance) from those of controls: *P >= 0,05. That marked with a dagger differs significantly from the group fed 0,1% NTA: t P <= 0,05

Table 2. Effect of 0.1 and 0.5 % dietary NTA on the reproductive performance and lactation of first- and second-generation rats (Continuous feeding of dietary levels)

Control 0,1% 0,5%
Parameter  Generation and litters   F1a+ F1b  F2a   F1a+ F1b  F2a   F1a+ F1b  F2a
Conception (%)    95,0 92,0 95,0 92,0 97,0 86,0
No. of stillborn    15,0 13,0 9,0 10,0 27,0 5,0
Average no. born alive/litter    11,3 12,3 11,9 12,3 11,9 12,2
Average no. alive 4 days post partum   11,1 11,1 11,3 10,9 11,0 11,3
Average no. weaned/litter+ +    7,2 7,0 7,1 6,5 7,3 7,6
Lactation index§    96,3 96,3 94,4 96,6 96,0 96,3
Average weaning weight of pups:               
Male    54,3  55,4 49,9 51,9  53,2 49,1 48,3* 51,9 48,7 t
Female    52,4  55,3 47,5 49,9  50,3 47,3 46,1* 50,4 46,6

++Litters containing more than eight pups were reduced to that number on day 4.

§No. of pups weaned/no, alive at 4 days after litters reduced × 100.

Values marked with an asterisk are significantly less (Analysis of Variance) than those of controls (*P <= 0,05) and that marked with a dagger is significantly less than that for the group fed 0.1 % NTA on days 6-15 of gestation (t P <= 0,05).

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.