Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
- Not all required organ weights were determined. - Sperm parameters were not assessed for all animals.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Test material: Ziram
- Lot/Batch number: V528/8331 AA
- Description: White powder
- Purity: 97.8%
- Stability: Not reported.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: ♂: 144-218 g, ♀: 117-174

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on mating procedure:
- M/F ratio: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Any other deviations from standard protocol: Litters were culled to 8 pups/litter on lactation day 4.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
F0 parents: from study initiation until scheduled sacrifice
F1 parents: from weaning (day 22) until scheduled sacrifice
F2 pups: from weaning (day 22) until scheduled sacrifice

Duration of exposure before mating (F0 / F1 parents): 10 weeks
Frequency of treatment:
Ad libitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60*, 180#, 540 ppm (Concentration was increased to 72 ppm (*)/ 207 ppm (#) to compensate losses during storage)
Basis:
nominal in diet
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
- Actual doses
F0 ♂:
5, 15, 37 mg/kg/day (prior to breeding)
3, 10, 25 mg/kg/day (after breeding)

F0 ♀: 6, 17, 43 mg/kg/day (prior to breeding)
5, 13, 33 mg/kg/day (gestation)
12, 33, 85 mg/kg/day (lactation)
5, 15, 37 mg/kg/day (after weaning)

F1 ♂:
6, 18, 46 mg/kg/day (prior to breeding)
3, 10, 26 mg/kg/day (after breeding)

F1 ♀:
7, 20, 51 mg/kg/day (prior to breeding)
5, 13, 32 mg/kg/day (gestation)
11, 30, 79 mg/kg/day (lactation)
5, 14, 34 mg/kg/day (after weaning)
Positive control:
none

Examinations

Parental animals: Observations and examinations:
MORTALITY
- Twice daily

CLINICAL SIGNS
- Twice daily

BODY WEIGHT
- Weekly during treatment and prior to terminal sacrifice for males.
- Confirmed mated ♀ were weighed on presumed gestation Days 0, 7, 10, 14, and 20. Nursing dams were weighed on Days 1, 4, 7, 14, and 21 post partum. After weaning (day 22) once weekly until scheduled sacrifice.

FOOD CONSUMPTION
- Daily until pairing for all animals.
- Male food consumption was measured after mating again daily until scheduled sacrifice.
- Female food consumption was measured daily throughout gestation and lactation period.
- No food consumption data was obtained during the mating period.






Oestrous cyclicity (parental animals):
- Daily during mating.
Sperm parameters (parental animals):
- Only performed on males which were paired, but failed to sire a litter.
Litter observations:
- Sex-determination, pup viability, body weight gain, clinical signs, necropsy on dead pups, behavioural testing (F2)
Postmortem examinations (parental animals):
ORGAN WEIGHTS
- Yes
- Organs: testes + epididymides/ovaries, brain, pituitary gland, kidneys, liver

HISTOPATHOLOGY
- All animals from control and high-dose group.
- Tissues: Cervix, coagulating gland, epididymides, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus, vagina, vas deferens, all internal gross lesions
Postmortem examinations (offspring):
- neuropathological examination (F2)
Statistics:
STATISTICAL TEST
- Chi-square test with Yates' correction factor
Pup Sex Ratios, Parental Mating and Fertility Indices, Numbers of Stillborn and Dead Pups, Pup Viability Indices

- One-way ANOVA with Dunnett's test
Parental Weekly Body Weights and Weight Changes, Gestation and Lactation Body Weights and Body Weight Changes, Parental Food Consumption, Mean Gestation Length, Pup Body Weights, Absolute and Relative Organ Weights, Live Litter Size

- Kolmogorov-Smimov test ( one-tailed test)
Histopathological fmdings

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

F0 (♂+♀):
- Mean body weights and body weight gains in the 540 ppm group males were reduced early in the treatment period. In females mean body weights and body weight gains in the 540 ppm group were generally reduced throughout gestation and lactation and after weaning.
- Food consumption in the 540 ppm group was generally reduced in males and prior to breeding, during gestation and lactation and after weaning in females.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day
Sex:
female
Basis for effect level:
other: (post-breeding intake )
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
NOAEL
Effect level:
ca. 10 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: (post-breeding intake)
Remarks on result:
other: Generation: neonatal (migrated information)
Dose descriptor:
NOAEL
Effect level:
ca. 25 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: (post-breeding intake)
Remarks on result:
other: Generation: reproduction (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

F1 (♂+♀):
- Mean pup body weights in the 540 ppm group litters were slightly reduced during lactation. Mean body weights and body weight gains in the 540 ppm group males were reduced throughout the treatment period. In females mean body weights and body weight gains in the 540 ppm group were generally reduced throughout gestation and lactation and after weaning.
- Food consumption in the 540 ppm group was generally reduced in males and prior to breeding, during gestation and lactation and after weaning in females.

F2 (♂+♀):
BODY WEIGHT
Mean pup body weights in the 540 ppm group litters were slightly reduced during lactation and throughout the remainder of the study until the postnatal day 70 neuropathology evaluation.

MOTOR ACTIVITY
No effects

AUDITORY STARTLE TEST
No effects

BIEL MAZE SWIMMING TRIALS
No adverse effects on swimming ability, learning and recall were noted in the treated groups when compared to the control group values. Swimming
ability on test day 1 of the first testing interval (days 19-23) was comparable in all study groups. Mean time to swim the Biel maze in the Path A direction
(forward route) decreased from day 2 to 3 for all study groups. Mean time to swim the Biel mazein the Path B direction (reverse of Path A) decreased from day 4 to 5 for all study groups. Similarly the mean number of errors recorded during the test decreased from test days 2 to 3 and 4 to 5. After a three day rest period, testing results indicated that rats in all study groups were able to recall the maze pattern at mean times similar to the day 3 (Path A) and 5 (Path B) results. During the second testing interval (days 58-62), mean swimming, learning and recall times for all study groups were initially lower than or comparable to the shortest times recorded during the first test interval, then were generally decreased further throughout the interval.

BALANOPREPUTIAL SEPARATION
Balanopreputial separation was not affected in any of the F2 pups. All male pups (100%) were observed with balanopreputial separation by postnatal day 50.

VAGINAL PERFORATION
Vaginal patency was not affected in any of the F2 pups. All female pups (100%) had vaginal opening by postnatal day 40.

BRAIN WEIGHTS AND BRAIN MEASUREMENTS
No effects were apparent on mean absolute and relative (to final body and whole brain weights) F2 pup brain, forebrain and hindbrain (including cerebellum) weights on PND 11 and 70.

HISTOMORPHOLOGICAL EXAMINATION
No remarkable changes were apparent at the qualitative histomorphological examination of brains in the F2 control and 540 ppm group male and female pups on postnatal day 11.
No microscopic lesions attributed to the administration of Ziram were observed in any central or peripheral nervous system tissues upon histopathological examination on postnatal day 70.


Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1-A1        Body weight changes in reproductive toxicity study

Parameter

Genera­tion

Controls

60 ppm

180 ppm

540 ppm

Dose-response

+/–

Body weight [g]

average weight on gestation
Day 14

F0 dams

/

348.4

/

338.6

/

356.0

/

343.8

/

F1 dams

/

357.1

/

339.1

/

344.7

/

316.7*

/

average pup weight on lactation Day 21

F1 pups

44.8

42.8

44.6

41.8

46.3

43.5

40.2*

37.7*

F2 pups

40.7

39.5

41.0

39.3

44.1

41.2

38.5

36.9

* statistically significant different from control p </= 0.05

Applicant's summary and conclusion

Conclusions:
In conclusion, parental toxicity in the F0 and F1 generations was exhibited at a concentration of 540 ppm by inhibition of body weight gain and reduced food
consumption. No parental toxicity was observed at concentrations of 72 and 207 ppm.
Reproductive performance was unaffected by test article administration at the 72, 207 and 540 ppm concentrations. Neonatal toxicity was expressed at a concentration of 540 ppm by slightly reduced pup body weights {F1 and F2). No neonatal toxicity was observed at concentrations of 72 and 207 ppm. Based on the results of this study, a concentration of 207 ppm was considered to be the NOAEL (no observable adverse effect level) for parental systemic toxicity, 207 ppm was considered to be the NOAEL for neonatal toxicity and 540 ppm was considered to be the NOAEL for reproductive and developmental neurotoxicity.