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Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1980/02 to 1981/11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report date:
1981

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
- Females were housed with males for up to three 10-day periods.
- Food consumption was measured during the pre-mating periods only.
- F1 offspring not selected for mating were as old as 30 days at post mortem examination. Cervix, vagina and seminal vesicles of all animals selected for histopathology were not listed in the report as being examined. Individual weights of the pups at termination have not been reported.
GLP compliance:
no
Remarks:
Study conducted before GLP requirements came into force
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-cyclopropyl-1,3,5-triazine-2,4,6-triamine
EC Number:
266-257-8
EC Name:
N-cyclopropyl-1,3,5-triazine-2,4,6-triamine
Cas Number:
66215-27-8
Molecular formula:
C6H10N6
IUPAC Name:
N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine
Test material form:
solid: particulate/powder
Remarks:
coarse cream-colored

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
COBS CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Rats were housed individually in galvanized hanging wire-mesh cages
- Source: The Charles River Breeding Laboratories, Portage, Michigan.
- Diet: Rodent feed was available ad libitum
- Water: Water was available ad libitum
- Acclimation period:15 days

ENVIRONMENTAL CONDITIONS
- Temperature: The animals were maintained in a room controlled for temperature.
- Humidity: The animals were maintained in a room controlled for humidity.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Fresh diets were prepared and offered weekly throughout the study with control animals receiving basal diet on the same regimen.
- The test article was premixed with the basal diet, Purina Certified Rodent Chow in a Hobart mixer. Five hundred grams of premix were added to additional Rodent Chow and blended in a twin-shell blender. During the F0 generation, an intensifier bar was used for two minutes at the beginning and end of the 10 minute blending period. On study week 25, the blending procedures were changed to improve the homogeneity of the diet. The volume of premix was increased to 2000 grams, the blending time was increased to 30 minutes and the intensifier bar was used for the entire period.

Details on mating procedure:
F0 rats were mated after 100 days of treatment. Each male was randomly mated with two females from the same group. During mating periods females were observed daily for evidence of copulation by means of vaginal smears for sperm and/or the appearance of a vaginal plug. This day was designated as gestational day 1 and females were transferred to an individual cage. Any females failing to exhibit evidence of mating after an initial 10-day mating period were remated with a different male of known fertility from the same treatment group for a second ten-day period. If the second mating provided negative results, a third similar mating with another male was attempted. At the end of the third and final mating, all mating sets were separated and returned to individual cages.

F1 rats were mated after 120 days of treatment following the same procedure as described above.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After four weeks of weekly sample collection, analyses of test material concentrations in the were conducted. This procedure was continued throughout the study.
Duration of treatment / exposure:
28 weeks
Frequency of treatment:
Continuous exposure via feed.
Details on study schedule:
Groups of 15 male and 30 female (F0 parents) weanling rats were fed diet containing 0 (control), 30, 1000 or 3000 (initially 4000 ppm, reduced to 3000 ppm after four weeks of feeding to the F0 parents) ppm cyromazine. After 100 days, the animals were mated and allowed to rear their F1 litters to weaning. From these weanlings, the F1 parents were selected (15 males and 30 females from each dosing group) and after a 120 day pre- mating period were mated to produce the F2 litters. In each generation, on lactation day 4, the litters were culled to 10 animals (where possible of equal sex ratio).
Doses / concentrationsopen allclose all
Dose / conc.:
3 000 ppm
Remarks:
High dose group, equivalent to a mean daily intake of 228 mg/kg bw/d for male F0 Parents, 169 mg/kg bw/d for male F1 Parents, 259 mg/kg bw/d for female F0 Parents and 202 mg/kg bw/d for female F1 Parents.
Dose / conc.:
1 000 ppm
Remarks:
Mid dose group, equivalent to a mean daily intake of 64.1 mg/kg bw/d for male F0 Parents, 51.3 mg/kg bw/d for male F1 Parents, 77.2 mg/kg bw/d for female F0 Parents and 66.3 mg/kg bw/d for female F1 Parents.
Dose / conc.:
30 ppm
Remarks:
Low dose group, equivalent to a mean daily intake of 1.97 mg/kg bw/d for male F0 Parents, 1.55 mg/kg bw/d for male F1 Parents, 2.34 mg/kg bw/d for female F0 Parents and 1.94 mg/kg bw/d for female F1 Parents.
No. of animals per sex per dose:
15 males and 30 female/dose
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
The parental rats and pups were observed twice each day for signs of overt toxicity, changes in general appearance and behavior and mortality. The nesting and nursing behavior of the females was observed. Detailed observations were recorded weekly on the parental rats and the pups after weaning. Examinations for gross deformaties of the pups were conducted at birth and on lactation days and 4, 7, 14 and 21.

Individual body weights were recorded weekly for adult rats from initiation of the F0 and F1 generations until mating. Males were weighed monthly after the mating period and just prior to sacrifice. Females were weighed prior to sacrifice. As specified by the protocol, weights were not measured during gestation and lactation. Pups were weighed individually on lactation days 0, 4, 7, 14 and 21 and individually by sex on lactation days 4 and 21.

Parental food consumption was measured weekly for F0 and F1 males and females from initiation of the generation until mating. Food consumption was not measured during or after the mating period. However, appropriate control or test diets were available ad libitum to all animals until sacrifice.
Sperm parameters (parental animals):
All F0 and F1 adult males were tested for spermatogenesis at sacrifice by examination of the epididymis for the presence of mature sperm. Upon sacrifice, the testes and epididymides were removed. Each epididymis was trimmed from the testis; testes were weighed. Semen was examined microscopically.
Litter observations:
After delivery (lactational day 0), all pups were examined for physical abnormalities and the number of viable and nonviable pups from each litter was recorded. On lactational day 4, male and female pups were weighed individually. Pup weights were also measured on days 7, 14 and 21.
Postmortem examinations (parental animals):
Complete gross postmortem examinations were conducted on all members of the F0 and F1 generations (parental rats) on lactation day 30.
Postmortem examinations included the external surfaces, all orifices, the cranial cavity, the carcass, the external and cut surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities and their viscera, and cervical tissues and organs. The epididymis of F0 and F1 males was examined for the presence and condition of sperm.
Complete histopathologic examination was conducted for the F1 generation (parental rats) on lactation day 30. For F0, only testis was examined histopathologically.
Postmortem examinations (offspring):
Complete gross and histopathologic postmortem examinations were conducted on five F1 weanlings per sex per group not selected as members of the F1 generation and on five F2 weanlings per sex per group on lactational day 30 (weaning at lactational day 21). The remaining F1 and F2 weanlings were given gross internal examinations only if appearing abnormal on external examination.
Postmortem examinations included the external surfaces, all orifices, the cranial cavity, the carcass, the external and cut surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities and their viscera, and cervical tissues and organs.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical condition of parental animals was not affected by treatment with cyromazine; however, there was some evidence of a viral infection affecting a small number of rats (Sialodacryoadenitis virus, SDV).
Mortality:
no mortality observed
Description (incidence):
Survival was 100% in all animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the end of the pre-mating period, F0 generation parental animals treated with 1000 and 3000 ppm cyromazine, had decreased body weights (decreased by 9.5 and 22.3% respectively, for males and 12.6 and 18.3% respectively, for females, compared with controls),
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the end of the pre-mating period, F0 generation parental animals treated with 1000 and 3000 ppm cyromazine, had a corresponding reduction in food consumption in both sexes.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no compound-related microscopic changes in the testes of F0 adult male rats.

Reproductive function / performance (P0)

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No differences occurred between control and treatment group males with regard to sperm motility or the frequency of cytoplasmic droplets.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The reproductive performance of treated animals at all dose levels was comparable with that of controls for both generations. Details are oncluded below. Male fertility at 3000 ppm cyromazine in the F0 generation was reduced (86.7%, 86.7%, 92.9% and 66% in the controls and males exposed to 30, 100 and 3000 ppm, respectively). No similar effects was seen for the F1 males (86.7%, 66%, 100% and 93.3% in the controls and males exposed to 30, 100 and 3000 ppm, respectively). Since there was no evidence of a dose-response in the two generations, the effect on fertility was not attributed to cyromazine administration.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
> 215 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
ca. 2 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical condition of parental animals was not affected by treatment with cyromazine; however, there was some evidence of a viral infection affecting a small number of rats (Sialodacryoadenitis virus, SDV).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female F1 parental animal died but the death was considered to be incidental. Survival was 100% in the remaining animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights were reduced in the F1 males at 3000 ppm cyromazine (at the end of the pre- mating period body weight decrease was approximately -17.1% compared with controls). There were no statistically significant differences between the 30 and 1000 ppm cyromazine groups and control in males. F1 females fed 1000 or 3000 ppm cyromazine showed a dose-related decrease in mean body weight gain compared with controls; body weights in the 30 ppm cyromazine group were only slightly lower than control over the generation and thus the effect at this dose was not considered to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was decreased at 1000 and 3000 ppm for males and females.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences between control and treated groups were noted in organ weight values. However, these changes were considered to be either sporadic or as a consequence of reduced body weights at 3000 ppm cyromazine, and of no toxicological significance.
There were no treatment-related microscopic findings in several organs of the F1 parents at the 30, 1000 and 3000 ppm levels at scheduled sacrifice.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of any effect of treatment on macroscopic changes on F1 parents.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings in several organs of the F1 parents at the 30, 1000 and 3000 ppm levels at scheduled sacrifice.

Reproductive function / performance (P1)

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Motile morphologically normal sperm was present in all males in control and treated groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
The reproductive performance of treated animals at all dose levels was comparable with that of controls for both generations. Male and female fertility at 30 ppm cyromazine in the F1 generation was reduced compared to controls. Since there was no evidence of a dose-response, the effect on fertility was not attributed to cyromazine administration.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
> 215 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
ca. 2 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No remarkable differences in appearance or behaviour were observed between treated and control pups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup viability at birth and mean litter size in the treated groups of the F1 litters were similar to control.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weight was statistically significantly reduced at 3000 ppm cyromazine from lactation day 0 until weaning (day 21).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences between control and treated groups were noted in organ weight values. However, these changes were considered to be either sporadic or as a consequence of reduced body weights at 3000 ppm cyromazine, and of no toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no evidence of any effect of treatment on macroscopic changes on F1 pups.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings. Focal pneumonia was observed among both male and female pups in all dose groups (including controls), this was considered not related to exposure to cyromazine.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
No remarkable differences in appearance or behaviour were observed between treated and control pups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In the F2 litters, pup viability (number of viable pups/ litter and mean number of pups/ litter) at birth was reduced in the 3000 ppm cyromazine group; subsequent survival was not affected. The groups exposed to 30 and 1000 ppm were comparable to the control litters with respect to these parameters.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weight was statistically significantly reduced at 3000 ppm cyromazine from lactation day 0 until weaning (day 21).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences between control and treated groups were noted in organ weight values. However, these changes were considered to be either sporadic or as a consequence of reduced body weights at 3000 ppm cyromazine, and of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of any effect of treatment on macroscopic changes on F2 pups.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

In the early part of the study, analytical results suggestive of non-homogeneity of the 30 ppm diet were noted; results ranged from 11% of the target (average of the duplicate assays at study week 23) to 133% (study week 15). Prepared diets of 1000 and 3000 ppm cyromazine contained 92 to 143% and 72 to 125% of the desired concentrations, respectively. The mean concentrations found from study weeks 1-25 were 78 ±  29%, 109 ±  12% and 104 ±  9.1% of the target levels in the 30, 1000 and 3000 ppm cyromazine groups respectively.


In diets prepared for study weeks 26 to termination, cyromazine concentrations ranged from (mean ±  SD) 47% to 155% (91% ±  23%), 83% to 122% (101% ±  9.6%) and 76% to 116% (97% ±  9.6%) of the target levels in the 30, 1000 and 3000 ppm cyromazine groups, respectively. Nevertheless, although there were notable variations in concentrations at the 30 ppm dose in the first part of the study, the overall exposure is considered acceptable.


Results are provided in the overall section below.

Applicant's summary and conclusion

Conclusions:
In a 2-generation study cyromazine did not affect reproductive performance or fertility.
The NOAEL(parental) was estimated to be 30 ppm (equivalent to approximately 2 mg/kg bw/day), based on decreased body weight from the dose of 1000 ppm. The NOAEL(reproductive) was estimated to be greater than 3000 ppm (equivalent to approximately 215 mg/kg bw/day) based on absence of adverse effects on the reproductive performance or fertility at all doses tested. The NOAEL(offspring) was set at 1000 ppm (equivalent to approximately 65 mg/kg bw/day), based on reduced pup body weight at 3000 ppm during lactation.
Executive summary:

Cyromazine was administered to rats over two generations to evaluate its effect on reproduction and the offspring of rats. Groups of 15 male and 30 female (F0 parents) weanling rats were fed diet containing 0 (control), 30, 1000 or 3000 (initially 4000 ppm, reduced to 3000 ppm after four weeks of feeding to the F0 parents) ppm cyromazine.


 


After 100 days, the animals were mated and allowed to rear their F1 litters to weaning. From these weanlings, the F1 parents were selected and after a 120 day pre- mating period were mated to produce the F2 litters. In each generation, on lactation day 4, the litters were culled to 10 animals (where possible of equal sex ratio). Test diets were fed continuously throughout the study. Parental food and body weights were measured throughout the study. Reproductive performance and selected offspring parameters were measured.


 


Under the conditions of this study, dietary administration of 30, 1000 and 3000 ppm cyromazine to Sprague-Dawley CD rats for two successive generations, did not affect reproductive performance or fertility. The NOAEL (parental) was estimated to be 30 ppm (equivalent to approximately 2 mg/kg bw/day), based on decreased body weight from the dose of 1000 ppm. The NOAEL (reproductive) was estimated to be greater than 3000 ppm (equivalent to approximately 215 mg/kg bw/day) based on absence of adverse effects on the reproductive performance or fertility at all doses tested. The NOAEL (offspring) was set at 1000 ppm (equivalent to approximately 65 mg/kg bw/day), based on reduced pup body weight at 3000 ppm during lactation.