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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed per OECD 422 and following GLP. A subset of the animals tested were evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. A justification for read-across is provided.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
This study exceeded the std OECD 422 design by following the F1 offspring to adulthood, with continued exposure and assessments of neurologic, immunologic, and reproductive structures and functions. The protocol also assessed F0 recovery males and 28-day
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Stability under test conditions: Stable in vehicle (corn oil) for at least 35 days
- Storage condition of test material: Ambient or refrigerated conditions.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: P = 63 days old
- Housing: singly in polycarbonate cages with bedding except during mating.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77 degrees F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light cycle

IN-LIFE DATES: From: 2003-09-2 To: 2004-01-06

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil

- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female
- Length of cohabitation: 14 days
- Proof of pregnancy: Observation of a vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dosing formulations were analyzed for concentration verification. Standards for acceptable accuracy of mixing were that the means of the analyzed samples was within +/- 10% of the nominal concentration and the relative standard deviation for triplicate samples did not exceed 10%.
Duration of treatment / exposure:
F0 females were dosed premating through the day prior to necropsy.
Recovery males, females and 28 days females were dosed for 28 days.
F1 animals were dosed post-weaning until the day before scheduled necropsy, at least 7 weeks duration.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100, and 200 mg/kg/day
Basis:
nominal conc.
at a dose volume of 5 mL/kg/day
No. of animals per sex per dose:
F0 generation: 10 males and 10 females plus 5 addition males and females each in the control and high dose groups designated recovery animals.

In addition, 10 females (5 control and 5 in the high dose group) designated as the 28 day females were dosed for 28 days and necropsied.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a dose range finding study, the test substance was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore, 300 mg/kg/day was considered too toxic for the OECD 422 study design.
- Rationale for animal assignment (if not random): Randomization stratified by body weight

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included. twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and included changes to skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity, and behavior patterns.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreeding, for F0 females during gestation (gd 0,7,14, and 21) and lactation (pnd 0, 4, 7, 14, and 21). Body weights of the 28 day females and recovery males and females were recorded on a weekly basis until termination. Body weight gains were computed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, euthanized, necropsied and completed external and visceral examinations performed.

PARAMETERS EXAMINED
The following parameters were examined in surviving F1 offspring: For the remaining F1 pups, survival indices were calculated at least weekly through weaning (pnd21).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

OTHER:
At weaning at least one male and one female from each litter (when possible) were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for retained F1 females included examination of vaginal patency and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last three weeks of the postwean exposure period prior to scheduled sacrifice. For each retained male offspring, observationsfor cleavage of the balanopreputial gland began at day 35 and continued until preputial separation. Androgenic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected F1 adult male and females per dose group.

Body weights for selected F1 offspring from weaning through scheduled sacrifice were taken.

Functional Observation Battery (FOB), including cage side observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations was performed on 5 F1 females and 5 F1 males once midway during the postwean exposure period. Grip strength was also assessed for the 5 F1 males and 5 F1 females per group selected for FOB during the last week of the post weaning exposure.
Postmortem examinations (parental animals):
GROSS NECROPSY and HISTOPATHOLOGY
-All F0 parental animals, 28-day females, recovery males and females, adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of F0 females were examined for the number of nidation (implantation) scars. All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. A full histopathology was performed on all retained organs from 5 randomly selected F0 males and females in the control and high-dose groups as well as for the 28-day females. Since no treatment-related findings were identified, no histopathology on lower dose groups or recovery groups was performed.
Postmortem examinations (offspring):
Retained F1 adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of F0 females were examined for the number of nidation (implantation) scars. All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. The retained F1 offspring were sacrificed at 70 days of age and subjected to the same assessments as the F0 parents.
Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females in the control and high-dose groups. Since no treatment-related findings were identified, no histopathology on lower dose groups was performed.

Statistics:
Appropriate statistical analyses were used throughout the assay.
Reproductive indices:
At the time of sacrifice, 1 testis from each F1 adult male was frozen at -20 degrees C for subsequent enumeration of testicular homogenization-resistant spermatid heads for high-dose and control males. If treatment-related changes in the number of testicular homogenization-resistant spermatid heads were observed in the high-dose group, then these evaluations were extended to the mid- and low-dose group animals (from retained frozen testes). In addition, 1 cauda epididymis from each F1 male was immediately removed, weighed, and seminal fluid from the cauda assessed for sperm number, motility, and morphology. Sperm motility (motile and progressively motile) was assessed immediately after necropsy for all males. The number and morphology (at least 500 sperm per male, if possible) was evaluated at a later date using appropriately retained sperm samples initially from the high-dose and control males. If treatment-related andrological changes were observed in the high-dose group, then these evaluations were extended to the mid- and then to the low-dose group animals if treatment-related changes were seen in the mid-dose group (from retained sperm samples).

The mating index, fertility index and pregnancy index were all calculated for males and females.

F1 female age acquisition of vaginal patency was evaluated and the estrous cycle length monitored for the last 3 weeks of the post wean period for the F1 females.
Offspring viability indices:
For the offspring, the live birth index, the 4, 7, 14 and 21 day survival index, as well as the lactation index were all calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
rooting post dosing -consistent with taste aversion
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

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 Reproductive Indices
There were no significant effects of exposure to the test substance on F0 mating, fertility, gestational, or pregnancy indices in F0 parental males and females during the production of F1 offspring. The precoital interval and gestational length were equivalent across all groups. There were also no changes across groups for the number of total implantation sites per litter and the number of total and live pups per litter at birth. There were also no significant differences across groups for percent postimplantation loss per litter or the number of dead pups at birth.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Treatment-related clinical observations of F0 males included rooting post-dosing in 5 males at 200 mg/kg/day. No other findings exhibited a treatment- or dose-related pattern of incidence or severity.
All 10 F0 females/group survived to scheduled sacrifice


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No treatment-related effects on F0 male body weights were observed. Significantly higher weights for sd 7, 14, 21, and 28 (but not sd 0) occurred at 10 mg/kg/day, with no effects 100 or 200 mg/kg/day. F0 male body weight change was significantly increased at 10 mg/kg/day for sd 0-7 and unaffected for all other intervals at this dose and for all intervals at 100 and 200 mg/kg/day. No treatment-related effects on feed consumption were observed. Feed consumption values, expressed as g/day, were significantly increased at 10 mg/kg/day for sd 0 to 7, 7 to 14, and 0 to 14, with no effects at 100 or 200 mg/kg/day. There were no significant effects of treatment on F0 male feed consumption.

There were no significant differences among groups for F0 female body weights or body weight changes during the prebreed period (sd 0-14) or for the few females in the control and low-dose groups (10 mg/kg/day) that were not identified as sperm positive during the mating (sd 0-28) or postmating (sd 14-42) periods. There were no significant effects on F0 female feed consumption, expressed as g/day or g/kg/day, in any group during the 2 weeks of the prebreed period.


ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 males (10/group) were necropsied after 28 days of dosing (2 weeks prebreed + 2 weeks mating). There were no treatment-related effects on organ weights. Sacrifice body weights and absolute weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles with coagulating glands were all unaffected. Absolute prostate weight was significantly reduced at 100 mg/kg/day but statistically equivalent at 10 and 200 mg/kg/day. Organ weights relative to terminal body weights were unaffected for the thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles plus coagulating glands. Relative brain weight was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day. Relative prostate weight was significantly reduced at 10 and 100 mg/kg/day but unaffected at 200 mg/kg/day. Organ weights relative to brain weights were unaffected for the thymus, heart, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, prostate, and seminal vesicles with coagulating glands. Relative liver weight was significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS)
At scheduled necropsy of F0 females, there were no differences among groups for terminal body weights and organ weights (absolute, relative to terminal body weights, or relative terminal brain weights) for the brain, heart, liver, spleen, paired kidneys, paired adrenal ,lands, uterus with cervix and vagina, and paired ovaries. Absolute and relative (to both body and brain weights) weights of the thymus were significantly increased at 100 mg/kg/day and unaffected at 10 or 200 mg/kg/day.
No treatment-related gross necropsy findings were observed. No treatment related microscopic findings in any females (of 5/group) at 200 mg/kg/day were observed.



OTHER FINDINGS (PARENTAL ANIMALS):
FOB: No parameters evaluated in the FOB exhibited any statistically significant or biologically relevant difference across male F0 groups. There was also no effect of treatment on motor activity, auditory startle, or grip strength which were performed in week 4 prior to scheduled termination.
For female F0 groups the week 1 pupil size score was significantly reduced in the 200 mg/kg/day dose group and in week 3 the tail pinch score was also reduced in this dose group. No other parameters were affected at any time period in any dose group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no indications of toxicity; no adult F0 parental toxicity in either sex, and no reproductive toxicity.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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)

There were 9, 7, 10, and 8 live litters on pnd 0 with 0, 10, 100, and 200 mg/kg/day, respectively. Live birth and stillbirth indices were unaffected, as were the survival indices for pnd 0-4, 4-7, 7-14, and 14-21 and the lactational index for pnd 4 (postcull) through 21. The mean number of live pups per litter for pnd 0, 7, 14, and 21 was unaffected across all groups. Mean F1 female and male anogenital distances (absolute or adjusted for body weight) per litter on pnd 0 were equivalent across all dose groups. Mean F1 pup body weights per litter (sexes combined or separately) were unaffected by treatment for all time points across all dose grou). Sex ratio (% males) per litter was also unaffected across all groups for all gestational intervals. There were no male pups in any litter with retained nipples on pnd 11-13. The number of areolae per pup and the number of pups with 1 or more areolae on pnd 11-13 were equivalent across all groups. F1 pup clinical observations during lactation indicated that the number of F1 pups found dead for pnd 0-21 was 2, 2, 5, and 2 pups at 0, 10, 100, and 200 mg/kg/day, respectively. In addition, 1 female pup at 100 mg/kg/day exhibited a herniated umbilicus on pnd 0.

Necropsy findings of F1 culled pups on pnd 4, and F1 pups found dead or euthanized moribund on pnd 0-21 were performed. Pups that died on pnd 0 exhibited closed ductus arteriosus (postnatal state), no air (fetal state) or air (postnatal state) in lungs, no or little milk in stomach, and autolysis of abdominal organs. Pups that died on pnd 1-21 included 1 at 100 mg/kg/day on pnd 1 with no findings and 1 pup on pnd 21 at 0 mg/kg/day that was cannibalized. These findings are typical of pups during early lactation and exhibited no treatment-or dose-related pattern. At weaning of the F1 litters on pnd 21, there were still 9, 7, 10, and 8 live litters at 0, 10, 100, and 200 mg/kg/day, respectively. There were 36, 23, 38, and 32 F1 male offspring and 32, 27, 41, and 27 F1 female offspring evaluated at scheduled sacrifice on pnd 21 for 0, 10, 100, and 200 mg/kg/day, respectively. There were no effects on F1 male or female body weights or organ weights at sacrifice on pnd 21 at any dose. Increases in relative thymus weight for females in the 100 and 200 mg/kg/day groups were not considered treatment related, because the absolute weights were not increased and thymus weight was not affected in F0 females or males. Necropsy findings for F1 male and female pups on pnd 21 included 1 male at 0 mg/kg/day with right undescended testis, with no findings in any other group and no findings in any female in any group.

Ten F1 males/group were necropsied as adults. There were no treatment-related effects observed for organ weights. Absolute paired kidney weights relative to body or brain weights) were significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day. Liver weight, relative to terminal body weight), was significantly increased at 200 mg/kg/day.
There were no effects across all 4 groups for percent motile sperm, epididymal sperm concentration, testicular homogenization resistant SHC’s, sperm production (per testis), efficiency of daily sperm production (per gram of testis), or percent abnormal sperm. The % abnormal sperm values at 0 mg/kg/day and 200 mg/kg/daywere within historical control values.

The absolute weights and weights relative to terminal body and brain weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenals, paired ovaries, and uterus with cervix and vagina were equivalent across F1 female control and dose groups.

No histopathologic findings were identified in F1 males or females.

Prior to the scheduled sacrifice of the F0 females at the weaning of the F1 litters on pnd 21, auditory startle, motor activity and grip strength were assessed/ No differences among any parameters were identified.

CLINICAL SIGNS (OFFSPRING): Treatment related clinical observations in F1 males included rooting postdosing occurred in 2,2,7 and 9 F1 males at 0,10, 100 and 200 mg/kg/day, respectively. Salivating pre-/post dosing was observed in 4 males only at 100 mg/kg/day.
In F1 females, 7 females each in the 100 and 200 mg/kg/day exhibited rooting and 1,4 and 2 females in the 10, 100 and 200 mg/kg/day exhibited salivation post dosing, respectively.

BODY WEIGHT (OFFSPRING): Body weight change values were unaffected for all F1 male groups for all intervals from pnd 22 through 78. There were no differences in feed consumption for any postwean interal in any group.
On pnd 22, the mean F1 female body weight at 200 mg/kg/day was significantly reduced with no effects at any later time points at this dose or any time points at any other doses.

SEXUAL MATURATION (OFFSPRING): F1 male age at PPS was unaffected across all dose groups. F1 female age acquisition of vaginal patency was equivalent across all groups. Estrous cycle length monitored for the last 3 weeks of the post wean period for the F1 females, were equivalent across all the dose groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no F0 offspring toxicity during lactation in either sex and the acquisition of puberty in F1 males and females was unaffected. F1 postweanlings exhibited no systemic toxicity in either sex at any dose.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the test substance, administered by gavage once daily at 0, 10, 100, and 200 mg/kg/day to parental rats, from prebreed through mating, gestation, and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice, resulted in no adult F0 parental toxicity and no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals.
Therefore, the F0 male and female systemic NOAEL was at least 200 mg/kg/day. The F1 male and female systemic NOAEL was also at least 200 mg/kg/day. The NOAELs for F0 reproductive toxicity were at or above 200 mg/kg/day for males and females. The NOAELs for F1 offspring toxicity were also at or above 200 mg/kg/day for males and females.
Justification for read-across has been provided.