Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive 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
Study period:
11 Jan - 30 Oct 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance CAS 16470-24-9. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Reference:
Composition 0
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approx 4 weeks of age
- Weight at study initiation: 52 to 78 g
- Housing: individually in suspended, stainless steel, wire-mesh type cages except during mating where females were individually housed in plastic cages containing wood chip bedding
- Fasting period before study: none
- Diet: certified meal Rodent chow #5002 from PMI Nutritionals International Inc. St. Louis, Missouri, available ad libitum
- Water: tap water, available ad libitum
- Acclimation period: during the 2-week acclimation period, all rats were observed daily for any clinical signs of disease, and all animals were given a detailed clinical examination prior to selection fur study

ENVIRONMENTAL CONDITIONS
- Temperature: 18.89 to 24.44 °C
- Humidity: 34 to 77 %
- Photoperiod: fluorescent lighting was provided for approx. 12 hours per day
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The required amount of test article was weighed and suspended in vehicle and mixed using a Polytron tissue homogenizer. The solutions were stirred with a magnetic stir bar, and then dispensed into amber glass containers using a syringe. Fresh suspensions were prepared for each dose group weekly, and stored refrigerated and protected from light for approximately 1 week.

ADMINISTRATION
- Volume: the test article was administered at a constant volume of 10 mL/kg/day.

VEHICLE
- Concentration: 0.5 % aqueous carboxymethylcellulose.
- Storage: the contents of the container were mixed thoroughly and stored refrigerated when not in use.
- Lot/batch no.: 108H0070.
Details on mating procedure:
After a minimum of 10 weeks of treatment, the males were cohabited, 1 male to 1 female, with the corresponding females from the same group. The pairing was not random. Instead, the first male in the group was paired with the first female in the same group. The pairings were assigned in sequential fashion until all animals in each group were cohabited. Mating took place in the cage of the male.

Vaginal smears (lavage) were performed daily on females during the mating period, and the presence or absence of sperm or vaginal plug was recorded. The day on which evidence of mating was observed was designated as Day 0 of gestation.
The rats were paired for a maximum of 14 consecutive days. When evidence of mating was noted, the female was removed from the cage of the male and individually housed. After the mating period, females with no positive evidence of mating were individually housed in a plastic cage with wood chip bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were diluted with methanol and analyzed by HPLC (UV detection) for verification of concentration.
Samples of vehicle and test article suspensions at each concentration were collected weekly for the first 4 weeks and every 4 wceks, thereaher, for analysis of test article concentration. The assayed concentrations ranged from 90 to 109 %.
Duration of treatment / exposure:
Treatment of the parental (P) generation began 10 weeks prior to mating and continued until euthanasia.
F1 and F2 generation offspring were potentially exposed in uterus and during lactation.
The F1 offspring selected to comprise the F1 parental group were exposed for ≥ 70 days prior to mating (beginning at age ≥ 28 days), until euthanasia.
Frequency of treatment:
Once daily.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
26 males and 26 females.
Control animals:
yes, concurrent vehicle
Details on study design:
SELECTION OF THE F1 GENERATION AND TERMINATION OF THE P GENERATION
At weaning (Day 21 of lactation), a minimum of 1 male and 1 female from each litter in each group was randomly selected to become parents oft he next generation (F1, parents). A total of 26 males and 26 females per group were selected, but due to deaths, 24 to 26/sex/group were paired.
All pups were available for selection except those that were not expected to survive because of physical abnormalities. Records were maintained on any pup excluded from the selection process. The parentage of each weanling was ascertained to avoid sibling pairings.

After selection, the remaining offspring from each litter were euthanized and subjected to a necropsy.

All P females, those that delivered litters and those that did not deliver, were continued on treatment until the F2, pups were weaned and following assessment as to whether a second mating was warranted. After the Sponsor, in consultation with the Study Director, decided not to conduct a second mating of the P parental animals, the P females were euthanized and subjected to a necropsy.

F1 GENERATION OBSERVATIONS
The selected F1, rats were individually housed and treated for a minimum of 10 weeks prior to mating. Dosing initiated when all rats were at least 28 days of age. During the premating period, the following indices of sexual maturity were assessed:
- Vaginal Opening: beginning on Day 28 of age, each female pup selected for the next generation was examined daily for the presence of vaginal opening until this developmental landmark was observed.
- Preputial separation: beginning on Day 35 of age, each male pup selected for the next generation was examined for preputial separation. Pups did not show complete retraction of the penile prepuce on Day 35 were examined daily until this developmental landmark was achieved.
- Mating, Gestation, and Lactation: following the procedures described in the P Breeding Procedures, the F1 parents were mated to produce the F2 generation. Care was taken to avoid sibling matings. Treatment continued through mating until termination.
After the mating period, the F1 males were individually housed and continued on treatment until euthanasia. All F1 females were continued on treatment until the F2 pups were weaned and following assessment as to whether a second mating was warranted. At weaning, the F2 offspring were euthanized and subjected to a necropsy.
Positive control:
Not used
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
All rats were observed twice a day, 7 days a week, for morbidity, mortality, and signs of injury. Any positive findings were recorded. Mortality or other signs of toxicity were recorded on the day they were observed.

DETAILED CLINICAL OBSERVATIONS
A detailed clinical examination of each rat was performed once during each study week. The examination included, but was not limited to, observations of the general condition, skin, fur, eye, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration. Modifiers were included in the clinical sign when necessary to describe the location, size, shape, color, or other characteristics.

BODY WEIGHT
Individual body weights were recorded on the day of arrival, prior to randomization, the first day of dosing, and weekly during the study, with the following exceptions. Mated female rats were weighed on Days 0, 7, 14, and 20 of gestation. Rats that delivered litters were weighed on Days 0, 4, 7, 14, and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE
Individual food consumption was measured and recorded weekly during the study, with the following exception. During the mating period, food consumption was not measured because the animals were cohoused. Food consumption for mated female mts was recorded on Days 0, 7, 14, and 20 of gestation. Food consumption for rats that delivered litters was recorded on Days 0, 4, 7, 14, and 21 of lactation.
Estrous cyclicity (parental animals):
Vaginal smears (lavage) were performed daily beginning 3 weeks prior to initiation of mating in all parental females to establish estrous cyclicity.
Sperm parameters (parental animals):
Sperm production, testes weighting, motility, morphology and analysis epididymal sperm count were performed following dissection.
Litter observations:
GESTATION PERIOD
Toward the end of the gestation period, females were examined twice daily for signs of parturllion. The females were allowed to give birth (F1).
The duration of gestation was calculated.

STANDARDISATION OF LITTERS
For each lilter, the day on which all pups were delivered was designated as Day 0 of lactation. ln 1 P and 1 F additional pups were found after the date initially designated as Day 0 of lactation. Day 0 of lactation was changed to the date these pups were found.

PARAMETERS EXAMINED
The litters were examined as soon as possible alter delivery, and the following parameters were recorded: litter size, nutnber of stillbom pups, number of live born pups, gross abnormalities ofthe pups, and pup weight by sex.
Litters were housed with their dams for 3 weeks alter birth. The dams and litters were observed twice daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded.
Pups were individually weighed by sex on Days 0, 4, 7, 14, and 21 of lactation. Detailed clinical observations were recorded at the same intervals.
Postmortem examinations (parental animals):
MORIBUNDITY
Any animal that showed signs of severe debility or toxicity, particularly if death appeared imminent, was euthanized for humane reasons and to prevent the loss of tissues through autolysis. All animals euthanized in exrremis or found dead were subjected to a necropsy.

SACRIFICE
Performed on:
- P, F1, and F2 animals dying spontaneously or euthanized in extremis
- P and F1 females that showed evidence of mating but failed to deliver
- P and F1 females that showed no evidence of mating and failed to deliver
- P and F1 animals surviving to scheduled termination
- F1 and F2 pups culled on day 4 of lactation
- three F1 pups not chosen to continue on study per sex from each litter at weaning
- all F2 pups at weaning

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues were prepared for microscopic examination: brain, adrenal glands, epididymis, kidney, liver, pituitary gland, prostate, seminal vesicles with coagulating glands, spleen, testis, and ovaries.
Postmortem examinations (offspring):
SACRIFICE
On Day 4 of lactation after weighing of each pup, litter size was reduced by random selection to 8 pups of equal sex distribution, whenever possible. The culled pups were euthanized and subjected to a necropsy. Any intact, dead pups were subjected to a necropsy.
The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age were subjected to postmortem examinations macroscopic and microscopic examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
Statistics:
The following parameters were analyzed statistically for significant differences: body weights, change in body weights, food consumption, fertility indices, reproductive organ weights, sperm parameters, follicle counts, number of uterine implantations, litter parameters (size, weight, and viability), and developmental indices.
Clinical signs:
no effects observed
Description (incidence and severity):
all observations noted are commonly seen in eaged rats of this age and strain and were not attributed to test article administration (P1 and F1 parent)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
sporadic, statistically signiticant, but not considered to be test article-related (P1 and F1 parent)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
sporadic, statistically signiticant, but not considered to be test article-related (P1 and F1 parent)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
change in kidney weight was considered to be test article related (P1 and F1 parent)
Gross pathological findings:
no effects observed
Description (incidence and severity):
the findings were not considered to be test article related (P1 and F1 parent)
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
findings comparable to the control (P1 and F1 parent)
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: sporadic, statistically signiticant, but no dose-related changes were evident (P1 and F1 parent)
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
(P1 and F1 parent)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
findings were not considered to be test article related (P1 and F1 parent)
Reproductive performance:
no effects observed
Description (incidence and severity):
no test article-related effects on reproductive performance noted (P1 and F1 parent)
P GENERATION
MORTALITY
Three females died or were euthanized in extremis during the P generation.
One female from the 1000 mg/kg bw/day group was found dead on Day 118 of the study. This female had red fluid and severe redness of the lungs at necropsy, and moderate congestion of the lungs at microscopic evaluation. Based on the macroscopic and microscopic evidence, technical (gavage) error may have been the cause of moribundity in this female.
One female from the 300 mg/kg bw/day group was euthanized in extremis on Day 87 of the study and had distended intestines, small spleen, enlarged mesenteric lymph nodes, and an ulceration of the palate. Microscopic evaluation did not reveal a cause of moribundity.
A second female from the 1000 mg/kg bw/day group was euthanized in extremis on Day 90 of the study. Other than distended intestines, no other findings were noted at necropsy and a cause of moribundity could not be ascertained from microscopic evaluation.
In the absence of any evidence of any toxicity at 1000 mg/kg bw/day, it is unlikely that these deaths were test article related.

CLINICAL SIGNS
No test article-related effects on survival or clinical observations were noted during the P parental generation.
Few clinical observations were noted during the premating, gestation, or lactation periods in P generation animals and the incidences were similar in all groups or occurred sporadically in 1 to several animals within a single group with no dose-related patterns discernible. All observations noted are commonly seen in rats of this age and strain and were not attributed to test article administration.

BODY WEIGHT and BODY WEIGHT CHANGES
No test article-related changes in body weight or body weight gain were noted during the premating, postmating (males), gestation, or lactation periods.
Sporadic, statistically significant increases or decreases were noted in the treatment groups when compared with controls, but there were no corresponding changes in food consumption during these intervals and a dose-related pattern was not evident. Consequently, these changes were not considered to be test article-related.

FOOD CONSUMPTION
No test article-related changes in food consumption were noted during the premating, postmating (males), gestation, or lactation periods.
Sporadic, statistically significant increases or decreases were noted in the treatment groups when compared with controls, but there were no corresponding changes in body weight and no dose-related patterns were evident. Therefore, these changes were not considered to be a result of test article administration.

MACROSCOPIC
No test article-related changes in macroscopic observations were noted during the P generation.
Very few abnormalities were noted at necropsy of the P generation animals. Since observations were only noted in 1 or 2 animals in any group, the observations noted were those commonly seen in rats of this age and strain and no dose-related patterns were evident, the findings were not considered to be test article related.

ORGAN WEIGHTS
A test article-related increase in absolute and relative kidney weight was noted in females at 1000 mg/kg/day. There were statistically significant increases in absolute and relative (to brain and body weight) kidney weight in females at 1000 mg/kg bw/day when compared with control values. Since similar changes in kidney weight were noted in F1 parents at 1000 mg/kg bw/day, the change in kidney weight was considered to be test article related.
Statistically significant decreases in absolute and relative (to brain weight) epididymides weight were noted at 300 and 1000 mg/kg bw/day when compared with controls. These changes were not correlated with any microscopic changes in the epididymis and there was no evidence of test article-related change in epididymal sperm concentrations. Since there was no microscopic correlation or a dose-related change in epididymal sperm concentrations, the decreases in epididymides weights were not considered to be test article related.
Also noted were statistically significant increases in absolute and relative (to brain weight) pituitary weight in females at 1000 mg/kg bw/day when compared with control values. Microscopic evaluation cf the pituitary did not reveal any changes, and consequently, these increases were not considered to be test article related.

MICROSCOPIC
No test article-related changes in microscopic incidences were noted following microscopic evaluation of tissues.
There were very few microscopic findings noted in either male or female parental animals.
Three males had abnormalities of the reproductive organs. One male from the 300 mg/kg bw/day group had small testes noted at necropsy with microscopic evidence of mild hypospermia noted in the left epididymis. Evaluation of sperm parameters in this animal revealed a reduced sperm count, 0 % motility, and 94 % abnormal sperm. This animal exhibited positive evidence of mating, but the female was not pregnant.
One male from the 1000 mg/kg bw/day group had small testes bilateral at necropsy and was diagnosed with aspermia of the epididymides. No sperm were found in this animal following sperm evaluation, and mating data indicated that this male did not mate or sire a litter.
A second male from the 1000 mg/kg bw/day group had small epididymides and flaccid left testis at necropsy. Microscopically, this animal had spermatid giant cells in the epididymides. Sperm evaluation of this animal revealed no abnormalities, and this animal sired a litter.
Occasionally, male rats have morphological problems and low sperm counts, as well as other changes in sperm parameters. Furthermore, there was 1 F1 control animal in this study that had no sperm. Since only 1 animal at 300 mg/kg bw/day and 1 animal at 1000 mg/kg bw/day were affected, these findings were not considered to be test article related.
These 2 animals contributed to the low fertility and mating indices noted during this generation.
The microscopic findings noted in females were similar in incidence among all groups, including controls, and none were considered to related to lest article administration.

REPRODUCTIVE PERFORMANCE
There were no test article-related effects on reproductive performance noted during the P generation.
There were slight decreases in the male and female fertility indices in all treatment groups when compared with controls. Historical control ranges for male and female fertility indices are 65.4 to 90.0 and 65.4 to 100.0 percent, respectively. Historical control ranges for both male and female mating indices are 88.5 to 100.0 percent. These values were neither statistically different from concurrent controls nor out of the range of historical control data for this laboratory and, consequently, were not considered to be test article related.
Copulatory interval was statistically higher at 1000 mg/kg bw/day when compared with controls. This was due mainly to 2 females who did not become pregnant until 8 and 13 days after pairing. These females appeared to be cycling normally and the 2 males with whom these females had been paired appeared to have normal sperm parameters. Since no morphological abnormalities were found, it is questionable that the change in copulatory index at 1000 mg/kg bw/day is toxicologically meaningful.
All other reproductive performance parameters, including female and male mating indices. female and male fecundity indices, oestrous cycle length and duration, in the treatment groups were comparable with controls, and no statistically significant or toxicologically relevant changes were obtained.
Slight, though statistically significant, decreases in the majority of sperm evaluation parameters were noted at 300 mg/kg bw/day when compared with controls. Since similar decreases were not noted at 1000 mg/kg/day, however, these changes were not considered to be toxicologically meaningful or test article related.

F1 GENERATION:
CLINICAL SIGNS AND MORTALITY
No test article-related changes in survival or clinical observations were noted during the F1 parental generation.
A total of 0, 2, 1, and 5 animals from the 0, 100, 300, and 1000 mg/kg bw/day groups died or were euthanized in extremis during the study.
Two females from the 100 mg/kg bw/day group died prior to initiation of dosing for the F1 parental generation. The macroscopic findings included distended intestines and stomach and/or red, tarry material on the stomach mucosa. Microscopically, immature ovaries and uteri were noted, but a cause of death was not ascertained. Since these animals were not dosed and were only potentially exposed to the test article via the milk during the lactation period and based on the lack of any test article-related findings in pups necropsied during lactation and weaning, the deaths of these animals were considered to be incidental and unrelated to test article administration.
One male from the 300 mg/kg bw/day group died on Day 16 of study. Clinical signs included skin cold to touch, decreased activity, and material around eyes and nose. Macroscopic findings included distention and reddish brown fluid in the stomach and intestines, and microscopic evaluation revealed no cause of death.
Two males and l female from the 1000 mg/kg bw/day group were euthanized in extremis due to moderately to severely swollen feet/hind limbs. No abnormalities were noted microscopically for these animals, although the feet (gross lesions) were not examined. The swollen limbs were the major reason for euthanasia of these animals and may have been a result of mechanical injury. There were no other animals at 1000 mg/kg bw/day exhibiting swollen limbs during the study. Based on this, the gross finding was not considered to be a result of test article administration, and consequently, the deaths of these animals were not attributed to test article administration.
An additional male and female from the high-dose group died during the study. Macroscopic and microscopic evaluation of the female suggested that the cause of death may have been technical error (gavage). The males had no macroscopic or microscopic findings to suggest a cause of death. In the absence of any evidence of toxicity at 1000 mg/kg/day, it is unlikely that this death was test article related.
There were no changes in clinical observations between control and treatment groups during the premating, postmating (males), gestation, or lactation periods. The incidences were comparable between control and treatment groups or the findings were those commonly seen in caged rats of this age and strain and were not attributed to test article administration.

BODY WEIGHT and BODY WEIGHT CHANGES
No test article-related changes in body weight or body weight gain were noted during the F1 premating, postmating (males), gestation, or lactation intervals.
Statistically significant changes (increases and decreases) in body weight gain were noted very sporadically in the treatment groups when compared with controls. Since there was no dose-related pattern and the changes were not corroborated by statistically significant changes in food consumption, however, these changes were not considered to be test article related. Body weight was comparable between controls and treatment groups during all intervals, and no statistically significant changes were noted.

FOOD CONSUMPTION
No test article-related changes in food consumption were noted during the F1 premating, postmating (males), gestation, or lactation intervals.
A statistically significant decrease in food consumption was noted in males at 300 mg/kg bw/day during the first 2 weeks of the premating period, but since a similar change was not noted at 1000 mg/kg bw/day and no other statistically significant changes in food consumption were seen, these changes were not considered to be test article related. No other statistically significant or toxicologically relevant changes in food consumption were noted in males or females during the remainder of the F1 parental generation.

MACROSCOPIC
No test article-related changes in necropsy findings were noted in the F1 parental generation.
Very few findings were noted at necropsy of the F1 parents. Since observations were only noted in 1 or 2 animals in any group, the observations noted were those commonly seen in rats of this age and strain, and no dose-related patterns were evident, the findings were not considered to be test article related.

ORGAN WEIGHT
Test article-related changes in kidney weight were noted at 300 and 1000 mg/kg/day.
There were statistically significant increases in absolute and relative kidney weight in both males-and females at 1000 mg/kg bw/day as well as relative kidney (to body weight) in females at 300 mg/kg/day. No microscopic evidence of kidney changes was noted. However, there was a clear change noted in both males and females at 1000 mg/kg/day, and this was considered to be test article related. The statistical change in 300 mg/kg bw/day females was considered to be spurious since no changes in absolute weight or the kidney weight relative to brain weight were affected, and similar decreases were not seen in 300 mg/kg bw/day males.
Absolute liver weight was statistically lower in males at 300 and 1000 mg/kg bw/day when compared with controls. Relative (to brain and body) liver weight was statistically lower than controls in males at 300 mg/kg/day, but this was not noted in males at 1000 mg/kg/day.
No changes in liver weight were noted in the females. Also noted were statistically significant increases in absolute and relative adrenal weight in females, but not males, at 1000 mg/kg/day. There were no corresponding macroscopic changes noted in these organs, and, consequently, the changes in the adrenal and liver weights were considered to be sporadic and unrelated to test article administration.

MICROSCOPIC
No test article-related microscopic changes were noted in parental males or females.
The incidences of all findings were either comparable between control and treatment groups or those commonly seen in rats of this age and strain and considered to be unrelated to test article administration. One control male had epididymal aspermia and testicular atrophy and one male at 1000 mg/kg bw/day had trace degeneration of the seminiferous tubules of the testis. Neither of these males sired a litter.

REPRODUCTIVE PERFORMANCE
No test article-related changes in F, parental reproductive performance were noted.
Slight decreases in male and female mating and fertility indices were noted at 1000 mg/kg/day. Historical control ranges for male and female fertility indices are 6544 to 90.0 and 65.4 to 100.0 percent, respectively. Historical control ranges for both male and female mating indices are 88.5 to 100.0 percent. These changes were not statistically different when compared with concurrent controls and/or out of the range of historical controls for this laboratory, however, and consequently were not considered to be toxicologically relevant or test article related. Fertility index was slightly, but not statistically higher in males and females at 1000 mg/kg bw/day when compared with controls. A statistically significant increase in the number of estrous cycles was noted in females at 300 mg/kg bw/day when compared with controls. Since no changes were noted in females from the high-dose group, this increase was not considered to be a result of test article administration. Length of estrous cycle was comparable between controls and all treatment groups.
No statistically significant or toxicologically relevant changes in primordial follicle counts were noted in the treatment groups when compared with controls. A slight decrease in follicle count was noted at 300 mg/kg/day, but since follicle count at 1000 kg/kg/day was comparable with controls, a dose response was not evident. Therefore, this decrease was considered to be spurious and unrelated to test article administration.
All sperm parameters, including motility, concentration, homogenization resistant sperm head count, daily sperm production, spermatogenic efficiency, and percent abnormal sperm, were comparable among all treatment groups when compared with controls, and no test article related changes were noted. lt should be noted that 1 male in the control group had no sperm found at evaluation of sperm parameters. This male also had morphological evidence of infertility.
Dose descriptor:
NOAEL
Remarks:
Toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
Growth and Development
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: F1 and F2 (migrated information)
Clinical signs:
no effects observed
Description (incidence and severity):
very few clinical observations were noted, but incidences were either comparable between control (F1 and F2)
Mortality / viability:
no mortality observed
Description (incidence and severity):
(F1 and F2)
Sexual maturation:
no effects observed
Description (incidence and severity):
comparable between the control and treatment groups (F1 and F2)
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no statistically signiticant or toxicologically meaningful changes were noted (F1 and F2)
Gross pathological findings:
no effects observed
Description (incidence and severity):
few observations not attributed to test article administration (F1 and F2)
F1 GENERATION - LITTER DATA
PARTURITION DATA
No test article related changes in litter parameters were noted for the F1 litters.
There were no statistically significant or toxicologically relevant changes in gestation length, litter size on Day 0, numbers of live bum or stillborn pups/litter on Day 0, or sex ratio in the treatment groups when compared with controls during the F1 litter generation.

F1 OFFSPRING SURVIVAL
No test article-related change in survival was noted for the F1 litters. Survival at birth and during lactation was comparable in control and treatment groups.

CLINICAL OBSERVATIONS
No test article related changes in observations were noted during the F1 lactation period.
Very few clinical observations were noted, but incidences were either comparable between control and treatment groups or commonly seen in rats of this age and strain, and no dose-related patterns were evident. Therefore, none of the observations noted were considered to be a result of test article administration.

OFFSPRING GROWTH
No test article-related changes in growth were noted for the F1 litters.
A statistically significant increase in body weight (males and total pups) was noted at birth in pups at 1000 mg/kg/day when compared with controlsThe increase was not considered to be adverse or biologically meaningful, however, since the difference from controls was less than 10%. Furthermore, pup weights at 1000 mg/kg/day were comparable with controls throughout the remainder of the lactation period and similar increases at birth were not noted in F1 pups. Consequently, no test article-related changes in growth were noted during the F1 lactation period.

MACROSCOPIC OBSERVATIONS
No test article-related changes in macroscopic observations were noted in F1 offspring euthanized after weaning.
Very few macroscopic observations were noted at necropsy. The findings observed were either seen in only 1 to 2 animals per group, were of similar incidence in control and treatment groups, or are commonly seen in rats of this age and strain and were not attributed to test article administration.

ORGAN WEIGHTS
No test article related changes in organ weights were noted in F1 offspring euthanized after weaning.
No statistically significant or toxicologically meaningful changes in organ weights were noted in treatment groups when compared with controls.

SEXUAL MATURATION
No test article-related changes in sexual maturation were noted in F1 offspring.
The mean day on which vaginal opening and preputial separation occurred was comparable between the control and treatment groups.


F2 GENERATION:
PARTURITION
No test article related changes were noted.
A statistically significant increase in the number of implant scars was noted at 100 mg/kg/day when compared with controls. Since a similar change was not noted at 300 or 1000 mg/kg/day and consequently no dose response relationship was evident, this change was not considered to be test article related. Also noted was a statistically significantly higher sex ratio (% males) at birth and Day 4 of lactation for pups in the 300 mg/kg/day group. This statistical change appeared to be due to the fact that the % males/litter in the concurrent control group was low (42.58% at Day 0 and 42.42% at Day 4), rather than an increase in the % males/litter at 300 mg/kg/day. Furthermore, a similar change in sex ratio was not evident at 1000 mg/kg/day. Consequently, this statistical change in sex ratio at 300 mg/kg/day was not considered to be test article related. All other parturition parameters, including number of females delivering litters, litter size (total and live born pups) at birth, gestation, live birth, and stillborn indices and gestation length were comparable between controls and treatment groups.

F2 OFFSPRING SURVIVAL
There were no test article-related changes in survival in F2 pups during lactation.
Pup survival during the first 4 days and throughout lactation was comparable between control and treatment groups, and no statistically significant changes in viability or lactation indices were noted.

CLINICAL OBSERVATIONS
Very few clinical observations were noted, and the incidences were either comparable between control and treatment groups or commonly seen in rats of this age and strain. Consequently, no clinical signs were considered to result from test article administration.

OFFSPRING GROWTH
No adverse, test article-related changes in offspring growth were noted in F2 pups during lactation.
Sporadic, statistically significant increases in pup body weights were noted in all the treatment groups when compared with controls during the lactation period. Based on the fact that all treatment groups were increased, this effect appeared to be test article related, but was not considered to be adverse.

MACROSCOPIC OBSERVATIONS
No test article-related changes were noted at necropsy.
As was noted in the F2 offspring, very few Endings were observed at necropsy. All observations noted were either seen with equal frequency in control and treatment groups or occurred in 1 or 2 animals within a group and were not considered to be toxicologically relevant. No dose related pattern in the incidence of findings was observed; thus, all findings were considered to be sporadic in nature or commonly seen in rats of this age and strain and unrelated to test article administration.

ORGAN WEIGHTS
No test article-related change in brain, spleen or thymus weights were noted in the treatment groups when compared with controls.
As was seen during the lactation period, terminal body weight of pups in the treatment groups was higher than controls, and the changes were statistically significant in males at 300 and 1000 mg/kg/day when compared with controls. In these same groups, brain weight was also statistically increased when compared with controls. Although not statistically significant, body weights in female pups were also increased and brain weight in females at 1000 m g/kg/day was statistically higher when compared with controls. As stated earlier, the increases in body weight were not considered to be adverse, even though they may have been test article related, and the increase in brain weight was considered to be a consequence of the increased body weight. In all cases, the increases in both brain and body weight were less than 10 % when compared with controls, and consequently, the changes were not considered to be biologically relevant.
Reproductive effects observed:
not specified
Conclusions:
Based on the results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg/day and for parental reproductive performance, the NOAEL was 1000 mg/kg/day. For offspring growth and development, the NOAEL was also 1000 mg/kg/day.
Executive summary:

Method

The effects of the test article on the integrity and performance of male and female reproductive systems, including gonadal function, estrous cycle, mating behaviour, conception, gestation, parturition, lactation, weaning and growth and development of the spring were assessed testing rats, according to the EPA OPPTS 870.3800 (Reproduction and Fertility Effects) guideline.

Animals were exposed to the test articloe daily, via oral gavage at the dose levels of 0, 100, 300 and 1000 mg/kg bw, at a constant volume of 10 ml/kg. The duration of the entiere study was approx. 9 months.

Results

Based on the results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg/day and for parental reproductive performance, the NOAEL was 1000 mg/kg/day. For offspring growth and development, the NOAEL was also 1000 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no data available for the reproduction toxicity of CAS 12004488-68-5. In order to fulfil the standard information requirements, a read-across from structurally related substances was conducted.

A screening on reproduction study in Sprague-Dawley rats as range finding dose for the 2 -generation is available (CAS 16470-24-9). 10 rats/sex/dose were dosed with 30, 100, 300 or 1000 mg/kg bw/day by oral gavage during premating, mating, gestation and lactation. Males were killed after mating and females and pups were killed on day 4 of lactation. No substance-related finding was noted in any of the parental animals or pups at any dose level. Therefore, a NOAEL of 1000 mg/kg bw/day for parental and offspring toxicity was established (Turk, 2000). In the definitive 2-generation rat study according to EPA Guideline OPPTS 870.3800 / OECD 416 and performed under GLP, 26 Sprague-Dawley rats per sex per group were administered 100, 300 or 1000 mg/kg bw/day by oral gavage. In parental animals, the only test substance-related effect noted was an increased kidney weight. In F0 animals, an increased kidney weight (absolute and relative to body and brain weight) was observed in females at 1000 mg/kg bw/day. In F1 parental animals, there was an increase in kidney weight in males (absolute and relative to body weight) and females (absolute and relative to body and brain weight) at 1000 mg/kg bw/day as well as an increase in kidney weight (relative to body weight) in females at 300 mg/kg bw/day. The statistical change in 300 mg/kg bw/day was considered to be spurious since no changes in absolute weight or kidney weight relative to brain weight were seen, and similar increases were not observed in 300 mg/kg bw/day males. There were no test substance-related effects on reproductive performance noted for either parental generation. No adverse, test substance-related changes in growth or development of offspring were observed in either the F1 or the F2 generations. Based on the results of this study, the NOAEL for parental toxicity was 300 mg/kg bw/day. For parental reproductive performance, the NOAEL was 1000 mg/kg bw/day. For offspring growth and development, the NOAEL was also 1000 mg/kg bw/day (Turk, 2001).


Short description of key information:
No effects concerning reproduction toxicity observed at highest dose tested in a two-generation study in rats performed according to OECD testing Guideline 416 and under GLP. The NOAEL was 300 mg/kg bw/day for parental toxicity and 1000 mg/kg bw/day for reproductive performance and offspring toxicity (Turk, 2001).

Justification for selection of Effect on fertility via oral route:
The most conservative NOAEL on Parental toxicity has been reported.

Effects on developmental toxicity

Description of key information
Two studies revealed no evidence of teratogenicity in rats and rabbits: rat: NOAEL = 1000 mg/kg bw/day (maternal and foetal toxicity); rabbit: NOEL = 100 mg/kg bw/day (maternal and foetal toxicity), NOAEL teratogenicity = 400 mg/kg bw/day GLP-compliant OECD 414 studies (Turk, 1999 and 2000).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09 Feb 1998 - 15 Jan 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance CAS 16470-24-9. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Reference:
Composition 0
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
not impairing the study results.
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Reason for the Species selection: the rabbit is an acceptable model for evaluating developmental toxicity of various classes of chemicals and for which there is a large historical database. This strain is susceptible to known developmental toxicants.
- Number of animals: 106 females
- Source: Covance Research Products, Inc., Kalamazoo, Michigan
- Age at study initiation: 7 months
- Weight at study initiation: 2 - 8 kg
- Housing: animals were individually housed in suspended, stainless steel, with deotized paper beneath the caging to control excretory odor
- Acclimation period: 6 days
- Observation during the acclimation period: daily for any clinical signs of disease, given a detailed clinical examination prior to selection
- Identification: unique identification includes cage number, group, applied individually by a metal ear tag
- Diet: Certified Rabbit Chow #53222, PMI Feeds, Inc., St. Louis, Missouri, limited at the day of arrival to 50 g and increased to 170 g per day beginning on the second day of acclimation (each diet lot were certified by the manufacturer, but no additional analysis was conducted)
- Water: tap water, ad libitum (monitored for specific contaminants at periodic intervals according to SOP by MPI Research, but no additional analysis was conducted)
No contaminants were known to have been in the water or feed which could have altered the outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4 to 21.1°C
- Humidity: 453 to 69 %
- Photoperiod: 12h day/12h night with an automatic timer
- Parameters monitoring and recording: daily
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %, low viscosity, white powder, Sigma Chemical Company.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Before the test no adjustments for purity were made. The test article was mixed with aqueous 0.5% CMC at a dose volume of 10 ml/kg/day to achieve the desired dose concentrations. Fresh suspensions were prepared weekly. The required amount of test article was weighed directly into a calibrated beaker and suspended in vehicle using a stainless steel spatula. The spatula was rinsed into container with vehicle. Additional vehicle was added to the cylinder to yield 9000 ml of prepared test article. The contents of the cylinder were mixed using a motor driven propeller and dispensed into amber glass containers using a syringe and stored refrigerated.

VEHICLE
185 g of CMC was weighted and mixed with deionized water using a Warning blender. The solution was transferred into a calibrated container and additional deionized water was added to yield 37 mL of prepared vehicle. The contents of the container were mixed thoroughly using a motor-driven propeller. The prepared vehicle was stored refrigerated when not in use.

ADMINISTRATION
Test and control articles were drawn into an appropriate sized, plastic disposable syringe attached to a 12-gauge, stainless steel rabbit gavage needle.
A magnetic stir bar and stir plate were used to mix dosing suspensions during administration.
All dosage volumes administered were based on most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test article was characterized by AvTech Laboratories, Inc., Kalamazoo, Michigan.

HOMOGEINICITY ANALYSIS
Prior to initiation of test article administration, test batches of the dose suspensions at the low and high concentration of test article were prepared employing the same method and batch size as used during the study to assess the homogeneity of the dose preparations. Following mixing, samples were collected and analysed. Remixing was necessary to obtain satisfactory homogeneity results.
The dosing suspensions prepared for all dose levels-were homogeneous and accurately prepared.

STABILITY ANALYSIS
Stability analysis was not conducted. The stability of the test article in the vehicle (0.5%carboxymethylcellulose) over the range of dosages to be used in this study was conducted in a concurrent rat study (MPI Research Study "No. 795-003).

CONCENTRATION ANALYSIS
Samples of test article suspension at each concentration were collected from the first study preparation.
Details on mating procedure:
Day 0 of gestation was designated as the day in which mating was observed. Females were mated to males and observed for evidence of mating.

Duration of treatment / exposure:
From gestational Day 7 to through Day 28.
Frequency of treatment:
Once per day.
Duration of test:
From gestational Day 7 until Day 29.
Remarks:
Doses / Concentrations:
100, 400, and 800 mg/kg bw
Basis:
actual ingested
constant volume of 10 ml/kg/day in 0.5 % of CMC.
No. of animals per sex per dose:
25 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
Rabbits considered suitable for study, based on acceptable health status, were randomized into treatment groups using a stratified, by weight, block rendomization procedure.
See the following details of the assignment procedure:
Group 1
Dose Level (mg/kg): 0, control
Initial females: 25
Uterine Exam/Necropsy: 25
Group 2
Dose Level (mg/kg): 100 TS
Initial females: 25
Uterine Exam/Necropsy: 25
Group 3
Dose Level (mg/kg): 400 TS
Initial females: 25
Uterine Exam/Necropsy: 25
Group 4
Dose Level (mg/kg): 800 TS
Initial females: 25
Uterine Exam/Necropsy: 25
Maternal examinations:
CLINICAL FINDINGS
- Observation: at least twice a day, 7 days each week.
- Clinical findings: morbidity, mortality, signs of injury.
- Registration: all findings on the day they were observed.
From Days 7 through 29 of gestation each rabbit was removed from the cage and given a detailed clinical examination. The detailed examination included observations of the general appearance and condition activity and behaviour, excretory matter, respiration body surface abnormalities (scabbing, hair loss), and oral, nasal, and ocular regions. Observations included location size, and colour when applicable.

BODY WEIGHT
- Time schedule for examinations: on gestational days 0 and daily on Days 7-29.
Body weight change was recorded for gestational days 0 to 7, 7 to 10, 10 to 13, 13 to 16, 16 to 19, 19 to 22, 22 to 25, 25 to 29, 7 to 29 and 0 to 29.

FOOD CONSUMPTION
- Food consumption reported on gestational days 0 to 7, 7 to 10, 10 to 13, 13 to 16, 16 to 19, 19 to 22, 22 to 25, 25 to 29, 7 to 29 and 0 to 29.

PREMATURE DELIVERY
Females that showed signs of abortion before scheduled euthanasia were subjected to a necropsy.

POST-MORTEM EXAMINATIONS
A necropsy was performed by trained personnel under the supervision of a veterinary pathologist on does that aborted, were found dead or were euthanized in extremis. Euthanized rabbits of gestation were subjected to a limited necropsy on Day 29, and special emphasis was placed on structural abnormalities or pathologic changes which may have influenced the pregnancy.
Foetuses from aborts or euthanized in extremis were examined externally to the fullest possible extent and placed in 10 % neutral buffered formalin for possible future examination.

GROSS PATHOLOGY
Gross lesions and/or target organs were saved in 100 % neutral buffered formalin and the carcasses were discarded due to excessive toxicity (16 of 25 rabbits found dead, aborted, or euthanized in extremis), the rabbits remaining in the 800 mg/kg/day dose group were euthanized and necropsied in September 16, 1998. Animals in this dose group were euthanized on Day 29 of gestation and treated as scheduled described in the protocol (standard uterine examination and teratologic examination of foetuses).
Females euthanized prior to Day 29 of gestation were treated as an interim death and their foetuses were examined externally to the fullest possible extent and placed in 10 % neutral buffered formalin for possible future examination.
Due to excessive maternal toxicity, the 800 mg/kg/day group was terminated. Six does were euthanized prior to Day 29 of gestation. On Day 29 of gestation each surviving female was euthanized by intravenous injection of approximately 2.0 ml of sodium pentobarbital euthanasia solution via a marginal ear vein followed immediately by exsanguination and laparohysterectomy.
The abdomen was opened by an incision to remove the skin and examine mammary tissue and locate any subcutaneous masses.
The abdominal cavity was opened, and the uterus was exposed.
The carcasses were discarded.

Ovaries and uterine content:
UTERINE EXAMINATION
The location of viable and nonviable foetuses, early and late resorptions for each uterine horn, position of the cervix, and the number of corpora lutea were recorded, beginning from the distal end of the left uterine horn. The uterus was excised, and gravid uterine weight was recorded.
The foetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each foetus was gently removed, and each foetus was pulled away from the placenta, fully extending the umbilical cord.
The placentae were grossly examined.
Uteri from females that appeared non-gravid were opened and placed in 10 % ammonium sulphide solution for detection of implantation sites (Kopf, et al., 1964).

IMPLANTATION
Each implant was categorized according to the following criteria: a viable foetus if it responded to touch; a nonviable foetus if there were no signs of autolysis and did not respond to touch; late resorption if a recognizable foetal form and underwent autolysis; and an early resorption if there was an implantation site, but the tissue had no recognizable foetal characteristics. A necropsy was conducted on each doe and maternal gross lesions were saved in 10 % neutral buffered formalin for possible microscopic examination.
Fetal examinations:
TERATOLOGIC EXAMINATION
Before the umbilical cord was cut for each foetus, it was momentarily clamped with forceps to prevent bleeding and promote clotting.
Each foetus was individually weighed and examined for external malformations and variations. Each foetus was deeply anesthetized by approximately
0.02 mL of a sodium pentobarbital euthanasia solution injected sublingually, making every attempt to avoid injection of internal organs and subjected to a fresh foetal soft tissue dissection (Smckhardt and Poppe, 1984). Any visceral abnormalities were recorded.
After dissection and examination of internal organs was complete, the heads from approximately one-half of the foetuses were removed and placed in Bouin's solution for subsequent soft tissue examination using the Wilson razor-blade sectioning technique (Wilson, 1965).
A mid-coronal head slice was made on the remaining foetuses and the brains were examined.
The carcasses and remaining foetuses were eviscerated, skinned, and fixed in alcohol, macerated in potassium hydroxide, stained with Alizarin Red S and Aldan blue, and cleared with glycerin for subsequent skeletal examination of bone and cartilage (Kimmel and Trammel, 1981).
Foetal findings were classified as malformations or developmental variations under the supervision of a developmental toxicologist.
Statistics:
Differences between groups were assessed using:
- Group pair-wise Comparison: Levene’s test, Dunnett’s test, Welch’s test with a Bonferroni correction (homogeneity)
- Arcsin-Square-Root Transformation
- Chi-square test (homogeneity)
- Kruskal-Wallis test, Mann-Whitney U-test (malformations)
- Parson Chi-Square, Fishers exact test (malformations and developmental variations)
- Descriptive statistics (means, SD)
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
An excessive mortality and maternal toxicity were observed at 800 mg/kg/day during this study.
As a result, this group was terminated as of September 16, 1998.
A total of 8 does in this group were found dead and an additional doe was euthanized in extremis.
Two does from the control group died during the study. These deaths were considered to be related to technical error or mechanical injury. One doe in the 400 mg/kg/day group was diagnosed as moribund, but died before it could be euthanized. The necropsy endings indicated that this dam was suffering from an esophageal laceration. Hence, this death was considered to be due to gavage error, and not test article toxicity.

CLINICAL OBSERVATIONS
Treatment related clinical signs observed in the 800 mg/kg/day group included convulsions, decreased defecation soft stool, discoloured faeces and reddish fluid in the refuse pan. In addition, 7 does from the 800 mg/kg/day group aborted during the study, and these abortions were considered to be treatment related. Furthermore, 2 does from the 400 mg/kg/day group delivered early and this was considered to be related to treatment. Slight increases in soft stool and discoloured faeces were also noted in the 400-mg/kg/day group when compared with the vehicle control group, and since these signs were also observed at 800 mg/kg/day, the findings were considered to be related to test article administration.
One doe each from the 100 and 400 mg/kg/day groups aborted during the study. The 400mg/kg/day doe that aborted had an oedematous stomach and liquid, bloody contents in the intestines at necropsy. This abortion, therefore, could have been secondary to these effects.
Furthermore, necropsy of the doe at 100 mg/kg/day that aborted revealed injuries consistent with gavage error, suggesting that this abortion also may have beensecondary to a physical trauma. For further details see Table 1.

BODY WEIGHT
Treatment-related, statistically significant decreases in body weight gain were noted at 800 mg/kg/day when compared with the vehicle controls during the study.
Significant losses in body weight occurred in the 800 mg/kg/day group when compared with the vehicle controls during several intervals of the gestation period.
No statistically significant changes in body weight or body weight gain were noted in does at 100 or 400-mg/kg/day during gestation.
The body weight gain of both treatment groups was comparable with vehicle controls during the study.
For further details see Table 2.

FOOD CONSUMPTION
Treatment-related reductions in food consumption were noted in the 800 mg/kg/day group when compared with the vehicle control group during the study. This was corroborated by the significant decreases in body weight gain observed in this group during gestation. No changes in food consumption were noted in the lower treatment groups when compared with the vehicle control group during the study.
For further details see Table 3.

NECROPSY OBSERVATION
Treatment-related necropsy findings appeared to be limited to the 800 mg/kg/day group with 1 exception and included discoloration of the liver, edematous and/or discoloured stomach red discolored and/or edematous intestines, bloody and/or mucoid contents in the intestines, and loci in the lung.
The rabbit in the 400 mg/kg/day group that aborted had an edematous stomach and liquid, bloody contents in the intestines at necropsy. Since these findings were also noted in the 800 mg/kg/day group, the observations at 400 mg/kg/day were considered to be treatment related. All other necropsy findings were considered to be spontaneous in nature or observations normally seen in rabbits of this age and strain and unrelated to test article administration.
One vehicle control rabbit (Animal No. 1986) that died appeared to have suffered from a mechanical injury since it had a subdural haemorrhage in the brain at necropsy as well as some hemorrhaging around the thymic region.
This rabbit exhibited decreased activity, labored breathing and white material around the nose prior to death.
For further details see Table 4.

UTERINE EXAMINATION
The 800 mg/kg/day group was terminated before completion of the study due to excessive maternal toxicity.
Six does were subjected to elective euthanasia prior to Day 29 of gestation. Three does from the 800 mg/kg/day group survived to Day 29 and were euthanized and subjected to the protocol-specified uterine examination, and their foetuses were examined as specified in the protocol. The data for these 3 does and litters are presented in the individual tables within the report, but were not included in the summary tables or in the statistical analysis. No treatment-related effects on uterine parameters were observed in the 100 or 400 mg/kg/day groups when compared with the vehicle control group. Numbers of corpora lutea, implantation sites, live and dead foetuses and resorptions, pre and post-implantation loss and sex ratio were comparable between the vehicle control and treatment groups.
The number of live foetuses/litter was statistically higher at 400 mg/kg/day than controls.
This was due to the lower (not statistically significant) pre-implantation loss at 400 mg/kg/day when compared with controls and was not considered to be related to test article administration.
There were no changes in uterine weight, adjusted body weight or body weight gain in the 100 or 400 mg/kg/day groups when compared with the vehicle control group. There were no changes in uterine weight, adjusted body weight or body weight gain in the 100 or 400 mg/kg/day groups when compared with the vehicle control group. For further details see Table 5a.
Treatment-related effects on foetal body weight, however, were noted in the 400 mg/kg/day group when compared with the vehicle control group. Statistically lower foetal (all viable foetuses) body weight and male foetal body weight were noted at 400 mg/kg/day when compared with the vehicle control group. These changes may have been secondary to the maternal toxicity observed in this study and were not considered to be an indication of developmental toxicity. No changes were observed at 100 mg/kg/day when compared with the vehicle control group.
For further details see Table 5b.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL EVALUATIONS
1) EXTERNAL EXAMINATION
No treatment-related effects were noted. The incidences of findings were low and sporadic among the treatment groups and were within historical control ranges for this laboratory.

2) VISCERAL EXAMINATION
There were no treatment-related increases in the incidence of visceral variations or malformations in this study. Slight, but not statistically significant, increases in several malformations were noted at 400 mg/kg/day when compared with the vehicle control group.
The litter incidence of haemorrhagic iris at 400 mg/kg/day was slightly above the historical control range for this laboratory. The litter incidences of gallbladder agenesis (malformation), hypoplasia of the gallbladder (variation) and azygous lobe of lung absent (variation) at 400 mg/kg/day were within historical control range (litter incidence).
Since all the above findings were within or only slightly above historical control range, the findings were considered to be spontaneous in nature and unrelated to test article administration.
For further details see Table 6.

3) SKELETAL EXAMINATION
No treatment-related increases in skeletal variations or malformations were noted at 100 or 400 mg/kg/day when compared with the vehicle control group. The incidences of all endings were either comparable with the concurrent control group or within historical range for this laboratory.
Slight increases in the litter incidence of hyoid arch bent and unilateral full rib were noted at 400 mg/kg/day when compared with the vehicle control group. These findings are relatively common in rabbits, however, and both litter incidences are well within the historical control range for this laboratory.
Furthermore, decreases in foetal weight were observed at 400 mg/kg/day, suggesting that foetal development was delayed as a result of maternal toxicity. These findings may have been a further indication of delayed development as a result of maternal toxicity rather than a direct result of the test article on skeletal development.
For further details see Table 7.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

TABLE 1: Clinical observations (n. animals affected)

Clinical signs Dose level (mg/kg/day)
0 100 400 800
Found dead 2 0 0 8
Euthanizedin extremis 0 0 1 (gavage error) 1
Elective euthanasia 0 0 0 6
Aborted 0 1 1 7
Delivered early 0 0 2 0
Convulsions 0 0 0 1
Decreased defecation 9 7 11 20
Soft stool 4 7 9 13
Discoloured faeces 0 0 1 15
Reddish fluid in refuse pan 1 0 1 13

TABLE 2: Gestational Body Weight Gain (g)

Study interval Dose level (mg/kg/day)
(Interval) 0 100 400 800
0-7 249 299 259 301
7 -29 240 289 244 -221
0-29 502 585 502 53

TABLE 3: Gestational Food Consumption Data (g/animal/day)

Study interval Dose level (mg/kg/day)
(Interval) 0 100 400 800
0-7 138.4 149.2 144.1 140.8
7 -29 129.0 140.6 128.8 109.5
0-29 131.2 142.6 134.5 117.2

TABLE 4: Necropsy Observations (n. Animals Affected)

Findings Dose level (mg/kg/day)
0 100 400 800
Foci in the lungs 0 0 0 3
Discoloration of the liver 1 0 0 4
Accentuated lobulation of the liver 0 0 1 1
Discoloration of the stomach 0 0 0 2
Edematous stomach 0 0 1 2
Red discoloration of the intestines 0 0 0 2
Liquid mucoid contents of the intestines 1 0 0 4
Liquid bloody contents of the intestines 0 0 1 4
Edematous intestines 0 0 0 1

TABLE 5a: Summary of Uterine Parameters

Uterine Parameters Dose level (mg/kg/day)
0 100 400
N. Pregnant 25 25 25
N. Corpora lutea/doe 10.10 10.17 10.14
N. Implantation sites/doe 8.83 9.04 9.57
N. Live foetuses 8.35 8.79 9.00
Sex ration (% males) 50.76 53.28 55.34
N. Total resorption/doe 0.48 0.25 0.57
Gravid uterine weight (g) 489.0 493.0 524.6
Adjusted body weight gain (g) 13.5 91.9 50.2

TABLE 5b: Foetal Body weight (g)

Uterine Parameters Dose level (mg/kg/day)
0 100 400
All viable foetus 41.75 41.56 38.13
N. Corpora lutea/doe 41.00 41.43 37.61
N. Implantation sites/doe 42.29 41.64 38.81

TABLE 6: Incidence of Visceral Findings (n. Foetus/litters)

Variation Dose level (mg/kg/day) Historical control (% Litters Affected)
0 100 400
Foetus/litters examined 192/23 211/24 205/23 NA
Hemorrhagic iris 0/0 1/1 (4 %) 2/2 (9 %) 0-6
Azygous lobe of lung absent 8/7 (30 %) 6/6 (25 %) 16/9 (9 %) 0-39
Hypoplasia of gallbladder 3/2 (9 %) 1/1 (4 %) 11/6 (26 %) 0-32
Agenesis of the gallbladder 0/0 0/0 1/1 (4 %) 0-10

TABLE 7: Skeletal Variations (n. Foetus/litters)

Variation Dose level (mg/kg/day) Historical control (% Litters Affected)
0 100 400
Fetuses/litters examined 192/23 211/24 205/23 NA
Hyoid arch bent 3/3 (13 %) 5/4 (17 %) 18/10 (43 %) 0-53
27 Presacral vertebrae 57/15 (65 %) 30/9 (38 %) 35/11 (48 %) 25 -85
Extra rib  16/9 (39 %) 22/11 (46 %)  20/14 (61 %)  0 -100
7th cervical rib 1/1 (4 %)  10/4 (17 %)  1/1 (4 %) 0 -42 
Conclusions:
The test item is considered under the study not teratogenic in rabbits.
Executive summary:

Method

The developmental toxicity, including the teratogenic potentialof the test article was assessedin rabbits in accordance with OPPTS Guideline 870.3700, EPA Guidelines, March 1997. The dose levels of 100, 400, and 800 mg/kg/day were administered via gavage, once at day, to 3 treatment groups of 25 females and 1 vehicle control group. The female rabbits were time mated at receipt. 

Exposure initiated on Day 7 of gestation and continued to and included Day 28 of gestation. Observations of does included clinical signs, gestational body weights and food consumption. Litters were delivered by caesarean section on Day 29 of gestation. Gravid uterine weights were recorded. Total number of corpora lutea, implantations, early and late resorptions, live and dead foetuses, and individual sex and body weights of foetuses were recorded. All foetuses were examined for external, visceral, and skeletal abnormalities (bone and cartilage).

Results

Excessive maternal toxicity, as evidenced by mortality, statistically significant decreases in body weight gain and food consumption as well as an increase in abortion, was observed at 800 mg/kg/day. As a result, this group was terminated prior to completion of the study. Less severe maternal toxicity was observed at 400 mg/kg/day.

Two does died in the control group, but these deaths were a result of technical gavage error or mechanical injury. A total of 8 rabbits from the 800 mg/kg/day group died during gestation and another high-dose doe was euthanized in extremis. An additional 7 does from the high-dose group aborted during the study. No treatment-related mortality was noted at 100 or 400 mg/kg/day. 

A doe in the 400 mg/kg/day group was considered to be moribund, but died prior to being euthanized. Necropsy findings suggested that this animal died due to gavage-related injury. Treatment-related clinical observations at 400 mg/kg/day included soft faeces and discoloured stool. No changes in body weight, body weight gain or food consumption were noted at 100 or 400 mg/kg/day. 

Necropsy findings in does from the 800 mg/kg/day group included discoloration of the liver, edematous and/or discoloured stomach, red discoloured and/or edematous intestines, bloody and/or mucoid contents in the intestines. The rabbit in the 400 mg/kg/day group that aborted had an edematous stomach and liquid, bloody contents in the intestines at necropsy which were also considered to be treatment related.

Foetal data collected from 3 litters that were examined prior to terminating the 800 mg/kg/day group are presented in individual tables, but were not summarized, analysed statistically or discussed in this report. No effects on uterine parameters were noted at 100 or 400 mg/kg/day.

Numbers of corpora lutea, implantations, live and dead foetuses and resorptions were comparable between the vehicle control and the 100 and 400 mg/kg/day groups.

Foetal body weights were statistically lower at 400 mg/kg/day when compared with the vehicle control group.

Based on treatment-related clinical observations and necropsy findings seen in does at 400 mg/kg/day, the No Observed Effect Level (NOEL) for maternal effects in this study was 100 mg/kg/day, the lowest dose tested. There were statistically significant decreases in foetal body weight at 400 mg/kg/day. These changes may have been secondary to the maternal toxicity observed in this study and were not considered to be an indication of developmental toxicity. 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no data available for the developmental toxicity of CAS 12004488-68-5. In order to fulfil the standard information requirements, a read-across from structurally related substances was conducted.

Two studies on developmental toxicity and teratogenicity according to EPA Guideline OPPTS 870.3700 and under GLP conditions have been performed in rats and rabbits with CAS 16470-24-9. In the rat study, 30 pregnant Sprague-Dawley rats per group were dosed with 100, 400 or 1000 mg/kg bw/day by oral gavage on gestation days 6-19. The only substance-related effect observed was discoloured faeces at 400 and 1000 mg/kg bw/day. At skeletal examination of foetuses, the incidence of misaligned sternebrae was slightly increased in all dose groups but was well within historical control range and not dose-related and therefore not considered to be test substance related. The incidence of rudimentary ribs was slightly above the historical control range at 100 and 1000 mg/kg bw/day. As the difference from the concurrent control group was not statistically significant and the increase was not dose-related, these findings were not considered biologically significant or test substance-related. The number of vertebral malformations at 1000 mg/kg bw/day (litter incidence 7.1 %) was very slightly above the historical control range (0 - 7 %) and not statistically different from the vehicle controls. Therefore, this border finding too was considered to be within normal variation and unrelated to test substance administration. As there were no adverse maternal or developmental effects seen at any dose level, the NOAEL for both maternal and foetal toxicity is the highest dose tested (1000 mg/kg bw/day) (Turk, 1999).

In the definitive rabbit study, 7 pregnant New Zealand White rabbits per group were dosed with 100, 400 or 800 mg/kg bw/day by oral gavage on gestation days 7 - 28. The application of 800 mg/kg bw/day resulted in excessive maternal toxicity as exhibited by death, abortion, increased incidence of clinical and gross pathological findings, and a marked decrease in food consumption and body weight gain. As a consequence this group was terminated prior to study. Abortion or early delivery and soft stool and discoloured faeces also occurred in some dams at 400 mg/kg bw/day. The fetal body weights were lower in the 400 mg/kg bw/day group than compared to controls which are considered to be secondary to maternal toxicity. At visceral examination of foetuses, the litter incidence of hemorrhagic iris at 400 mg/kg bw/day was slightly above the historical control range while the slightly increased incidences of gallbladder agenesis, hypoplasia of the gallbladder and azygous lobe of lung absent were within historical control range. Since all the above findings were within or slightly above historical control range, the findings were considered to be spontaneous in nature and unrelated to test substance. Also, no substance-related effects were noted at external and skeletal examinations. At a dose level of 100 mg/kg bw/day no substance-related effects were seen in dams or at foetal examinations.

The NOEL for both maternal and foetal toxicity therefore was established as 100 mg/kg bw/day and teratogenicity NOAEL = 400 mg/kg bw/day (Turk, 2000).


Justification for selection of Effect on developmental toxicity: via oral route:
Two developmental studies are available on rats and rabbits. Rabbits have been revealed as more sensitive, since in the same testing conditions demonstrated systemic effects and toxicity at maternal level.

Justification for classification or non-classification

Based on read-across from the structurally similar substances, the available data on toxicity to reproduction do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.