Registration Dossier

Toxicological information

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Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

The purpose of this study was to evaluate the toxicity of the test chemical on reproductive performance of CD-1 mice. CD-1 mice were exposed to the test chemical in drinking water at dose concentration of 0, 0.25, 2.5, and 5.0% (w/v) during a 7 day premating period, after which they were randomly paired (one male: one female) within each dose group and cohabited for approximately 14 weeks (100 days). Newborn litters were evaluated and immediately sacrificed.Exposure to the test chemical in the drinkinq water did not appear to seriously affect the reproductive performance of CD-l mice. No siqnificant differences in fertility, averaqe number of litters, proportion of pups born alive or proportion of male pups were observed in Task 2. Averaqe litter size and averaqe adjusted pup weight in the high dose group was significantly decreased. Although the decrease in overall pup weiqht was statistically siqnificant, it represented only a 5% decrease, relative to controls.An assessment of the effects of the test chemical on mid dose offsprinq relative to control offsprinq was performed in Task '4. No siqnificant differences were observed in fertility, proportion of pups born alive or proportion of male pups. Mid dose pairs were found to have sliqhtly smaller litters and decreased pup weiqhts, but the differences were not statistically siqnificant. Based, on all of the above observesations, the No Observed Adverse Effect Level of the test chemical on male and female CD-1 mice was found at dose concentration of 25 mg/ml.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Principles of method if other than guideline:
Reproductive and fertility effect of the test chemical was asessed in CD-1 mice.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Molecular formula:C7H5NO3S•Na
- Molecular weight : 205.2 g/mol
- Substance type: Organic
- Physical state: Solid
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
No Data Available
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animal
TEST ANIMALS
- Source: Charles River Breeding Laboratory Inc
- Weight at study initiation: 25 to 32 g
- Housing: Cage: Polycarbonate shoebox type cages (5" xII" x 7") with stainless steel wire bar lids
Bedding: Ab-Sorb-Dri
-Age at study initiation: Eleven-week old
- Diet (e.g. ad libitum): Purina certified Rodent Chow animal diet 5002 (ad libitum)
- Water (e.g. ad libitum): Distilled water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 deg C
- Humidity (%): 20 to 70%.
- Air changes (per hr): 10 or more air changes per hour
- Photoperiod (hrs dark / hrs light): 14 hour light cycle











Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Treatment solutions will be prepared in quantities adequate for two weeks of treatment period.The test chemical stock solution will be prepared by dissolving the test article in distilled water. The remaining dose levels will be prepared by appropriate dilutions or independently formulated. An aliquot of
each formulation will be sent to Midwest Research Institute (Kansas City, MO) prior to onset of exposure at week 1, 5, 11, and 17 to confirm dose levels and certify stability of the test article.
Details on mating procedure:
- M/F ratio per cage : 1:1 ratio
- Length of cohabitation : 14 weeks (100 days)
- Proof of pregnancy : Vaginal plug
- After successful mating each pregnant female was caged : individually
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No Data Available
Duration of treatment / exposure:
No Data Available
Frequency of treatment:
Daily
Details on study schedule:
The study was performed in 4 stages
Task 1: Dose finding study:
In dose range-finding study, sodium saccharin was tested at the following dose levels: 0, 0.25, 0.50, 1.0, 2.5, and 5.0% (w/v)

Task 2: Reproduction and fertility study:
Based on dose finding study this task 2 was performed, male and female CD-1 mice exposed to sodium saccharine at dose level 0, 0.25, 2.5, and 5.0% (w/v) during a 7 day premating period, after which they were randomly paired (one male: one female) within each dose group and cohabited for approximately 14 weeks (100 days). Newborn litters were evaluated and immediately sacrificed.

Task 3: Determination of affected sex:
Based on the results obtained from Task 2, the experimental animals will be assigned to Task 3 which will determine which sex is affected

Task 4: Offspring assessment:
Based on the results obtained from Task 2, the experimental animals will be assigned to Task 4 which will determine the reproductive capacity of the ,1st generation offspring.
Remarks:
Doses / Concentrations:
0, 1.25%, 2.5%, 5% (0, 12.5, 25, 50 mg/ml)
Basis:
nominal in water
No. of animals per sex per dose:
Treatment group: 20 males and 20 females
Control group: 40 male and 40 females
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available
Positive control:
No Data Available
Parental animals: Observations and examinations:
Task 2: Body Weight, Number of litters produced, Number and percent of live pups per litter, Mean body weight of live offspring, Percent of infertile pairs were observed

Task 3: For males: Body weight, Liver weight, Fixed Brain Weight, Right Testicular Weight, Ventral Prostate Weight (pair), Seminal vesicle weight, Right epididymal weight, Left testis with attached epididymis weight.

For females: Body weight, Liver weight, Fixed brain weight, Fixed pituitary weight,Weights of complete reproductive tract (ovaries,
oviducts, uterus, and vagina).
Oestrous cyclicity (parental animals):
Task 4: Vaginal smears were prepared for 7 consecutive days from 5 second generation female mice
which failed to deliver any pups at the end of Task 4. These slides were evaluated for the relative frequency of estrous stages and approximate estrous cycle length
Sperm parameters (parental animals):
Task 4: Sperm morphology studies were also conducted at necropsy. Sperm motility, sperm density, and the incidence of abnormal sperm were counted
Litter observations:
Task 4:
Second generation pups will be examined on postnatal day 0, humanely sacrificed, and the following parameters evaluated: Number and percent fertile pairs, Litter sizes, Litter weight, males and females weighed separated, Number and percent of live pups per litter
Postmortem examinations (parental animals):
No Data Available
Postmortem examinations (offspring):
No Data Available
Statistics:
The Kruskal-Wallis test is the nonparametric analysis of the parametric one-way analysis of variance. Pairwise comparisons of sroup medians are performed usins the Mann Whitney U test. Pairwise comparisons of proportionsare performed using Fisher's exact test.
Reproductive indices:
The fertility index in the control and various treatment groups varied between 97 to 100%: all breeding pairs except one in the control group delivered at least one litter
Offspring viability indices:
No Data Available
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the low dose (1.25%) group and only 1 animal died in the 2.5% dose group. Eight out of 40 animals died in the 5.0% dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in body weight observed in females of highest dose group significant increase in food consumption observed in dose group (1.25% and 2.5%)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in body weight observed in females of highest dose group significant increase in food consumption observed in dose group (1.25% and 2.5%)
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Two out of 5 animals (one from each group) were in diestrus phase during all 7 days of smearing.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, sperm density, and the incidence of abnormal sperm were not affected by the test chemical treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive performance
Number of litters per fertile pair in the control and three treatment groups was essentially the same live offspring in the 5.0% dose group was significantly lower (p<0.05) than the control group. Proportion of pups born alive, and sex ratio values were not affected

Dose descriptor:
NOAEL
Effect level:
25 other: mg/ml
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality, body weight , food consumption, water consumption, no. of litters, pups born alive, and sex ratio
Critical effects observed:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When the live pup weight was adjusted for the total number of live and dead pups per litter, the average male and the combined pup weight values in the 5.0% dose group were significantly decreased
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not examined
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 other: mg/ml
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: no adverse effects observed
Reproductive effects observed:
not specified
Conclusions:
Based, on all of the above observesations, the No Observed Adverse Effect Level of the test chemical on male and female CD-1 mice was found at dose concentration of 25 mg/ml.
Executive summary:

The purpose of this study was to evaluate the toxicity of the test chemical on reproductive performance of CD-1 mice. CD-1 mice were exposed to the test chemical in drinking water at dose concentration of 0, 0.25, 2.5, and 5.0% (w/v) during a 7 day premating period, after which they were randomly paired (one male: one female) within each dose group and cohabited for approximately 14 weeks (100 days). Newborn litters were evaluated and immediately sacrificed. Exposure to the test chemical in the drinkinq water did not appear to seriously affect the reproductive performance of CD-l mice. No siqnificant differences in fertility, averaqe number of litters, proportion of pups born alive or proportion of male pups were observed in Task 2. Averaqe litter size and averaqe adjusted pup weight in the high dose group was significantly decreased. Although the decrease in overall pup weiqht was statistically siqnificant, it represented only a 5% decrease, relative to controls. An assessment of the effects of the test chemical on mid dose offsprinq relative to control offsprinq was performed in Task '4. No siqnificant differences were observed in fertility, proportion of pups born alive or proportion of male pups. Mid dose pairs were found to have sliqhtly smaller litters and decreased pup weiqhts, but the differences were not statistically siqnificant. Based, on all of the above observesations, the No Observed Adverse Effect Level of the test chemical on male and female CD-1 mice was found at dose concentration of 25 mg/ml.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Data is from a Klimisch 2 database.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive Toxicity Study:

The toxicity of the test chemical was assesed and evaluated on the basis of the following studies:

Reproductive Toxicity Study 1:

The purpose of this study was to evaluate the toxicity of the test chemical on reproductive performance of CD-1 mice. CD-1 mice were exposed to the test chemical in drinking water at dose concentration of 0, 0.25, 2.5, and 5.0% (w/v) during a 7 day premating period, after which they were randomly paired (one male: one female) within each dose group and cohabited for approximately 14 weeks (100 days). Newborn litters were evaluated and immediately sacrificed.Exposure to the test chemical in the drinkinq water did not appear to seriously affect the reproductive performance of CD-l mice. No siqnificant differences in fertility, averaqe number of litters, proportion of pups born alive or proportion of male pups were observed in Task 2. Averaqe litter size and averaqe adjusted pup weight in the high dose group was significantly decreased. Although the decrease in overall pup weiqht was statistically siqnificant, it represented only a 5% decrease, relative to controls.An assessment of the effects of the test chemical on mid dose offsprinq relative to control offsprinq was performed in Task '4. No siqnificant differences were observed in fertility, proportion of pups born alive or proportion of male pups. Mid dose pairs were found to have sliqhtly smaller litters and decreased pup weiqhts, but the differences were not statistically siqnificant.Based, on all of the above observesations, the No Observed Adverse Effect Level of the test chemical on male and female CD-1 mice was found at dose concentration of 25 mg/ml.

Reproductive Toxicity Study 2:

In a one generation study, male C3Hx101 mice were exposed to 0, 200 or 500 mg/kg/day of the test chemical by gavage for 2 successive weeks. The dose-dependent effect of the test chemical on the induction of chromosomal translocation in directly treated male mice and their transmission to F1 male offspring was investigated. The test chemical-treated animals and controls, as well as their F1 offspring, did not show any statistically significant changes relating to body and testis weight. The test chemical induced no chromosomal translocations in directly treated animals or in their offspring. However, the cells analyzed in diakinesis-first metaphase in the parental generation were affected on the spermatogonial stage and in the F1 generation there was a significant fertility reduction after a dose of 500 mg/kg. Therefore, NOAEL was considered to be 200 mg/kg/day in the parental generation, whereas LOAEL was considered to be ≤ 500 mg/kg/day in the F1 generation when exposed to the test chemical.

Reproductive Toxicity Study 3:

The Purpose of this study was to evaluate the toxicity of the test chemical in CBA mice. Twenty male mice of CBA strain were administered 1.72% the test chemical in distilled water for 30 days. Twenty four hours after trenatment ceased each treated male was mated with three normal females.Vaginal plug was considered day 0 of pregnancy. The mated females were dissected on 15thday of gestation and the numbers of live and dead embryos were recorded. Induction of Dominant lethal mutations in treated males was evident by statistically significant increase in the incidence of intrauterine deaths in females mated with treated males. Also,Statistically significant decrease in Total number of implants, Implants per  female were observed in treated as compared to control. Statistically significant increase in number of dead, percent dead and dead per female was  observedin treated as compared to control.Thus, the Low Observed Adverse Effect Level (LOAEL) of the test chemical in mice in 30 days study was observed at dose concentration of 1.72% (2866 mg/kg bw).

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

In a one-generation study, dose response changes associated with the test chemical treatment was evaluated in Sprague-Dawley rats at 30 and 90 days post-birth. The test chemical in the dietary levels of 0, 1, 3 or 7.5% was administered to rats by feed. Body weights in the parental generation and in the offspring decreased with increased test chemical concentrations. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at 1% and 3% test chemical per day but were significantly increased with 7.5% of the test chemical. The increases in lipids and vitamin E were reversible. The reversibility of the effects of 7.5% of the test chemical was examined in 90-day-old rats. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, however, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% of dietary test chemical on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumors. Therefore, the NOEAL for the offspring was considered to be 1% of the test chemical per day and the LOAEL in the parental generation was considered to be 1% of the test chemical per day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
other:
Principles of method if other than guideline:
Effects of in utero and postnatal test chemical exposure exposure were assessed in one generation of Sprague-Dawley Rats.
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
- Molecular formula:C7H5NO3S•Na
- Molecular weight : 205.2 g/mol
- Substance type: Organic
- Physical state: Solid
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Details on test animal & Environmental conditions
- Source: Charles River Breeding Laboratories Inc.,
- Age at study initiation: 4 wk old
- Housing: Polycarbonate cages with corncob bedding
- Diet (e.g. ad libitum): Purina Rodent Chow No. 5002
- Water (e.g. ad libitum): distilled water
- Acclimation period: 2 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71 +_5°F
- Humidity (%): 50 +_ 20%.
- Photoperiod (hrs dark / hrs light): 12-hr light/dark schedule.




















Route of administration:
oral: feed
Vehicle:
other: Purina Rodent Chow No. 5002
Details on exposure:
The test chemical was mixed in the diet at concentrations of 0, 1, 3 or 7.5% by weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dietary levels of the test chemical were analyzed by HPLC. Before each batch of test diet was fed.
Details on mating procedure:
Mating was started after 4-5 wk of test diet feeding.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 1, 3 or 7.5% by weight (0,1000,3000,7500 mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
Parental generation
Control: 33 females, 17 males
7.5% per day: 33 females, 17 males

F1-generation
Control: 14 litters
1% per day: 14 litters
3% per day: 14 litters
7.5% per day: 14 litters
7.5% per day (30 days) and then basic diet (60 days): 14 litters
Basic diet (30 days) and then 7.5% per day (60 days): 14 litters

Importantly, litters were randomly culled on day 4 after birth to four males and four females when possible.
Control animals:
yes, concurrent vehicle
Details on study design:
Male and female rats were placed on the appropriate test diet at 6 weeks of age. Mating was started after 4-5 weeks of test diet feeding. Pups were weaned at 21 days of age and fed the same diet as the parental rats. Parental males were killed at the end of the mating period and female rats were killed after their litters had been weaned. Seven litters from each of groups 1-4 were killed when they were 30 days old to evaluate the dose-response effects of the test chemical. Also at 30 days, the diet for group 5 was changed from 7.5% of the test chemical to control, and the diet for group 6 was changed from control to 7.5% of the test chemical. Seven litters from each of groups 1, 4, 5 and 6 were then killed at 90 days of age to evaluate the reversibility of the effects of the test chemical.
Maternal examinations:
Food and water consumption were measured for 1 week prior to mating and for 3 weeks during gestation. Body-weight measurements of parental rats were obtained on the same days as consumption determinations, and detailed physical examinations were performed before mating. The urinary bladder was removed and processed for light microscopy.
Ovaries and uterine content:
No Data Available
Fetal examinations:
Food and water consumption were determined weekly after weaning except during week 5 post-birth when some litters were killed and others had their experimental diets changed. Pups in each litter were weighed as a group by sex after birth, at 4, 7 and 14 days after birth, and then individually at weekly intervals until the time of killing. The health status of the pups was also determined on these days. Blood specimens were collected for haematology. Serum was analyzed for the presence of cholesterol, triglycerides, folate, vitamin A and E, and iron and iron-binding capacity. The adrenals, liver and full and emptied caecum was removed at the time of killing and weighed. Bladders from one male and one female in each litter were examined by light microscopy.
Statistics:
Statistical analyses were performed with a generalized linear model procedure (SAS Institute, Inc., USA). Duncan's multiple range test was used for multiple comparison of means. Comparisons were made between group 1 and each of the other groups and between group 4 and groups 5 and 6.
Indices:
No Data Available
Historical control data:
No Data Available
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The test chemical treatment causes lower body-weight gains. Significant differences were noted only between the control and the high-dose groups, with the exception of a significant increase in water consumption by the females treated with 1% of the test chemical.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Unchanged basal diet consumption in males were observed. Significant differences were noted only between the control and the high-dose groups, with the exception of a significant increase in water consumption by the females treated with 1% of the test chemical.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The test chemical caused increase in water consumption in females. Significant differences were noted only between the control and the high-dose groups, with the exception of a significant increase in water consumption by the females treated with 1% of the test chemical.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No Data Available
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Dose descriptor:
LOAEL
Effect level:
1 000 other: mg/kgbw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
For male and female rats at week 4 post-birth, the values of body weight, food consumption (basal diet) and water consumption decreased with increased test chemical concentrations. Rats treated with 7.5% of the test chemical weighed about 60% as much as control rats, and males fed 3% of the test chemical weighed significantly less (approx. 10%) than controls. Decreases in diet consumption were also observed at 3 and 7.5% dietary test chemical. At week 12 post-birth, body weights of 7.5% of the test chemical-treated males and females remained significantly lower than controls.


Details on embryotoxic / teratogenic effects:

At week 12 post-birth, body weights of 7.5% NaS-treated males and females remained significantly lower than controls. All male rats fed NaS, including those switched from NaS to control diet, and consumed significantly less diet than controls during wk 6-12 post-birth. Female rats in all NaS-treated groups consumed similar amounts of diet as did controls. Water consumption during week 6-12 post birth was significantly greater, compared with controls in groups 4 (7.5% NaS) and 6 (control switched to 7.5% NaS).




Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): For male and female rats at week 4 post-birth, the values of body weight, food consumption (basal diet) and water consumption decreased with increased test chemical concentrations. Rats treated with 7.5% of the test chemical weighed about 60% as much as control rats, and males fed 3% of the test chemical weighed significantly less (approx. 10%) than controls. Decreases in diet consumption were also observed at 3 and 7.5% dietary test chemical. At week 12 post-birth, body weights of 7.5% of the test chemical treated males and females remained significantly lower than controls.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
At 30 days post-birth, a decrease in both absolute and relative liver weights related to the dose of the test chemical was observed. Male rats switched from test chemical to control diet also had lower absolute liver weights than control rats. For female rats liver weights were lower than those of controls when fed 7.5% test chemical diet throughout the study. For those switched from the control to test chemical diet, there were no differences at 30 days in the absolute weight of the empty caecum. Animals exposed to 7.5% test chemical were anemic at 30 days post-birth, as indicated by the decrease in red blood cells (RBC), hemoglobin (Hgb) and haematocrit (HCT). Mean values for mean corpuscular volume, hemoglobin and hemoglobin concentration were consistent with a diagnosis of iron deficiency. Rats of both sexes fed 3% test chemical had significantly decreased Hgb and HCT. In addition, no difference was observed in hematological parameters between the group switched from the 7.5% test chemical to control diet at 30 days post-birth and the control group. Compared with the control group, serum cholesterol, triglycerides and vitamin E were increased and serum vitamin A and folate were decreased in both males and females. t 90 days post-birth, males on the 7.5% test chemical diet had lower absolute and relative liver weights than controls. All animals exposed to 7.5% test chemical had increased caecal weights compared with controls. At 90 days post-birth, the values of haemoglobin (Hgb) and haematocrit (HCT) in rats exposed to 7.5% test chemical throughout the study were still significantly below control values .The effects in the female rats were similar to those in the males. With continuous test chemical treatment serum triglyceride levels in males and females, and serum cholesterol levels in males, were lower than in control. Serum folate was similar to control animals, except in two groups 7.5% test chemical switched to control and control switched to 7.5% test chemical) which showed elevated and reduced values. Serum vitamin A remained significantly depressed in all male groups treated with 7.5% test chemical. The microscopic effects of test chemical treatment on the bladder were negligible. Simple hyperplasia was noted in the bladders of some 7.5 % test chemical-treated rats at 90 days. Morphological changes in the urothelium were also observed by SEM in these groups, but the severity and incidence were low.
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body-weight gain, food consumption water consumption, Hematological parameters, serum Lipid and vitamin concentration,
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
LOAEL for the parental generation was considered to be 1% of the test chemical per day and the NOEAL for the F1 generation was considered to be 1% of the test chemical per day.
Executive summary:

In a one-generation study, dose response changes associated with the test chemical treatment was evaluated in Sprague-Dawley rats at 30 and 90 days post-birth. The test chemical in the dietary levels of 0, 1, 3 or 7.5% was administered to rats by feed. Body weights in the parental generation and in the offspring decreased with increased test chemical concentrations. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at 1% and 3% test chemical per day but were significantly increased with 7.5% of the test chemical. The increases in lipids and vitamin E were reversible. The reversibility of the effects of 7.5% of the test chemical was examined in 90-day-old rats. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, however, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% of dietary test chemical on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumors. Therefore, the NOEAL for the offspring was considered to be 1% of the test chemical per day and the LOAEL in the parental generation was considered to be 1% of the tet chemical per day.

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The data is from a Klimisch 2 database.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

Developmental Toxicity Study:

The toxicity of the test chemical was assesed and evaluated on the basis of the following studies:

Developmental Toxicity Study 1:

In a one-generation study, dose response changes associated with the test chemical treatment was evaluated in Sprague-Dawley rats at 30 and 90 days post-birth. The test chemical in the dietary levels of 0, 1, 3 or 7.5% was administered to rats by feed. Body weights in the parental generation and in the offspring decreased with increased test chemical concentrations. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at 1% and 3% test chemical per day but were significantly increased with 7.5% of the test chemical. The increases in lipids and vitamin E were reversible. The reversibility of the effects of 7.5% of the test chemical was examined in 90-day-old rats. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, however, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% of dietary test chemical on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumors. Therefore, the NOEAL for the offspring was considered to be 1% of the test chemical per day and the LOAEL in the parental generation was considered to be 1% of the test chemical per day.

Developmental Toxicity Study 2:

The purpose of this study was to evaluate the toxicity of the test material in two generation of Sprague-Dawley Rats.In a two-generation lifetime feeding study, 50 male and 50 female weanling Sprague-Dawley rats were given test material at dose of 5% for 90 days. After 3 months on test, the F0 rats were bred and 50 pups of each sex, were weaned onto the parental diets which both generations received for their lifetime. Rats from both generations fed diets 5% of the test material had decreased growth rates, but only the former two had lowered feed consumption. There were no treatment related effects upon reproduction, longevity, or hematological parameters. The animals were free of the bladder parasite Trichosomoides crussicauda. A few animals developed grossly visible bladder and kidney stones but the incidence of these was not treatment related. The incidence of bladder tumors in male rats fed the 5% test material diet was significantlyincreased in both generations compared to their respective control groups. An evaluationof individual feed consumption data for animals fed test material-containing diets did notreveal any statistical difference between the amount of feed consumed by animals whichhad bladder tumors and those that did not. The incidence of bladder tumors in the female rats fed the 5% saccharin diet was not significantly different from that in control animals.Thus, based on all the above observations, the Low Observed Adverse Effect Level (LOAEL) of the test material in Rats in 90 days study was observed at dose concentration of 5% (2500mg/kg bw/day).

Developmental Toxicity Study 3:

The development toxicity study oftest material was performedVirgin adult female albino CD-l outbred mice.The test material in dose concentration0,40, 100 and 240,600 mg/kg bw/day was administered via oralintubationon Day 6 and continuing daily through Day 15 of gestation. Sham treated group administered only vechicle and positive control Aspirin in dose concentration 150mg/kg also administered . Body weights were recorded on Days 0,6, II, 15, and 17 of gestation. All animals were observed daily for appearance and behavior withparticularattention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal. On Day 17 all dams were subjected to Caesarean section under surgical anesthesia, and the nuniliers of implantation sites, resorption sites, and live and dead fetuses were recorded. The body weights of the live pups were also recorded.Theurogenital tract of each darn was examined in detail for anatomical normality. All fetuses were examined grossly for the presence of external congenital abnormalities.One-third of the fetuses of each litter underwent detailed visceral examinations employing lOX magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects. No effects onNo effects on total pregnant mice, Number of implant sites, Number of fetuses alive, Number of resorptions were observed . no clearly dis-cernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham treated controls. HenceThe No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 600 mg/kg, when female micewere treated with test chemical orally.

Developmental Toxicity Study 4:

The development toxicity study oftest material was performedVirgin adult femalealbino rats (Wistar derived stock).The test material in dose concentration0,40, 100 and 240,600 mg/kg bw/day was administered via oralintubationon Day 6 and continuing daily through Day 15 of gestation. Sham treated group administered only vechicle and positive control Aspirin in dose concentration 250mg/kg also administered . Body weights were recorded on Days 0,6, II, 15, and 17 of gestation. All animals were observed daily for appearance and behavior withparticularattention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal. On Day 20 all dams were subjected to Caesarean section under surgical anesthesia, and the nuniliers of implantation sites, resorption sites, and live and dead fetuses were recorded. The body weights of the live pups were also recorded.Theurogenital tract of each darn was examined in detail for anatomical normality. All fetuses were examined grossly for the presence of external congenital abnormalities.One-third of the' fetuses of each litter underwent detailed visceral examinations employing 10X magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects. No effects onNo effects on total pregnant rats , Number of implant sites, Number of fetuses alive, Number of resorptions were observed. No clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham treated controls. Hence, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 600 mg/kg, when female ratswere treated with test chemical orally.

 

Developmental Toxicity Study 5:

The development toxicity study oftest material was performed ongolden hamsters from an outbred strain. The test material in dose concentration0,40, 100 and 240,600 mg/kg bw/day was administered via oralintubation on Day 6 and continuing daily through Day 10 of gestation. Sham treated group administered only vechicle and positive control Aspirin in dose concentration 250mg/kg also administered . Body weights were recorded on Days 0,8,10and 14 of gestation. All animals were observed daily for appearance and behavior withparticularattention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal. On Day 15 all animals were subjected to Caesarean section under surgical anesthesia, and the nuniliers of implantation sites, resorption sites, and live and dead fetuses were recorded. The body weights of the live pups were also recorded.Theurogenital tract of each darn was examined in detail for anatomical normality. All fetuses were examined grossly for the presence of external congenital defects and one-third of each litter underwent detailed visceralexaminationunder 10x magnification. The remaining two-thirds of the pups were cleared in potassium hydroxide, stained with alizarin red dye, and examined for the presence of sketal abnormalities.No effects onNo effects on total pregnant animals , Number of implant sites, Number of fetuses alive, Number of resorptions were observed . no clearly dis-cernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham treated controls. Hence, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 600 mg/kg, when female hamster were treated with test chemical orally.

Developmental Toxicity Study 6:

The development toxicity study oftest material was performedDutch-belted female rabbits.The test material in dose concentration0,40, 100 and 240,600 mg/kg bw/day was administered via oralintubation on Day 6 and continuing daily through Day 18 of gestation. Sham treated group administered only vechicle and positive control2.5mg of 6-amino nicotinamide dosed on Day 9. On Day 0 each doe was given an injection of 0.4 ml of human chorionic gonadotropin(400 IU) via the marginal ear vein. Three hours later, each doe was inseminated artificially with 0.3 ml of diluted semen froma proven donor buck using approximately 20 x 10 6 motile sperm according to the procedure described by Vogin et al.Body weights were recorded on Days 0,6,12,18, and 29 of gestation.All animals were observed daily for appearance and behavior, withparticular attention to food consumption and body weight in order torule out any abnormalities which may have occurred as a result ofanorexic effects in the pregnant female animal.On Day 29 all does were subjected to Caesarean section under surgical anesthesia, and the numbers of corpora lutea, implantationsites ,resorption sites and live and dead fetuses were recorded. Body weights of the live pups were also recorded. The urogenital tract of each animal was examined in detail for normality. In addition all fetuses underwent a detailed gross examination for the presence of external congenital abnormalities. The live fetuses of each Ltitter were ,then placed in an incubator for 24 hours for the evaluation of neonatal survival. All surviving pups were sacrificed, and all pups examined for visceral abnormalities (by dissection). All fetuses were thenclearedin potassium hydroxide (KOH), stained with alizarin red'S dye and examined for skeletal defectsNo effects onNo effects on total pregnant animals , Number of implant sites, Number of fetuses alive, Number of resorptions were observed . no clearly dis-cernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham treated controls. Hence,The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 600 mg/kg when female rabbits were treated with test chemical orally.

Justification for classification or non-classification

From the NOAEL and LOAEL values obtained from the above experimental data for the test chemical for toxicity to reproduction as well as developmental toxicity, it is considered that the chemical is likely to be classified as reproductive/developmental toxicant in Category 2 according to CLP regulations.