Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity Study:

In a reproductive toxicity study, the effect of the test chemical was evaluated in male Sprague-Dawley rats for 10 days. The seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with the test chemical (0.25% in diet). No mortalities were observed in the males. All organs were weighed at time of sacrifice before being subjected to microscopic examination or analysis for protein and DNA contents. Terminal mean body weight was 5.1% higher in the treated group compared to the control group. Food intake was not measured. Absolute liver weight was significantly increased in the 2500 ppm treatment group compared to the control group. No statistically significant differences in absolute organ weights were observed for the kidneys, adrenal glands or testes. Accessory sex organs were significantly enlarged in the 2500 ppm treatment group, with seminal vesicles weighing 42% more, ventral prostate 37% more, LABC 136% more, and glans penis 35% more. Protein and DNA contents in ventral prostrate, LABC muscle and glans penis were significantly elevated in the 2500 ppm treatment group. There were no gross abnormalities of any of the accessory sex glands, testes or penis among the treated animals. Histologically, all tissues appeared normal and there were no consistent histological differences between the two groups.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: reproductive toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from handbook or collection of data.
Qualifier:
no guideline available
Principles of method if other than guideline:
A reproductive toxicity study to investigate the effect of the test chemical in Sprague-Dawley rats.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Details on test animal

TEST ANIMALS
- Source: No data available
- Age at study initiation: 48-52 days
- Weight at study initiation: 223.2±14.9 to 226.7±20.9
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): Standard rat chow
- Water (e.g. ad libitum): No data available
- Acclimtization period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: Standard rat chow
Details on exposure:
Details on exposure
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Standard rat chow
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard rat chow
- Concentration in vehicle: 0 or 0.25% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
- M/F ratio per cage: Not available
- Length of cohabitation: Not available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Not available
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. Not available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not available
- After successful mating each pregnant female was caged (how): Not available
- Any other deviations from standard protocol: Not available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No Data
Duration of treatment / exposure:
10 days
Frequency of treatment:
Daily
Details on study schedule:
No data available
Remarks:
Doses / Concentrations:
0 or 0.25% per day
Basis:
nominal in diet
No. of animals per sex per dose:
Control: 12 animals
0.25% per day: 12 animals
Control animals:
yes, concurrent vehicle
Details on study design:
No of animals: Twenty-four Sprague-Dawley rats
Details of controls: 12 rats
- Dose selection rationale: The dose of the test chemical was based on our previous in vitro work and previous study evaluating effects of oral exposure to the test chemical in castrated animals.
- Rationale for animal assignment (if not random): Twenty-four Sprague-Dawley rats (at the age of 48-52 days) were randomly assigned to 2 treatment groups, each consisting of 12 animals: control group (standard diet) and the test chemical group (0.25% TCC by weight in diet of standard rat chow).
- Fasting period before blood sampling for clinical biochemistry: No Data Available
- Other: No Data Available
Positive control:
No data
Parental animals: Observations and examinations:
Blood was collected by cardiac puncture prior to euthanasia.
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
No data available
Postmortem examinations (parental animals):
Liver, kidney, adrenal glands, testes, levator ani-bulbocavernosus muscle (LABC), glans penis, ventral prostate, and seminal vesicles were surgically removed and weighed.

Organs of half of the animals from each group were fixed and assessed histologically. Organs of the other half of the animals were freeze-dried, weighed, and protein and DNA content determined.

Histoloy and Immunohistochemicstry Protein and DNA determinations were performed, and in the serum levels of luteinizing hormone was measured and a T assay was conducted. From the serum the detection and quantification of TCC was also measured and recorded.
Postmortem examinations (offspring):
No data available
Statistics:
All data were expressed as mean+SE. Data were analyzed, as appropriate, either by Student t test or, in the absence of normal distribution, by Wilcoxon rank-sum test using JMP statistical package for Macintosh computer.
Reproductive indices:
No data available
Offspring viability indices:
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):
Pretreatment body weights were comparable in both groups; however, during the 10-day course of the study, animals exposed to the treatment gained significantly more weight than control animals (on average, 85.6 vs 67.0 g). This resulted in an average 5.1% greater terminal weight in the treatment group.
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment, livers in the treatment group were also bigger, weighing on average 13.3% more than in the control group. Kidneys, adrenals, and testes in both groups had comparable weight. In contrast, all accessory sex organs were significantly enlarged in treated group, with seminal vesicles weighing 42% more, ventral prostate 37% more, LABC 136% more, and glans penis 35% more. To determine whether the differences in organ weight persisted after accounting for greater total body weight in the treated group, organ weights were also expressed as percentage of terminal body weight. Following this calculation, liver, seminal vesicles, ventral prostate, LABC, and glans penis remained significantly larger in the treated group compared to control group.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no visible abnormalities of any of the accessory sex glands, penis or testes, in treated animals and no histologically distinguishable difference between specimens from the control and treated animals. The vesicular glands were variably distended with fluid, the epithelium was simple or pseudostratified and thrown into numerous, complex, primary and secondary folds extending into and sometimes obliterating the lumen. Lobes were surrounded by connective tissue and a thick layer of smooth muscle but appeared similar in treated and control tissues. The acini of the prostate gland were also distended, lined by a simple epithelium, and surrounded by a thin connective tissue and smooth muscle layer.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Lutenizing Hormones: No significant differences in either of circulating LH were noted between treated and control groups.
Effect of the test chemical on Androgen Induced Transcriptional Activity in Human Prostate Cell: Testosterone and DHT treatments induced luciferase activity in LNCaP cells transfected with probasin or simple ARE promoters. Cotreatment of androgen with the test chemical (1.0 nmol/L) further increased luciferase activity by 221% (Probasin promoter) and 175% (ARE promoter) in LNCaP cells compared to androgen treatment alone (P < .01,). Similarly, in C4–2B cells, the test chemical further potentiated androgen-induced luciferase activity by 25.9% (Probasin promoter) and 38.5% (ARE promoter), compared to androgen treatment alone, although the amplification was less substantial than that observed in LNCaP cells, which have higher expression of AR (P < .05). In both cell lines, the amplification enhanced by the test chemical was significantly suppressed by the strong AR binding inhibitor, bicalutamide.
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Increased body weight and organ enlargement.
Remarks on result:
other: Not Specified
Critical effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
System:
male reproductive system
Organ:
testes
seminiferous tubules
seminal vesicle
ventral prostate gland
Levatorani plus bulbocavernous muscle complex
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
not specified
Sexual maturation:
not specified
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
Based on all the observations and conclusions, the LOAEL was considered to be 0.25% per day i.e. 250 mg/kg /day when male rats were exposed to test chemical.
Executive summary:

In a reproductive toxicity study, the effect of the test chemical was evaluated in male Sprague-Dawley rats for 10 days. The seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with the test chemical (0.25% in diet). No mortalities were observed in the males. All organs were weighed at time of sacrifice before being subjected to microscopic examination or analysis for protein and DNA contents. Terminal mean body weight was 5.1% higher in the treated group compared to the control group. Food intake was not measured. Absolute liver weight was significantly increased in the 2500 ppm treatment group compared to the control group. No statistically significant differences in absolute organ weights were observed for the kidneys, adrenal glands or testes. Accessory sex organs were significantly enlarged in the 2500 ppm treatment group, with seminal vesicles weighing 42% more, ventral prostate 37% more, LABC 136% more, and glans penis 35% more. Protein and DNA contents in ventral prostrate, LABC muscle and glans penis were significantly elevated in the 2500 ppm treatment group. There were no gross abnormalities of any of the accessory sex glands, testes or penis among the treated animals. Histologically, all tissues appeared normal and there were no consistent histological differences between the two groups.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The data is Klimisch 2 source and provides robust summary.
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 summary of the data for the reproductive toxicity is as follows:

Reproductive Toxicity Study 1:

In a reproductive toxicity study, the effect of the test chemical was evaluated in male Sprague-Dawley rats for 10 days. The seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with the test chemical (0.25% in diet). No mortalities were observed in the males. All organs were weighed at time of sacrifice before being subjected to microscopic examination or analysis for protein and DNA contents. Terminal mean body weight was 5.1% higher in the treated group compared to the control group. Food intake was not measured. Absolute liver weight was significantly increased in the 2500 ppm treatment group compared to the control group. No statistically significant differences in absolute organ weights were observed for the kidneys, adrenal glands or testes. Accessory sex organs were significantly enlarged in the 2500 ppm treatment group, with seminal vesicles weighing 42% more, ventral prostate 37% more, LABC 136% more, and glans penis 35% more. Protein and DNA contents in ventral prostrate, LABC muscle and glans penis were significantly elevated in the 2500 ppm treatment group. There were no gross abnormalities of any of the accessory sex glands, testes or penis among the treated animals. Histologically, all tissues appeared normal and there were no consistent histological differences between the two groups.

Reproductive Toxicity Study 2:

In this study, the authors exposed male rats daily to vehicle solution by injection and normal diet (control group); to testosterone propionate (TP) by injection and normal diet; to vehicle solution by injection and diet containing 2500 ppm of the test chemical; and to TP by injection and diet containing 2500 ppm of the test chemical. All animals were sacrificed and examined after 10 days of consecutive exposure. Treatment with the test chemical without TP injections produced a significant increase in absolute liver and ventral prostate weight compared to control data. TP injection alone produced a significant increase in the absolute weight of accessory sex organs (i.e. seminal vesicles, ventral prostrate, cowper’s gland and LABC muscle) compared to control data. The co-treatment of TP and the test chemical resulted in a significant increase in the absolute weights of the above accessory sex organs and glans penis compared with TP treatment alone, indicating a synergism between TP and test chemical in vivo. Organs were not subjected for microscopic examination. Based on all the observations and results, it was concluded that the LOAEL of the test chemical was observed to be 2500 ppm.

Reproductive toxicity study 3:

In the present study three successive generations of Sprague-Dawley rats were administered test chemical  via the diet to assesed the reproductive developmental toxicity of the test chemical. Dosing of P0 rats began 60 days prior to mating and was continuous thereafter. F1and F2 rats were dosed for an 80-day period prior to mating and continuously thereafter. Each parental generation was mated twice.Body weights and food consumption were measured weekly during the study. Observations for mortality and adverse effects were done twice daily. Detailed physical exams were done weekly on all generations. All animals dying spontaneously or killed in a moribund condition were examined and tissues preserved in 10% formalin. Dead or stillborn pups were given a gross postmortem exam and preserved in 70% ethanol. All adult males and females were given a gross postmortem exam and tissues preserved. The results of the study revealed, no treatment-related effect on mortality or physical in-life evaluations. Body weight and food consumption were not adversely affected by treatment throughout the study. Mating indices and male fertility were not adversely affected by treatment for all generations. Pregnancy rates were comparable to controls for dose groups 250 -1000 ppm. The pregnancy rate was unusually low for the high dose group 3000 ppm during the second litter interval of the F1 generation. Gestation length, pup viability, litter size at birth, litter survival indices, pup growth, and survival to weaning were comparable to controls for dose groups 250 - 1000 ppm. The mean number of live pups at birth was lower than controls for both litter intervals of only the F1 generation of the high dose group (3000 ppm). From the observations and results,The NOAEL for the parental (P0), F1 and F2 generation was found to be 150 mg/kg; 50 mg/kg & 150mg/kg (3000, 1000 and 3000 ppm) respectively.

Teproductive Toxicity Study 4:

In the present study the Sprague Dawley rats were administered with test chemical to critical growth period of gestation to determine the susceptible windows of exposure for reproductive toxicity. Starting on GD 5, dams were fed either with control  or with test chemical-supplemented chow (0.2% and 0.5%). On GD 19, maternal blood was collected prior to sacrifice. At necropsy, amniotic fluid was collected and total number of fetuses and the number of implantation sites were counted. Systemic organs (liver, kidney, and adrenal) and sex organ (ovary) were dissected and weighed. Tissue sections were examined with routine hematoxylin– eosin staining, and histological changes were evaluated . Serum and amniotic fluid samples were frozen at -80 dergree centigrate. The results of the study revealed no change in maternal body weight of treated animals compared to control animals at 0.5% and 0.2%. The mean concentrations of test chemical in the serum collected from treated dams were significantly higher than controls. No significant effects were noted in systemic  and  sex organ weights at necropsy and histopathological examinatiion  between any group. No effects on implantation numbers and circulating harmonal levels such as estradiol, progesterone, testosterone, T4, and TSH were measured. However, significantly higher levels of test chemical were detected in amniotic fluid from the test chemical-treated dams group compared to control dams. From the observations and results the NOAEL for the test chemical was considered to be 500mg/kg/day when pregnant Sprague Dawley rats exposed to test chemical from GD5 to GD19.

Reproductive Toxicity Study 5:

In the present study the Sprague Dawley rats were administered with test chemical to critical growth period of gestation and lactation to determine the susceptible windows of exposure for reproductive and developmental toxicity. On GD 5,  Dams were fed either rat chow or chow supplemented with 0.5% w/w (500mg/kg) test chemical from GD 5 until weaning at postnatal day (PND) 21. On the day of delivery (PND 0), total neonate number was recorded and the survival of pups was monitored daily during the study period. Dams were terminated either on PND 21 or on the day when remaining pups died and mammary tissue was removed for histological analysis.The results of the study revealed, no statistical food intake difference was noted in treated  and control dams on GD19.Treated group at 0.5% w/w did not affect the ability of dams to carry offspring to term. Mammary glands collected from 0.5% w/w dams had evidence of involution showing increased lobule separation by interstitial mature fat, thinning epithelial height, and increased epithelial vacuolation with fat. At birth, no statistical difference in number of live births or average birth weight per litter between group (0.5%) was noted. Neonates born to and nursed by 0.5% w/w test chemical-treated dams could not survive beyond PND 8 as comapared to control. Milk bands were observed in pups raised by dams treated with 0.5% w/w test chemical and histology of mammary tissue collected from both control and 0.5% w/w test chemical supplemented dams revealed evidence of retained secretory material in the tissues. From the observations and results the LOAEL for the test chemical was considered to be 0.5%w/w (500mg/kg) when pregnant female sprague Dawley rats administered with test chemical in gestationa and lactation period.

Reproductive Toxicity Study 6:

The aim of the study was to evaluate potential histopathologic changes resulting from exposure to the test chemical in male albino rat. In the present study, Male albino rats were  treated with oral doses  of corn oil suspension containing test chemical at dose levels of either 2.5g/kg (2500mg/kg) or 5.0g/kg (5000mg/kg). Group of animals received 2500mg/kg daily for either for 30days (T-I-A), for 60 days(T-I-B) for 30days followed by 30days recovery (T-I-D). Two additional groups of animals were received 5000mg/kg daily for 30days (T-II-A) or for 30days followed by 30days recovery period (T-II-B). Concurrent groups of animals (C-A untreated and C-B and C-C trewated with corn oil)  were employed for comparative purposes. All animals were sacrifised following the end of respective treatment schedule and histologic evaluation of the brain, tested, prostate, seminal vesicles and pituitary gland was conducted. Haematological, urologic and clinical; chemistry parameters were studied during the experiment and growth, food consumption and behavioral reactions were monitered. The result of the study revealed, no deaths were attributed directly due to test chemical administartion in rats. Animals treated with 2500mg/kg/day showed slightly reduced weight gains during the exposure period, During 30days of recovery period (Test Days 30 to 60)  animals in this group gained more weight than corresponding  control animals (68 g compared to 62 g), Animals allowed 60 days of recovery  (T-I-D) showed total weight gains comparable to those of the controls. although a slight weight  depression 3% was observed at the end of the 30-day exposure period. Animals treated with 5000mg/kg/day showed reduced weight gains during the exposure period (approximately 10%reduction in total body weight. 30 to 55% reduction in weight gain). During the recovery period, these animals gained 65% as compared to 62 g by the concurrent control group. Food consumption was reduced in all animals receiving oral doses of either test chemical in corn oil or corn oil  alone. The haematological findings showed some statistical differences in the control and test chemical treated groups, however it was concluded that test chemical treatment did not affect the red blood cells count haemoglobin concentration, white blood cell counts and packed cell volume. Clinical chemistry parameters revealed statistically significant differences in BUN (Blood Urea Nitrogen) and SAP (Serum Alkaline Phosphatase Activity) values were observed but they were not considered to be treatment related.  Lower blood glucose level conncentrations in T-I-B(60days treatment with 2500mg/kg) and T-I-B (30days at 5000mg/kg) groups may be related to lower food consumption levels. SGPT (Serum Glutamic-pyruvic transaminase) activity was not affected by treatment. No significant differences in urinalysis parameters among any groups were observed. In the postmortem study, liver weights (and liver to body weight ratios) of test chemical treated animals of both 30 and 60 days treatment with 2500mg/kg and and 30 days treatement with 5000mg/kg were elevated. No signnificant difference were observed in the kidneys, heart and brain when treated animlas compared to control animals. Testicular weights were lower in animlas treated with 5000mg/kg, this effects were not observed in the 2500mg/kg animlas. Elevation in seminal vesical weight was not observed following 30 days of testing.  Statistical significant differences were observed at 60 days in 2 groups both groups allowed 30 days od recovery following 25000=mg/kg and 5000mg/kg of test chemical.   At  each sacrifice in gross pathological examination, changes were noted in alll treatment group animals, including yellow nodules in the epididymis and reduced testicular size  (sometimes associated by bluish color suggesting necrosis), these changes were not observed in control animals. Histopathological examination of the treated animals from all groups include, changes in animals treated for 30 days with 2500mg/kg or 5000mg/kg of test chemical for 60 days with2500mg/kg test chemical and in animals allowed 30 or 60 days of recovery.The treatment related lesions involved the testes and epididymis. These changes are characterized by impaired sprematogenesis,  degeneration of the germinal epithelium of the seminiferous tubules. and inflammatory responses of seminiferos tubules and epididymal ducts to cellular-debris and non-viable spernlatozoa. From the observations and results, the LOAEL for the test chemical was considered to be 2500mg/kg/day when test chemical administrered to male albino rat.

Reproductive Toxicity Study 7:

In a reproductive and teratogentoxicity, ranging over two generations, the effect of the test chemical were evaluated in male and female CD rats. The test chemical was given to the different generations of rats by feed in the concentrations 0, 0.05, 0.1, 0.2 or 0.25% per day. In the parental generation, there were significant reductions as compared to controls and the number of animals that conceived when exposed to 0.25% per day of the test chemical. There were also a reduction in the numbers of pups born to those that did conceive. The numbers of pups that survived until weaning and their body weights at weaning were reduced in treated animal. However, a dietary concentrations of 0.2% and less of did not cause these effects. Rats of the F0 generation receiving 0.25% per day of the test chemical, had significantly higher liver/body weight ratios than controls, and this ratio was somewhat elevated in the 0.05% per day group as well. There were no histological lesions were seen in either the young or the old rats of either generation, and no effect on embryo toxicity and teratogenicity was evident in the third pregnancy of the F1b generation. Therefore, based on all the observations and results, it was concluded that the NOAEL for the parental generation was considered to be 200 mg/kg bw/d (0.2% per day) while NOAEL for the F1b generation was considered to be 200 mg/kg bw/d (0.2% per day) when exposed to the test chemical.

Reproductive Toxicity Study 8:

Repeated dose chronic/carcinogenicity study was conducted to determine the toxic nature of the test chemical. The study was performed using male and female CD rats for 2 years. The test chemical was mixed with feed and used at dose level of 0, 25, 75 or 250 mg/Kg/day. During the study period, the animals were observed for mortality, clinical signs, body weight and food consumption changes, ophthalmology, hematology, clinical chemistry, urinalysis, gross pathology and histopathology. During the study, no treatment related mortality was noted. No physical observations were noted wich could be attributed week, to treatment. During 64-86 weeks, there was an overall increase in the number of control and treated males which exhibited varying degrees of emaciation, labored breathing and rales. The mean body weights of 250 mg/Kg/day males and 75 and 250 mg/Kg/day females were lower than the controls during most of the study. However, differences from control never exceeded 9 and 12% in the females and 6% in the males. The mean food consumption displayed normal variability, and was comparable to that of control. No ocular abnormalities attributed to test chemical treatment were noted. Slight decreases were observed in mean hemoglohin, hematocrit and erythrocyte counts in the hiqh-dose males throughout the study. These decreases were associated with an increased mean reticulocyte count at termination. Less marked decreases in mean hemoglohin, hematocrit and erythrocyte counts were noted in the mid-dose males at 6 and l2 months. The mean erythrocyte counts of the low-dose males was also slightlv reduced at 12 months. Mean reticulocyte counts were considered unremarkable in the low- and mid-dose males at termination. Mean total leukocyte counts were slightly elevated in the 25 and 75 mg/Kg/day dose males at 6 months, in the 25, 75 and 250 mg/Kg/day dose males at 12 months and in the high-dose males at termination. In the females, mean hemoglobin values and erythrocyte counts were reduced for the 75 and 250 mg/Kg/day dose groups at 6 months and for the 250 mg/Kg/day group at 12 and 20 months and termination. Mean hematocrit values were slightly reduced in the high-dose females at 6 and 20 months and termination. Mean alkaline phosphatase levels were slightly elevated in the 75 and 250 mg/Kg/day dose males at 12 months and at termination. Mean blood urea nitrogen levels were slightly increased in the high-dose males at all study intervals. Mean total bilirubin values were slightly increased in the high-dose females at termination. Mean absolute testes weights, testes/body weight ratios and testes/brain weight ratios were elevated in the high-dose males at 6 and l2 months, but not at termination. Mean absolute and relative (to body and brain weights) spleen weights were elevated in the 75 and 250 mg/Kg/day dose males at all necropsy intervals. Increased Spleen weights were observed in the 75 and 250 mg/Kg/day dose females at 6, 12 and 20 months and at termination and in the 25 mg/Kg/day dose females at termination. Mean liver weights (absolute, liver/body weight and liver/brain weight ratios) were increased in the mid- and high-dose males at 6 and 12 months and in the 25, 75 and 250 mg/Kg/day dose males at termination. Mean liver weight increases were also observed in the 75 and 250 mg/Kg/day dose females at 6 and 12 months and in all treated females at 20 months and termination. Mean absolute and relative adrenal weights were increased in the high-dose females at 12 months, in the 25, 75 and 250 mg/Kg/day dose females at 20 months and termination and in the high-dose males at termination. Mean absolute and relative heart weights were increased in the 250 mg/Kg/day males at 12 months and at termination. Compound-related pathological changes were seen grossly in the 250 mg/Kg/day males only, and microscopically in the 75 and 250 mg/Kg/day dose males and females. Grossly, flaccidity and decrease in size of the testes were shown by a large number of 250 mg/Kg/day dose males. Microscopically significant findings were seen in the testes/epididymides. liver, kidneys, spleen, bone marrow and mesenteric lymph nodes. The microscopic pathological changes consisted of degeneration of seminiferous tubules; enlargement of epididymal secretory epithelium; decrease or absence of sperm in epididymal ducts; hepatocellular hypertrophy; brown pigment in Kupffer‘s cells; cholangiofibrosis; brown pigment in cytoplasm of proximal convoluted tubules; splenic congestion; hypercellularity and congestion of bone marrow. The most common findings in male rats dying at 17-19 months of study were pulmonary consolidation and edema with pus-like material on the pleural surface grossly, and acute multifocal suppurative bronchopneumonia microscopically. No morphologic evidence of the carcinogenic potential of the test chemical was observed. Based on findings, the no observed adverse effect level (NOAEL) is considered to be 25 mg/Kg/day when male and female rats were exposed to the test chemical for 2 years.

Reproductive Toxicity Study 9:

The aim of the study was to evaluate potential histopathologic changes due to male reproductive organ toxicity resulting from exposure to the test chemical in male albino rat. In the present study, Male albino rats were  treated with oral doses  of corn oil suspension containing test chemical at dose levels of either 2.5g/kg (2500mg/kg) or 5.0g/kg (5000mg/kg). Group of animals received 2500mg/kg daily for either for 30days (T-I-A), for 60 days(T-I-B) for 30days followed by 30days recovery (T-I-D). Two additional groups of animals were received 5000mg/kg daily for 30days (T-II-A) or for 30days followed by 30days recovery period (T-II-B). Concurrent groups of animals (C-A untreated and C-B and C-C trewated with corn oil)  were employed for comparative purposes. All animals were sacrifised following the end of respective treatment schedule and histologic evaluation of the brain, tested, prostate, seminal vesicles and pituitary gland was conducted. Haematological, urologic and clinical; chemistry parameters were studied during the experiment and growth, food consumption and behavioral reactions were monitered. The result of the study revealed, no deaths were attributed directly due to test chemical administartion in rats. Animals treated with 2500mg/kg/day showed slightly reduced weight gains during the exposure period, During 30days of recovery period (Test Days 30 to 60)  animals in this group gained more weight than corresponding  control animals (68 g compared to 62 g), Animals allowed 60 days of recovery  (T-I-D) showed total weight gains comparable to those of the controls. although a slight weight  depression 3% was observed at the end of the 30-day exposure period. Animals treated with 5000mg/kg/day showed reduced weight gains during the exposure period (approximately 10%reduction in total body weight. 30 to 55% reduction in weight gain). During the recovery period, these animals gained 65% as compared to 62 g by the concurrent control group. Food consumption was reduced in all animals receiving oral doses of either test chemical in corn oil or corn oil  alone. The haematological findings showed some statistical differences in the control and test chemical treated groups, however it was concluded that test chemical treatment did not affect the red blood cells count haemoglobin concentration, white blood cell counts and packed cell volume. Clinical chemistry parameters revealed statistically significant differences in BUN (Blood Urea Nitrogen) and SAP (Serum Alkaline Phosphatase Activity) values were observed but they were not considered to be treatment related.  Lower blood glucose level conncentrations in T-I-B(60days treatment with 2500mg/kg) and T-I-B (30days at 5000mg/kg) groups may be related to lower food consumption levels. SGPT (Serum Glutamic-pyruvic transaminase) activity was not affected by treatment. No significant differences in urinalysis parameters among any groups were observed. In the postmortem study, liver weights (and liver to body weight ratios) of test chemical treated animals of both 30 and 60 days treatment with 2500mg/kg and and 30 days treatement with 5000mg/kg were elevated. No signnificant difference were observed in the kidneys, heart and brain when treated animlas compared to control animals. Testicular weights were lower in animlas treated with 5000mg/kg, this effects were not observed in the 2500mg/kg animlas. Elevation in seminal vesical weight was not observed following 30 days of testing.  Statistical significant differences were observed at 60 days in 2 groups both groups allowed 30 days od recovery following 25000=mg/kg and 5000mg/kg of test chemical.   At  each sacrifice in gross pathological examination, changes were noted in alll treatment group animals, including yellow nodules in the epididymis and reduced testicular size  (sometimes associated by bluish color suggesting necrosis), these changes were not observed in control animals. Histopathological examination of the treated animals from all groups include, changes in animals treated for 30 days with 2500mg/kg or 5000mg/kg of test chemical for 60 days with2500mg/kg test chemical and in animals allowed 30 or 60 days of recovery.The treatment related lesions involved the testes and epididymis. These changes are characterized by impaired sprematogenesis,  degeneration of the germinal epithelium of the seminiferous tubules. and inflammatory responses of seminiferos tubules and epididymal ducts to cellular-debris and non-viable spermatozoa. From the observations and results, the LOAEL for the test chemical was considered to be 2500mg/kg/day when test chemical administrered to male albino rat.

Effects on developmental toxicity

Description of key information

The developmental toxicity study:

The objective of the present study was to identify the susceptive windows of gestational and postnatal test chemical exposure to offspring survival. In the present study, Dams were then fed with rat chow or chow supplemented with either 0.2% (200mg/kg) or 0.5%(500mg/kg) w/w test chemical. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD). Litter size was culled to 6 females by random removal of pups on PND 0 right after sexing. After culling, on PND 0, a cross-fostering design was implemented within each litter. Briefly, each dam carried and nursed 2 female pups from their own original litter and fostered 2 female pups from each of the 2 other treatment groups. In this manner, each control dam raised 2 of their own pups, 2 pups born to 0.2% w/w-treated dams and 2 pups born to 0.5% w/w-treated dams. Each 0.2% w/w-treated dam raised 2 of their own pups, 2 pups born to 0.5%w/w-treated dams, and 2 pups born to control dams. Finally, each 0.5% w/w-treated dam raised 2 of their own pups, 2 pups born to control dams, and 2 pups born to 0.2%w/w-treated dams. The treatment regimen continued from GD5 throughout lactation until the dams were sacrificed either on weaning/PND 21 or on the same date when all pups died. The RER(respiration excahnge ratio) in maternal animal were determined. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD) and a cross-fostering design was implemented within each litter. Pup mortality and change in body weight were monitored daily throughout the experiment. The Vaginal opening and estrous cyclicity assessment were performed. RER were determined. Gross pathological examination in 0.5%w/w treated group animals were performed and organ weights were also recorded.

The results of the study revealed, no significant difference in RER in maternal animals at dose level 200 and 500mg/kg/day compared to control animals. At birth, no statistical difference in number of live births or average birth weight per litter between groups was noted. There was no initial statistical body weight difference in female pups born to control dams or pupsborn to either groups of 200 and 500mg/kg of treated dams prior to culling on PND 0. Average body weight was significantly less in pups nursed by test chemical supplemented dams at PND 3 with an average 16% decrease found in pups raised by 0.2% w/w test chemical-treated dams and a 25% decrease observed among pups raised by 0.5% w/w test chemical-treated dams compared to control raised pups. Within each dam treatment group, however, no statistical body weight difference was observed among the pups with different in utero exposure status (ie, born to a 0.5% w/w test-treated dam, 0.2% w/w test-treated dam, or a control dam) at PNDs 3, 6, and 9, respectively.

A significant reduction in pup number over time was observed between pups raised by 0.5% w/w or 0.2% w/w treated dams compared to those raised by controls. No pups raised by 0.5% w/w test chemical-treated dams survived beyond PND 5 compared to all pups raised by control dams survived throughout the study period. The abdomens of all pups exposed to test chemical were distended and all pups had diarrhea. Gross pathological examination of randomly selected pups (n =3) raised by the 0.5% w/w dams on PNDs 4 and 5 showed small acute gastric ulcers and fatty vacuolation of hepatocytes. No statistical difference in AGD indexed by cube root of body weight (at the time AGD was acquired) was detected on PND 3 among offspring raised by different dam treatment groups. The average RER measured on PND 41 from the 4 surviving offspring raised by 0.2% w/w dams was similar compared to the RER measured from offspring raised by control dams. The average age of VO in the 4 surviving offspring raised by 0.2% w/w test chemical-treated dams was 38.5 days, while the average age of VO from offspring raised by the control dams was 37.17 days. No significant difference were seen in vaginal opening.  No significant difference in organ weight noted between any groups (200 and 500mg/kg) for any organ analyzed. No significant differece were observed in first date of estrus in test chemical treated groups and control group. From the observations and results, the LOAEL for the maternal animals and  NOAEL for the fetuses considered to be500mg/kg and 200mg/kg respectively when test chemical exposed to Sprague Dawley rats during gestational and lactation period

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data is from publication
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
a cross-fostering design was implemented
Principles of method if other than guideline:
The objective of the present study was to identify the susceptive windows of gestational and postnatal test chemical exposure to offspring survival
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Source: Harlan Laboratory, Dublin, Virginia
- Age at study initiation: (P) x wks; (F1) x wks (Animals were pregnant)
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: housed individually with Harlan Teklad laboratory grade 7087 soft cob bedding (Harlan Laboratories, Madison, Wisconsin) in clear plastic cages

- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum): Ad libitum assess to water
- Acclimation period: Harlan ground 2020X chow

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20_C to 22 0C
- Humidity (%):of 40% to 50%.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12:12-hour photoperiod
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: feed
Remarks on MMAD:
No data available
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test chemical supplement was prepared by first weighing the correct amount of test chemical and mixing the compound with small amounts of powdered chow using a mortar and pestle.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered chow to obtain the required concentration.
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Details on mating procedure:
No mating were performed
Duration of treatment / exposure:
from GD 5 to on weaning/PND 21
Frequency of treatment:
Daily
Duration of test:
No data available
Remarks:
Control=0%(0mg/kg)
0.2%=(200mg/kg)
0.5%=(500mg/kg)
No. of animals per sex per dose:
Control=5 females
0.2% (200mg/kg)= 5females
0.5% (500mg/kg)= 5females
Control animals:
yes, plain diet
Details on study design:
Dams were then fed with rat chow or chow supplemented with either 0.2% (200mg/kg) or 0.5%(500mg/kg) w/w test chemical. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD). Litter size was culled to 6 females by random removal of pups on PND 0 right after sexing. After culling, on PND 0, a cross-fostering design was implemented within each litter. Briefly, each dam carried and nursed 2 female pups from their own original litter and fostered 2 female pups from each of the 2 other treatment groups. In this manner, each control dam raised 2 of their own pups, 2 pups born to 0.2% w/w-treated dams and 2 pups born to 0.5% w/w-treated dams. Each 0.2% w/w-treated dam raised 2 of their own pups, 2 pups born to 0.5%w/w-treated dams, and 2 pups born
to control dams. Finally, each 0.5% w/w-treated dam raised 2 of their own pups, 2 pups born to control dams, and 2 pups born to 0.2%w/w-treated dams. The treatment regimen continued from GD5 throughout lactation until the dams were sacrificed either on weaning/PND 21 or on the same date when all pups died.
Maternal examinations:
Body weight and average RER were measured.
Ovaries and uterine content:
No data available
Fetal examinations:
Body weight difference in female pups born before and after the crossover manipulation were recorded on PND 0,3,6,9 and 21.
Pup mortality was determined throughout the study. The average RER measured on PND 41 in offspring. Vaginal opening and estrous cyclicity assessed with AGD measurement.
Statistics:
Data were presented as group mean + standard error of the mean. Data were analyzed using SPSS (version 20, IBM, Armonk, New York) by analysis of variance (ANOVA; ie, organ weights, body weight, AGD, test chemical) or ANOVA with repeat measurements (ie, changes of AGD and body weight over time). In addition, data were analyzed with a covariate of PND 21 body weight (offspring) or pretreatment body weight (dams) when appropriate. Mortality measurements were analyzed by Kaplan-Meier survival analysis with JMP Pro 10 (SAS Institute Inc, Cary, North Carolina), followed by pairwise Student-Newman- Keuls post hoc test when appropriate. Statistical significance was considered P <.05. Data were transformed if either normality or the equal variance assumption was invalid. If transformation did not correct normality or equal variance assumption, Kruskal-Wallis one-way ANOVA on ranks was used.
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:
not specified
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
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:
no effects observed
Description (incidence and severity):
There was no significant difference in RER (Respiration excahnge ratio) in maternal animals at dose level 200 and 500mg/kg/day compared to control animals
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
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
At birth, no statistical difference in number of live births or average birth weight per litter between groups was noted
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: RER and number of live births or average birth weight per litter
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no initial statistical body weight difference in female pups born to control dams or pupsborn to either groups of 200 and 500mg/kg of treated dams prior to culling on PND 0. Average body weight was significantly less in pups nursed by test chemical supplemented dams at PND 3 with an average 16% decrease found in pups raised by 0.2% w/w test chemical-treated dams and a 25% decrease observed among pups raised by 0.5% w/w test chemical-treated dams compared to control raised pups.

Within each dam treatment group, however, no statistical body weight difference was observed among the pups with different in utero exposure status (ie, born to a 0.5% w/w test-treated dam, 0.2% w/w test-treated dam, or a control dam) at PNDs 3, 6, and 9, respectively.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
A significant reduction in pup number over time was observed between pups raised by 0.5% w/w or 0.2% w/w treated dams compared to those raised by controls.
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
No pups raised by 0.5% w/w test chemical-treated dams survived beyond PND 5 compared to all pups raised by control dams survived throughout the study period
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Description (incidence and severity):
The abdomens of all pups exposed to test chemical were distended and all pups had diarrhea. Gross pathological examination of randomly selected pups (n =3) raised by the 0.5% w/w dams on PNDs 4 and 5 showed small acute gastric ulcers and fatty vacuolation of hepatocytes.

No statistical difference in AGD indexed by cube root of body weight (at the time AGD was acquired) was detected on PND 3 among offspring raised by different dam treatment groups.

The average RER measured on PND 41 from the 4 surviving offspring raised by 0.2% w/w dams was similar compared to the RER measured from offspring raised by control dams

Vaginal opening(VO): The average age of VO in the 4 surviving offspring raised by 0.2% w/w test chemical-treated dams was 38.5 days, while the aver
age age of VO from offspring raised by the control dams was 37.17 days. No significant difference were seen

Organ weight: no significant difference noted between any groups (200 and 500mg/kg) for any organ analyzed.

Estrus cycle assessment: No significant differece were observed in first date of estrus in test chemical treated groups and control group.
Details on embryotoxic / teratogenic effects:
No data available
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in postnatal survival
other: The abdomens of all pups were distended and all pups had diarrhea, small acute gastric ulcers and fatty vacuolation of hepatocytes.
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Developmental effects observed:
not specified
Treatment related:
not specified
Conclusions:
From the observations and results, the LOAEL for the maternal animals and NOAEL for the fetuses considered to be 500mg/kg and 200mg/kg respectively when test chemical exposed to Sprague Dawley rats during gestational and lactation period
Executive summary:

The objective of the present study was to identify the susceptive windows of gestational and postnatal test chemical exposure to offspring survival. In the present study, Dams were then fed with rat chow or chow supplemented with either 0.2% (200mg/kg) or 0.5%(500mg/kg) w/w test chemical. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD). Litter size was culled to 6 females by random removal of pups on PND 0 right after sexing. After culling, on PND 0, a cross-fostering design was implemented within each litter. Briefly, each dam carried and nursed 2 female pups from their own original litter and fostered 2 female pups from each of the 2 other treatment groups. In this manner, each control dam raised 2 of their own pups, 2 pups born to 0.2% w/w-treated dams and 2 pups born to 0.5% w/w-treated dams. Each 0.2% w/w-treated dam raised 2 of their own pups, 2 pups born to 0.5%w/w-treated dams, and 2 pups born to control dams. Finally, each 0.5% w/w-treated dam raised 2 of their own pups, 2 pups born to control dams, and 2 pups born to 0.2%w/w-treated dams. The treatment regimen continued from GD5 throughout lactation until the dams were sacrificed either on weaning/PND 21 or on the same date when all pups died. The RER(respiration excahnge ratio) in maternal animal were determined. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD) and a cross-fostering design was implemented within each litter. Pup mortality and change in body weight were monitored daily throughout the experiment. The Vaginal opening and estrous cyclicity assessment were performed. RER were determined. Gross pathological examination in 0.5%w/w treated group animals were performed and organ weights were also recorded.

The results of the study revealed, no significant difference in RER in maternal animals at dose level 200 and 500mg/kg/day compared to control animals. At birth, no statistical difference in number of live births or average birth weight per litter between groups was noted. There was no initial statistical body weight difference in female pups born to control dams or pupsborn to either groups of 200 and 500mg/kg of treated dams prior to culling on PND 0. Average body weight was significantly less in pups nursed by test chemical supplemented dams at PND 3 with an average 16% decrease found in pups raised by 0.2% w/w test chemical-treated dams and a 25% decrease observed among pups raised by 0.5% w/w test chemical-treated dams compared to control raised pups. Within each dam treatment group, however, no statistical body weight difference was observed among the pups with different in utero exposure status (ie, born to a 0.5% w/w test-treated dam, 0.2% w/w test-treated dam, or a control dam) at PNDs 3, 6, and 9, respectively.

A significant reduction in pup number over time was observed between pups raised by 0.5% w/w or 0.2% w/w treated dams compared to those raised by controls. No pups raised by 0.5% w/w test chemical-treated dams survived beyond PND 5 compared to all pups raised by control dams survived throughout the study period. The abdomens of all pups exposed to test chemical were distended and all pups had diarrhea. Gross pathological examination of randomly selected pups (n =3) raised by the 0.5% w/w dams on PNDs 4 and 5 showed small acute gastric ulcers and fatty vacuolation of hepatocytes. No statistical difference in AGD indexed by cube root of body weight (at the time AGD was acquired) was detected on PND 3 among offspring raised by different dam treatment groups. The average RER measured on PND 41 from the 4 surviving offspring raised by 0.2% w/w dams was similar compared to the RER measured from offspring raised by control dams. The average age of VO in the 4 surviving offspring raised by 0.2% w/w test chemical-treated dams was 38.5 days, while the average age of VO from offspring raised by the control dams was 37.17 days. No significant difference were seen in vaginal opening.  No significant difference in organ weight noted between any groups (200 and 500mg/kg) for any organ analyzed. No significant differece were observed in first date of estrus in test chemical treated groups and control group. From the observations and results, the NOAEL for the maternal animals and  LOAEL for the fetuses considered to be 500mg/kg and 200mg/kg respectively when test chemical exposed to Sprague Dawley rats during gestational and lactation period.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is Klimisch 2 level and provides a robust summary.
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

Developmental toxicity study (Study 1):

The objective of the present study was to identify the susceptive windows of gestational and postnatal test chemical exposure to offspring survival. In the present study, Dams were then fed with rat chow or chow supplemented with either 0.2% (200mg/kg) or 0.5%(500mg/kg) w/w test chemical. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD). Litter size was culled to 6 females by random removal of pups on PND 0 right after sexing. After culling, on PND 0, a cross-fostering design was implemented within each litter. Briefly, each dam carried and nursed 2 female pups from their own original litter and fostered 2 female pups from each of the 2 other treatment groups. In this manner, each control dam raised 2 of their own pups, 2 pups born to 0.2% w/w-treated dams and 2 pups born to 0.5% w/w-treated dams. Each 0.2% w/w-treated dam raised 2 of their own pups, 2 pups born to 0.5%w/w-treated dams, and 2 pups born to control dams. Finally, each 0.5% w/w-treated dam raised 2 of their own pups, 2 pups born to control dams, and 2 pups born to 0.2%w/w-treated dams. The treatment regimen continued from GD5 throughout lactation until the dams were sacrificed either on weaning/PND 21 or on the same date when all pups died. The RER(respiration excahnge ratio) in maternal animal were determined. On PND 0, female pups were weighed and sexed based on anogenital distance (AGD) and a cross-fostering design was implemented within each litter. Pup mortality and change in body weight were monitored daily throughout the experiment. The Vaginal opening and estrous cyclicity assessment were performed. RER were determined. Gross pathological examination in 0.5%w/w treated group animals were performed and organ weights were also recorded. The results of the study revealed, no significant difference in RER in maternal animals at dose level 200 and 500mg/kg/day compared to control animals. At birth, no statistical difference in number of live births or average birth weight per litter between groups was noted. There was no initial statistical body weight difference in female pups born to control dams or pupsborn to either groups of 200 and 500mg/kg of treated dams prior to culling on PND 0. Average body weight was significantly less in pups nursed by test chemical supplemented dams at PND 3 with an average 16% decrease found in pups raised by 0.2% w/w test chemical-treated dams and a 25% decrease observed among pups raised by 0.5% w/w test chemical-treated dams compared to control raised pups. Within each dam treatment group, however, no statistical body weight difference was observed among the pups with different in utero exposure status (ie, born to a 0.5% w/w test-treated dam, 0.2% w/w test-treated dam, or a control dam) at PNDs 3, 6, and 9, respectively. A significant reduction in pup number over time was observed between pups raised by 0.5% w/w or 0.2% w/w treated dams compared to those raised by controls. No pups raised by 0.5% w/w test chemical-treated dams survived beyond PND 5 compared to all pups raised by control dams survived throughout the study period. The abdomens of all pups exposed to test chemical were distended and all pups had diarrhea. Gross pathological examination of randomly selected pups (n =3) raised by the 0.5% w/w dams on PNDs 4 and 5 showed small acute gastric ulcers and fatty vacuolation of hepatocytes. No statistical difference in AGD indexed by cube root of body weight (at the time AGD was acquired) was detected on PND 3 among offspring raised by different dam treatment groups. The average RER measured on PND 41 from the 4 surviving offspring raised by 0.2% w/w dams was similar compared to the RER measured from offspring raised by control dams. The average age of VO in the 4 surviving offspring raised by 0.2% w/w test chemical-treated dams was 38.5 days, while the average age of VO from offspring raised by the control dams was 37.17 days. No significant difference were seen in vaginal opening.  No significant difference in organ weight noted between any groups (200 and 500mg/kg) for any organ analyzed. No significant differece were observed in first date of estrus in test chemical treated groups and control group. From the observations and results, the NOAEL for the maternal animals and  LOAEL for the fetuses considered to be500mg/kg and 200mg/kg respectively when test chemical exposed to Sprague Dawley rats during gestational and lactation period

Study 2:

Three successive generations of Sprague-Dawley rats were administered test chemical to assessed the developmental toxicity associated with test chemical exposure during developmental phase. Dose levels of 250, 500, 1000 and 3000 ppm (12.5, 25, 50 and 150mg/kg/day) were choosed for the study. In the present study, In F0 generation dosing began 60 days prior to mating, then continuously thereafter. In F1 and F2 generations dosing for 80-day growth period before mating, then continuously thereafter  The first litters were raised to weaning, the second litter was used to continue the study. Body weights and food consumption were measured weekly during the study. Observations for mortality and adverse effects were done twice daily. Detailed phy sical exams were done weekly on all generations. All animals dying spontaneously or killed in a moribund condition were examined and tissues preserved in 10% formalin. Dead or stillborn pups were given a gross postmortem exam and preserved in 70% ethanol. All adult males and females were given a gross postmortem exam and tissues preserved. At weaning (day 21), pups not chosen as future parents were sacrificed and examined and only grossly abnormal tissues preserved. Data were analyzed between control and treated groups.  The results of the study revealed, No change in maternal clinical signs of toxicity, mortality, food consumption and body weight change. Litter viability were similar to control animals and survival rate was similar to control animals. The examination of fetuses revealed no changes with all fetal parameteres except One dead female F1b pup had clubbed legs and a filamentous tail (250 ppm (12.5mg/kg); one dead female F1b pup had a spina bifida malformation (1000 ppm (50mg/kg)); in the F2b litter, one dead pup was edematous (250 ppm (12.5mg/kg)) and one had a kinked tail (250 ppm (12.5mg/kg)); no malformations seen in the F3 litters.

Therefore, the NOAEL for both the maternal and fetal population was considered to be 150mg/kg/day.

Study 3:

In the present study aim to investigated whether exposure to an environmentally relevant concentration of test chemical results in transfer from mother to offspring in CD-1 mice during gestation and lactation. Females were housed with males (4 females, 1 male per cage) and monitored daily for vaginal plugs a nd considered as gestation day 0. For in utero exposure, dams were given 14C-test chemical drinking water starting on the day of plug visualization through gestation day 18 (GD18). At GD18, dams were euthanized by CO2 asphyxiation. Fetuses were removed and separated from the placenta and studied. For the long-term lactation study, dams were dosed with 14C-test chemical drinking water similar to the lactation exposure, starting on the day of litter delivery to PND10. An untreated control group was run in parallel for comparison, At PND21, offspring were separated into males and females and sex specific body weights were measured through PND56. Dams were euthanized on the day of offspring weaning (PND21).subset of offspring (n = 20 for test chemical, n = 20 control) was sacrificed at PND42 for bio-distribution analysis using AMS; these offspring were randomly chosen from all litters in both groups.  At PND56, the remaining offspring were euthanized after weighing. Tissues (liver, spleen, kidney, lungs, heart, brain, inguinal fat, gonadal/ovarian fat, retroperitoneal fat, uterine fat, uterus, muscle, gastrointestinal tract, colon, adrenals, thymus, ovaries and testes) were isolated and studied. The result of the study revealed that higher concentration of test chemical were observed in maternal placenta tissue. The fetuses examination reealed, no significance differences  in fetus weight were observed for the GD18 in utero tets chemical exposed group compared to control but for PND21-56, test chemical exposed offspring had a statistically significant increase in weight compared to the control group. Significant differences in litter sizes for the lactation exposure groups were observed. Visceral malformation of offspings results,all test chemical exposed groups was reduction in brain size. Uterine weight was reduced in test chemical female offspring by ~14%. Additionally, a 13.56% increase in thymus weight was noted for female offspring. A 3-fold higher concentration of test chemical was detected in offspring homogenates at PND10 after exposure solely through lactation compared to the in utero exposure demonstrating that test chemical transfers readily through breast milk. From the observations and result, the LOAEL for the test chemical was considered to be 100nM (0.03155861mg/kg/day) when test chemical exposed  to CD-1 mice during gestation and  lactation period.  

Study 4:

An experiment was performed wherein the effect of the test chemical was observed on the developmental parameters and on the reproductive parameters of the maternal animals. In this study, the test chemical was dosed to the CD rats of 21 -23 days old obtained from Charles River laboratories in the concentration of 0.05% and 0.25%. The animals were dosed with the test chemical via oral route through feeding. These animals were housed in an individual stainless-steel cages, fed with the basal diet; of ground Purina Chow feed for 3 days. The animals were divided in 5 groups namely A, B, C, D and E, respectively. Group A, the control group, received only ground Purina Chow throughout the experiment. Groups B and C also received this basal diet except during Days 6-15 of pregnancy, when the females received chow containing 0.05 % (Group B) or 0.25 % (Group C) of the 2 : 1 mixture of the test chemical. Groups D and E continuously received chow containing 0.05 and 0.25 %, respectively, of the test chemical. Rats diets were prepared weekly and dosing solutions were prepared daily. During mating,20 pairs were housed together as pairs in breeding cages (1:1). Cytological examination of the vaginal smears was used to determine the estrous cycle of each female and the day of conception, which was counted as Day 0 of gestation. The rats were then distributed among the groups so that the groups mean weights were similar and so that littermates were not concentrated in any group.The maternal animals were sacrificed after impregnation for the third time. The maternal animals were mated twice before they were sacrificed by ether inhalation Day 13, and the remainder on Day 21. Implantations, and resorptions were recorded for all the dams. For those sacrificed at Day 21, the numbers of live and dead fetuses and their positions in the uterine horns were recorded. In fetal parameters, the numbers of live and dead fetuses and their positions in the uterine horns were recorded. The fetuses were removed, dried of amniotic fluid, weighed, sexed, and examined for gross anomalies. One-third of the fetuses in each litter were cleared with KOH and stained with alizarin red S for skeletal examination; the other two-thirds were fixed in Bouin’s solution, razor blade sectioned, and examined for soft tissue anomalies. The continuous (measurement) data was treated by analysis of variance and partitioned by the Tukey minimum significant difference test. The discrete (enumeration) data were analyzed by theχ2method. In maternal parameters, females did not show significant differences in weight gain. After the growth study, and up until the final sacrifice, both male and female rats receiving 0.25 % test chemical weighed significantly less than controls; the differences generally amounted to 10-15 % of body weight. In the second-generation (F1b) rats there were no significant differences in body weights, but the animals receiving 0.20% test chmical (about 135 mg/kg/day) ate less feed than controls and had higher feed efficiencies, again suggesting that the diet was unpalatable. Also, the first generation rats did not significantly differ in the pattern of the food consumption, Females did not show significant differences in feed consumption. No effects on organ weights were observed in the animals dosed from the gestation day 6 to 15. No gross pathological and histological anomalies were observed in either groups and in either generations. However, significant increase in the number of abortions were obsrved in the high dose group of the test chemical. Also, significant increase in the number of implantations were obsrved in the high dose group of the test chemical. High number of fetuses were found dead in the high dose group (500 mg/kg bw) and the number of pregnant rats were found to be significantly decreased in the high dose group (500 mg/kg bw). In fetal parameters, no significant changes in the fetal body weights were observed in either of the dose groups. The number of the live pups decreased in the high dose group (500 mg/kg bw) treated dams. No effects were observed in skeletal, visceral and external malformations of pups in either dose groups. Thus, Based on all the above observation and results, it was concluded that the NOAEL of the test chemical for maternal and fetal parameters was found to be 100 mg/kg bw.

Study 5:

An experiment was performed in New Zealand doe's (rabbits) to examine and evaluate the toxicity to the maternal animals and to observe the developmental toxicity in the fetuses, after dosingthe maternal animals with the test chemical during gestation day 7 through 18. In this study, the test chemical was orally administered to the rabbits through gavage at doses of 50, 100 and 250 mg/kg bw. The test chemical was mixed in the corn oil before dosing. After being randomly distributed, the rabbits were artificially inseminated with 0.25 ml of semen that had been collected from proven bucks in an artificial vagina. Immediately before insemination, each doe was given 1 mg/kg pituitary luteinizing hormone to induce ovulation. Two does from each group were inseminated each day for 10 days. The day of insemination was counted as Day 0. After successful mating each pregnant female was caged individually. The animals were randomly distributed in 4 groups of 20 animals. Maternal animals were observed for clinical signs, mortality or morbidity, body weight and gross pathological and histopathological changes. The animals were sacrificed on day 29, by overdosing with sodium thiopental and their fetuses were removed by Caesarean section. The numbers of live and dead fetuses, resorptions, implantations, and corpora lutea of pregnancy were.The number of implantations, and corpora lutea of pregnancy were recorded, as well as the weights of the fetuses and the numbers and descriptions of malformed fetuses. In fetal examinations, the fetuses were observedfor any skeletal, visceral or external malformations. Also, the viability and the postnatal survival was also examined in fetal parameters. The continuous (measurement) data was treated by analysis of variance and partitioned by the Tukey minimum significant difference test. The discrete (enumeration) data were analyzed by the χ2 method. The administration of the test chemical caused maternal toxicity and morbidity. The administration of the test chemical caused moratality in the maternal animals. Fourteen of the dams died; 13 deaths (1 at 50 mg/kg, 5 at 100 mg/kg, and 7 at 250 mg/ kg) were attributed to the test chemical; while one death was accidental. Rabbits given each of the doses lost significant amounts of body weight. No test chemical related gross pathological and histopathological changes were observed in does. In ovarian and uterine examination, the incidence of abortions increased with increasing dosage, apparently being related to maternal toxicity. Although the overall conception was 85 %; there were many abortions in dams administered with the test chemical. When data from the dams that aborted were excluded, there were no significant differences in implantation losses, number of corpora lutea and resorptions amongst the animals. No dose related effects on skeletal, visceral and external anomalies were observed in any animals. Thus, based on all the observations and results, it was concluded that the LOAEL for the maternal animals was found to be 50 mg/kg bw while the NOAEL for the fetuses were found to be 250 mg/kg bw.

Toxicity to reproduction: other studies

Description of key information

Reproductive Toxicity Study (Other Studies):

The  experiment designed to differentiate whether the decreased neonate survival was secondary to the effect of test chemical on the reduction in the lactational capacity of the mammary glands. Pregnant (GD5) dams were weight ranked and randomized by body weight into groups fed either rat chow (n =6) or chow supplemented with 0.5% w/w test chemical (n = 3) from GD 5 until PND 14. After delivery at PND 0, litter size was culled to 6 from all 3 of 0.5% w/w-treated dams and only 2 control dams. No culling was conducted for the rest of the control dams (n = 4) which  served as reserve controls to provide healthy pups Starting on PND 1, healthy age-matched pups (n = 3) born to the 4 reserve control litters were added to replace half (n =3) the pups raised by test c hemical treated dams to maintain normal suckling activity. Therefore, by PND 1, all 0.5% w/w-treated dams carried 6 pups (3 born to 0.5% w/w test chemical-treated dams and 3 born to reserve control dams). On PND 3, the same procedure was conducted as PND 1, except 3 healthy control pups born to the 4 reserve control dams were added to treated dams to replace the pups previously transferred on PND 1 from reserve control dams. Therefore, on PND 3, all 0.5% w/w test chemical-treated dams carried 3 of their own pups and 3 new pups transferred from reserve control dams. At PND 6, the procedure was again conducted except that 3 healthy age-matched pups born to the reserve control dams were added to each treated dam to replace the remaining 3 pups originally born to 0.5% w/wtreated dams. After the above-mentioned manipulation on PND 6, pups nursed by the treated dams were all born to reserve control dams. The same substitution procedure was conducted once again on PND 9, and this time the 3 pups transferred from reserve control dams to treated dams on PND 3 were replaced. Milk band quantification comparison was only conducted between control born/raised animals and 0.5% w/w born/raised animals on PNDs 1, 3, and 6, the last day before all the pups born to test chemical-treated dams were replaced with pups born to reserve control dams.All dams were terminated on PND 14 and mammary tissue was removed for histological analysis. Milk bands were rated as described by Ruppert and colleagues. Briefly, 0 indicates no band visible; 1, small band visible on the side of pup; 2, small band visible across pup’s abdomen, and 3, large band visible across the pup’s abdomen The result o fthe study revealed, milk band size decreased over time after PND 3. On PND 6, the median milk band score was 0 in 0.5% w/w born/raised pups and 2 among pups born/raised by control dams  Compared to results from control dams. Histology evaluation revealed that mammary tissue collected from treated dams on PND 14 was not involuted when additional healthy pups were continuously provided on PNDs 3, 6, and 9 to maintain normal suckling activity. The dose level of the test chemical in pregnant Sprague Dawley rats was considered to be 500mg/kg when test chemical exposed during gestation and lactation period results in decreased milk band size overtime.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data is from publication
Qualifier:
equivalent or similar to
Guideline:
other: 414- prenatal develpmental toxicity studies
Deviations:
yes
Principles of method if other than guideline:
The experiment designed to differentiate whether the decreased neonate survival was secondary to the effect of test chemical on the reduction in the lactational capacity of the mammary glands
GLP compliance:
yes
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
- Source: Harlan Laboratory, Dublin, Virginia
- Age at study initiation: (P) x wks; (F1) x wks (Animals were pregnant)
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: housed individually with Harlan Teklad laboratory grade 7087 soft cob bedding (Harlan Laboratories, Madison, Wisconsin) in clear plastic cages

- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum): Ad libitum assess to water
- Acclimation period: Harlan ground 2020X chow

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20_C to 22_C
- Humidity (%):of 40% to 50%.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12:12-hour photoperiod
Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test chemical supplement was prepared by first weighing the correct amount of test chemical and mixing the compound with small amounts of powdered chow using a mortar and pestle.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): powdered chow to obtain the required concentration.
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
GD5 to PND14
Frequency of treatment:
daily
Remarks:
0.5% =500mg/kg
based on concentration in diet
No. of animals per sex per dose:
Control=6 pregnant female
0.5% (500mg/kg)= 3 preganant female
Control animals:
yes, plain diet
Details on study design:
Pregnant (GD5) dams were weight ranked and randomized by body weight into groups fed either rat chow (n =6) or chow supplemented with 0.5% w/w test chemical (n = 3) from GD 5 until PND 14. After delivery at PND 0, litter size was culled to 6 from all 3 of 0.5% w/w-treated dams and only 2 control dams. No culling was conducted for the rest of the control dams (n = 4) which served as reserve controls to provide healthy pups Starting on PND 1, healthy age-matched pups (n = 3) born to the 4 reserve control litters were added to replace half (n =3) the pups raised by test c hemical treated dams to maintain normal suckling activity. Therefore, by PND 1, all 0.5% w/w-treated dams carried 6 pups (3 born to 0.5% w/w test chemical-treated dams and 3 born to reserve control dams). On PND 3, the same procedure was conducted as PND 1, except 3 healthy control pups born to the 4 reserve control dams were added to treated dams to replace the pups previously transferred on PND 1 from reserve control dams. Therefore, on PND 3, all 0.5% w/w test chemical-treated dams carried 3 of their own pups and 3 new pups transferred from reserve control dams. At PND 6, the procedure was again conducted except that 3 healthy age-matched pups born to the reserve control dams were added to each treated dam to replace the remaining 3 pups originally born to 0.5% w/wtreated dams. After the above-mentioned manipulation on PND 6, pups nursed by the treated dams were all born to reserve control dams. The same substitution procedure was conducted once again on PND 9, and this time the 3 pups transferred from reserve control dams to treated dams on PND 3 were replaced. Milk band quantification comparison was only conducted between control born/raised animals and 0.5% w/w born/raised animals on PNDs 1, 3, and 6, the last day before all the pups born to test chemical-treated dams were replaced with pups born to reserve control dams.All dams were terminated on PND 14 and mammary tissue was removed for histological analysis.

Milk bands were rated as described by Ruppert and colleagues.
Briefly, 0 indicates no band visible; 1, small band visible on the side of pup; 2, small band visible across pup’s abdomen, and 3, large band visible across the pup’s abdomen
Statistics:
Data were presented as group mean + standard error of the mean. Data were analyzed using SPSS (version 20, IBM, Armonk, New York) by analysis of variance or ANOVA with repeat measurements. In addition Milk band rating was analyzed with a nonparametric Mann-Whitney U test. Statistical significance was considered P <.05. Data were transformed if either normality or the equal variance assumption was invalid. If transformation did not correct normality or equal variance assumption, Kruskal-Wallis one-way ANOVA on ranks was used.
Dose descriptor:
dose level:
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overtime decresed milk band size
Remarks on result:
other: Not Specified
The milk band size decreased over time after PND 3. On PND 6, the median milk band score was 0 in 0.5% w/w born/raised pups and 2 among pups born/raised by control dams Compared to results from control dams.

Histology evaluation revealed that mammary tissue collected from treated dams on PND 14 was not involuted when additional healthy pups were continuously provided on PNDs 3, 6, and 9 to maintain normal suckling activity
Conclusions:
The dose level of the test chemical in pregnant Sprague Dawley rats was considered to be 500mg/kg when test chemical exposed during gestation and lactation period results in decreased milk band size overtime
Executive summary:

The  experiment designed to differentiate whether the decreased neonate survival was secondary to the effect of test chemical on the reduction in the lactational capacity of the mammary glands. Pregnant (GD5) dams were weight ranked and randomized by body weight into groups fed either rat chow (n =6) or chow supplemented with 0.5% w/w test chemical (n = 3) from GD 5 until PND 14. After delivery at PND 0, litter size was culled to 6 from all 3 of 0.5% w/w-treated dams and only 2 control dams. No culling was conducted for the rest of the control dams (n = 4) which  served as reserve controls to provide healthy pups Starting on PND 1, healthy age-matched pups (n = 3) born to the 4 reserve control litters were added to replace half (n =3) the pups raised by test c hemical treated dams to maintain normal suckling activity. Therefore, by PND 1, all 0.5% w/w-treated dams carried 6 pups (3 born to 0.5% w/w test chemical-treated dams and 3 born to reserve control dams). On PND 3, the same procedure was conducted as PND 1, except 3 healthy control pups born to the 4 reserve control dams were added to treated dams to replace the pups previously transferred on PND 1 from reserve control dams. Therefore, on PND 3, all 0.5% w/w test chemical-treated dams carried 3 of their own pups and 3 new pups transferred from reserve control dams. At PND 6, the procedure was again conducted except that 3 healthy age-matched pups born to the reserve control dams were added to each treated dam to replace the remaining 3 pups originally born to 0.5% w/wtreated dams. After the above-mentioned manipulation on PND 6, pups nursed by the treated dams were all born to reserve control dams. The same substitution procedure was conducted once again on PND 9, and this time the 3 pups transferred from reserve control dams to treated dams on PND 3 were replaced. Milk band quantification comparison was only conducted between control born/raised animals and 0.5% w/w born/raised animals on PNDs 1, 3, and 6, the last day before all the pups born to test chemical-treated dams were replaced with pups born to reserve control dams.All dams were terminated on PND 14 and mammary tissue was removed for histological analysis. Milk bands were rated as described by Ruppert and colleagues. Briefly, 0 indicates no band visible; 1, small band visible on the side of pup; 2, small band visible across pup’s abdomen, and 3, large band visible across the pup’s abdomen The result o fthe study revealed, milk band size decreased over time after PND 3. On PND 6, the median milk band score was 0 in 0.5% w/w born/raised pups and 2 among pups born/raised by control dams  Compared to results from control dams. Histology evaluation revealed that mammary tissue collected from treated dams on PND 14 was not involuted when additional healthy pups were continuously provided on PNDs 3, 6, and 9 to maintain normal suckling activity. The dose level of the test chemical in pregnant Sprague Dawley rats was considered to be 500mg/kg when test chemical exposed during gestation and lactation period results in decreased milk band size overtime.

Additional information

Reproductive Toxicity Study (Other Studies):

The  experiment designed to differentiate whether the decreased neonate survival was secondary to the effect of test chemical on the reduction in the lactational capacity of the mammary glands. Pregnant (GD5) dams were weight ranked and randomized by body weight into groups fed either rat chow (n =6) or chow supplemented with 0.5% w/w test chemical (n = 3) from GD 5 until PND 14. After delivery at PND 0, litter size was culled to 6 from all 3 of 0.5% w/w-treated dams and only 2 control dams. No culling was conducted for the rest of the control dams (n = 4) which  served as reserve controls to provide healthy pups Starting on PND 1, healthy age-matched pups (n = 3) born to the 4 reserve control litters were added to replace half (n =3) the pups raised by test c hemical treated dams to maintain normal suckling activity. Therefore, by PND 1, all 0.5% w/w-treated dams carried 6 pups (3 born to 0.5% w/w test chemical-treated dams and 3 born to reserve control dams). On PND 3, the same procedure was conducted as PND 1, except 3 healthy control pups born to the 4 reserve control dams were added to treated dams to replace the pups previously transferred on PND 1 from reserve control dams. Therefore, on PND 3, all 0.5% w/w test chemical-treated dams carried 3 of their own pups and 3 new pups transferred from reserve control dams. At PND 6, the procedure was again conducted except that 3 healthy age-matched pups born to the reserve control dams were added to each treated dam to replace the remaining 3 pups originally born to 0.5% w/wtreated dams. After the above-mentioned manipulation on PND 6, pups nursed by the treated dams were all born to reserve control dams. The same substitution procedure was conducted once again on PND 9, and this time the 3 pups transferred from reserve control dams to treated dams on PND 3 were replaced. Milk band quantification comparison was only conducted between control born/raised animals and 0.5% w/w born/raised animals on PNDs 1, 3, and 6, the last day before all the pups born to test chemical-treated dams were replaced with pups born to reserve control dams.All dams were terminated on PND 14 and mammary tissue was removed for histological analysis. Milk bands were rated as described by Ruppert and colleagues. Briefly, 0 indicates no band visible; 1, small band visible on the side of pup; 2, small band visible across pup’s abdomen, and 3, large band visible across the pup’s abdomen The result o fthe study revealed, milk band size decreased over time after PND 3. On PND 6, the median milk band score was 0 in 0.5% w/w born/raised pups and 2 among pups born/raised by control dams  Compared to results from control dams. Histology evaluation revealed that mammary tissue collected from treated dams on PND 14 was not involuted when additional healthy pups were continuously provided on PNDs 3, 6, and 9 to maintain normal suckling activity. The dose level of the test chemical in pregnant Sprague Dawley rats was considered to be 500mg/kg when test chemical exposed during gestation and lactation period results in decreased milk band size overtime.

Justification for classification or non-classification

Based on all the observations and results, the test chemical has found to induce testicular toxicity after exposed to a prolonged period. Also, the test chemical is found to cross the placental barrier in females and may cause fetal abnormalities. Hence the test chemical is likely to classify in 'Category 2' as reproductive and developmental toxicant as per the criteria mentioned in CLP classification.