Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Justification for type of information:
Please see read across justification document.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Dowfax C6L Surfactant Solution
Purity: 43.4% active ingredient
Reference: Lot # M03112491 (39611), TMC # 20365
Appearance: clear liquid
Supplier: The Dow Chemical Company
Characterization: The IR spectrum matched that from a previous Dowfax C6L, the sample contained 1.05 wt% sulfate, 0.13 wt% chloride, 4.81 wt% sodium, and 50.66 wt% water. The purity was found to be (by difference) 43.4%. The purity was determined by using ion chromatography, Neutron Activation Analysis (NAA) and Karl Fischer volumetric titration. Infrared spectroscopy (FTIR) and liquid chromatographic mass spectroscopy (LC/MS) were used to confirm the proposed structure of Dowfax C6L.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Animal Acquisition and Acclimation
A total of 56 male and 56 female Sprague-Dawley [Crl: CD (SD)IGS BR] rats (7-8 weeks of age upon arrival) were received on March 17, 2005 fiom Charles River Laboratories, Portage, Michigan. Body weights were measured and recorded the day after arrival, and the sex of each animal was verified upon arrival. During the 11-day acclimation period, the animals were weighed and observed twice daily with respect to general health and any signs of disease. All animals were given a detailed clinical examination prior to selection for study.

Randomization, Assignment to Study, and Maintenance
Prior to assignment to study, the animals were weighed and examined for evidence of disease and other physical abnormalities. Animals assigned to study had body weights within +/-20% of the mean body weight for each sex. Extra animals obtained for this study, but not placed on study, were euthanized via carbon dioxide inhalation and discarded. Using a standard, by weight, block randomization procedure, 48 male and 48 female rats (weighing 249 to 278 g and 198 to 221 g, respectively, at randomization) were assigned to the control or treated groups. Each animal was assigned an animal number to be used in Provantis and implanted with a microchip bearing a unique identification number. Each cage was identified by the study number, animal number, group number, and sex. The individual animal number plus the study number comprised a unique identification for each rat. Pups were identified by tattoo on LD 0. Animal identification was verified during the conduct of the study, as documented in the study data.

From acclimation until euthanasia, the rats were individually housed in suspended, stainless steel, wire-mesh type cages, except during pairing, near parturition, and during lactation. During pairing, animals were cohabited, one male and one female within the same treatment group. On approximately GD 20, females were individually housed in plastic cages containing wood chip bedding. Fluorescent lighting was provided for approximately 12 hours per day. Temperature and humidity were monitored and recorded daily. The protocol- designated ranges were 64 to 79°F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported, but were reviewed and are maintained in the study file.

Diet (meal Lab Diet Certified Rdodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number fiom each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water analyses are retained in the Archives. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
other: distilled water
Details on exposure:
Vehicle and Test Article Preparation
The test article was adjusted for purity (43.4% active ingredient). The required volume of vehicle, distilled water, was dispensed into amber glass containers for use on study weekly, prior to handling the test article, and stored at room temperature. To prepare the test article formulations, the required amount of test article, DOWFAX C6L, was weighed directly into a beaker. Vehicle was added to the beaker, and the contents were stirred using a magnetic stir bar and stir plate until dissolved. The contents of the beaker were transferred into a graduated cylinder. The beaker was rinsed with vehicle, and the rinse was transferred into the graduated cylinder. More vehicle was added to the cylinder to yield the required amount of prepared test article formulation. The cylinder was shaken thoroughly, and the contents were transferred into the beaker. The contents of the beaker were stirred using a magnetic stir bar and stir plate. The contents of the beaker were dispensed, while stirring, into amber glass containers, using a syringe.

To prepare the first weekly preparation of the 60 mg/kg/day formulation, the required amount of test article, DOWFAX C6L, was weighed directly into a beaker. Vehicle was added to the beaker, and the contents were stirred using a magnetic stir bar and stir plate until dissolved. The contents of the beaker were transferred into a graduated cylinder. Vehicle was added to the cylinder to yield the required amount of prepared test article formulation. The cylinder was shaken thoroughly, and the contents were transferred into an amber glass container.

Formulations of the test article were prepared for each concentration weekly and stored at room temperature.

Administration
The vehicle control and test article were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period after all females had delivered) and up to 54 days in females dependent on reproductive performance (14 days premating, 14 days of mating [maximum], 22 days gestation and 4 days postpartum). The test article was administered to the treated groups via oral gavage once per day at dose levels of 60, 200, and 1000 mg/kg/day of DOWFAX C6L (corresponding to 125, 417, and 2083 mg/kg/day bulk material dose levels, based 48% purity) at a dose volume of 4 mL/kg. The control animals received the vehicle, distilled water, at the same volume, duration, and dosing regimen as the treated animals. Individual doses were based on the most recent body weight.

The vehicle and test article were administered via oral gavage using an appropriately-sized plastic disposable syringe attached to a Fuchigami dosing needle. The test article formulations were stirred continuously during test article administration using a magnetic stir bar and stir plate.
Details on mating procedure:
Breeding Procedures
After 14 days of treatment, the males were randomly cohabited, one male to one female, with the corresponding females in the treatment and control groups. Each female was housed in the cage of the male during pairing. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or sperm in the vaginal lavage. The day on which positive evidence of copulation was observed was considered GD 0. The rats were paired for a maximum of 14 days. If evidence of mating was confirmed, the female was removed fiom the cage of the male and individually housed. After the mating period, any unmated females were individually housed in a plastic cage with wood chip bedding in anticipation of undetected pregnancies.

After the mating period, the males were individually housed and continued on treatment for approximately 2 weeks or until all deliveries were completed and it was determined that an additional pairing was not necessary. At the end of this period, the males were euthanized and necropsied according to procedures indicated in the postmortem Study Evaluations section.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All samples were analyzed for Dowfax C6L concentration using high erformance liquid chromatography with ultraviolet absorbance detection (HPLC/UV). Multiple aliquots of samples mixed in Milli-Q water were diluted further with Milli-Q water 25, 100 or 10000- fold depending on concentration.

Analysis conditions:
HPLC system: HP1100
Binary pump: DE03007092
Autosampler: DE03013232
Detector: JP92112458
Eluent A: Milli-Q water plus 0.1% phosphoric acid
Eluent B: Acetonitrile plus 0.1% phosphoric acid
Gradient time (min) %B
0.0 5
20.0 95
25.0 95
30.0 5
Flow rate: 1ml/min
Injection volume: 100ul/injection/sample
Column: YMC Octyl (C8),5u, 2x50 mm S/N 4776132091K 04 with YMC Octyl guard column
Wavelength: 254 nm
Sensitivty: 2000mAU
Response time: 2 sec
Detector output: 1V
Data system: PE Nelson Turbochrom Labratory Data System

Homogeneity and Concentration Verification
Duplicate samples (5 mL/sample) of the first weekly preparation of the 60 and 1000 mg/kg/day formulations were collected fiom the top, middle, and bottom of the container, using a syringe, and placed in amber glass containers to confirm test article homogeneity in the vehicle. Concentration verification was obtained for the first preparation by using the mean of the 60 and 1000 mg/kg/day formulation homogeneity results. Samples (5 mL/sample) of the 0 and 200 mg/kg/day formulations were also collected fiom the container, using a syringe, and placed in amber glass bottles to be analyzed for concentration verification.

Stability
The Sponsor has confirmed 14-day stability of the test article in the vehicle at ambient conditions for the proposed dosage range. Analysi by LC/UV indicated stability at 0.250, 2.50 and 250 mg/mL for at least 2 weeks.

Concentration
Samples (5 mL/sample) of each test article formulation were collected weekly for the first 4 weeks and the last full week of study fiom the middle of the container, using a syringe, and placed in amber glass bottles for analysis of test article concentration. Dose solution of 0.250 and 250 mg/mL were homogeneous with relative standard deviations of 0.602 and 1.52%, respectively. Targeted dose concentrations, of 0, 34.6, 115, and 576 mg/ml were analyzed to be >/= 89.8% of targeted concentration.




Analyses
Samples were stored at room temperature until shipped at ambient temperatures to the Sponsor for analysis. All analytical work was conducted by the Sponsor.
Duration of treatment / exposure:
The vehicle control and test article were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period after all females had delivered) and up to 54 days in females dependent on reproductive performance (1 4 days premating, 14 days of mating [maximum], 22 days gestation and 4 days postpartum).
Frequency of treatment:
daily
Details on study schedule:
No additional data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 125,417, and 2083 mg/kg/day
Basis: other: nominal based on bulk material
Remarks:
Doses / Concentrations:
0, 54, 181, and 904 mg/kg/day
Basis: other: nominal based on active ingredient ( 43.4% purity)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted for The Dow Chemical Company to generate limited information concerning the effects of the test article, DOWFAX C6L, on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Three treatment groups of 12 male and 12 female Sprague-Dawley [Crl: CD® (SD)] rats were administered the test article at dose levels of 54, 181, and 904 mg/kg/day of DOWFAX C6L (corresponding to 125, 417, and 2083 mg/kg/day bulk material dose levels, based on 43.4% purity) at a dose volume of 4 mL/kg. One additional group served as the control and received the vehicle, distilled water, at the same volume and dosing regimen as the treated groups. The vehicle control and test article were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period after all females had delivered) and up to 54 days in females dependent on reproductive performance (14 days of premating, 14 days of mating [maximum], 22 days gestation and 4 days postpartum).

Observations included clinical signs, body weights, and food consumption during the premating, gestation, lactation, and postmating periods. After 2 weeks of vehicle or test article administration, the females were cohabited with males, one male to one female, from the same treatment group, for up to 14 days. Vaginal smears (lavage) were evaluated daily in females during the 14-day period to establish estrous cyclicity. Once mating was confirmed on Gestation Day 0 (GSD 0), females were separated from the male for the remainder of gestation, and allowed to deliver and nurse litters until Lactation Day (LD) 4. Observations of the F1 (first generation offspring) pups included survival at birth and during lactation, individual pup body weights and sex, and clinical examinations on LD 0 and 4. Pups were euthanized and externally examined on LD 4. Complete necropsies were performed on all adult animals and protocol-designated organs and tissues were weighed and microscopically examined.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Mortality and Cageside Observations
All rats were observed twice daily for morbidity, mortality, signs of injury, and availability of food and water.

Detailed Clinical Examinations
Detailed clinical examinations were conducted once prior to initiation of test article administration and daily during the study. During test article administration, these examinations were conducted approximately 60-90 minutes post-dose. The observations were carefully recorded and an effort was made to ensure that variations in the test conditions were minimal. The observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypics (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Pretest clinical observations are not reported, but are maintained in the study file.

Body Weights and Body Weight Changes
Individual body weights for the males and females were recorded the day after arrival, prior to randomization, at initiation of test article administration, weekly during the premating, mating, and postmating periods for males and unmated females through to termination. All mated females were weighed on GD 0,7, 14, and 20, and on LD 0 and 4, and at termination. Pre-test body weights are not reported, but are maintained in the study file.

Food Consumption
Individual food consumption was measured and recorded weekly for all animals prior to, but not during, pairing. Following the cohabitation period, food consumption was recorded weekly for males and unmated females until euthanasia. Food consumption was recorded for all mated females on GD 0,7, 14, and 20 and LD 0 and 4.
Oestrous cyclicity (parental animals):
Estrous Cyclicity
Vaginal smears (lavage) were evaluated daily in females during the 2-week premating period to establish estrous cyclicity. The stage of estrous continued to be recorded during the mating period until evidence of mating was confirmed.
Sperm parameters (parental animals):
No data
Litter observations:
Parturition and P1 Litter Observations
Beginning on GD 19, females were examined twice daily for signs of parturition. Females were allowed to give birth with the young designated F1 . The duration of gestation was calculated, and any difficulties occurring at pamuition were recorded. The day on which all pups were delivered was designated as LD 0. The litters were examined as soon as possible after delivery and parameters including litter size, number of still born and live born pups, pup body weights and external sexing, and gross external abnormalities of the pups were recorded. Pups were weighed and sex confirmed on LD 0 and 4, and any abnormal behavior observed in the pups was recorded. On LD 4, the pups were euthanized via intraperitoneal injection of sodium pentobarbital solution and necropsied as described in the Postmortem Study Evaluations section.
Postmortem examinations (parental animals):
Postmortem Study Evaluations
Macroscopic
At the termination of the study, all surviving adult animals were euthanized and examined. Males were euthanized by carbon dioxide inhalation after at least 4 weeks of treatment. Females were euthanized by inhalation of carbon dioxide according to the following schedule. Females that delivered were euthanized on LD 4. Females that failed to deliver (with evidence of mating) were euthanized 25 days after evidence of mating was detected. Females that failed to deliver (with no evidence of mating) were euthanized 25 days after completion of the mating phase. The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) were counted and recorded in females. All tissues were fixed in neutral buffered formalin.

Organ Weights
Body weights and protocol-specified organ weights were recorded at scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weights). Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.

Microscopic
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides were examined by a veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups. Normally developing implants from one female at 1000 mg/kg/day (animal number 287) that died pregnant were saved in 10% neutral buffered formalin for possible future examination.

A full list of organs and tissues were collected, weighed, and examined.
Postmortem examinations (offspring):
Pup Evaluations
F1 pups found dead at birth or during the lactation period were given a gross external examination for abnormalities. The pups found dead at birth were given a lung flotation test to determine stillborn status. If the lungs floated, the pups were determined not stillborn. Pups that survived to scheduled euthanasia (LD 4) were examined externally and subjected to a complete necropsy performed under procedures approved by a veterinary pathologist. Gross lesions were saved in 10% neutral buffered forrnalin for possible future histopathologic examination.
Statistics:
The table below defines the sets of comparisons used in the statistical analyses described in
this section.
Statistical Comparisons
Control Group Treatment Groups
1 2,3,4
The endpoints that were analyzed and the methods of analysis that were employed are presented in the table Statistical Analysis Methods
Endpoint Analysis
Parental In-life Data
Premating body weights Group Pair-wise Comparisons
Premating body weight change Group Pair-wise Comparisons
Mating and postmating body weight - Group Pair-wise Comparisons
males
Postmating body weight change - males Group Pair-wise Comparisons
Postmating food consumption - males Group Pair-wise Comparisons
Gestation body weights Group Pair-wise Comparisons
Gestation body weight change Group Pair-wise Comparisons
Gestation food consumption Group Pair-wise Comparisons
Lactation body weights Group Pair-wise Comparisons
Lactation body weight change Group Pair-wise Comparisons
Lactation food consumption Group Pair-wise Comparisons
Premating estrous cycling (number of Group Pair-wise Comparisons
cycles/period and mean cycle time)

Fertility Indices
Gestation Length Group Pair-wise Comparisons
Copulatory Interval (i. e., days-to-mating) Group Pair-wise Comparisons
Male Fertility Index Fisher's Exact Test
Female Fertility Index Fisher's Exact Test


Reproductive indices:
See stats above and below
Offspring viability indices:
See stats above and below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Common clinical observations noted for the high dose (904 mg/kg/day) males (animal numbers 238 and 239) just prior to unscheduled deaths included activity decreased, brown material around mouth and soft feces. There were no common clinical observations noted for the high dose (904 mg/kg/day) females (animal numbers 287, 288, 290 and 293) just before unscheduled deaths. A greater incidence of adverse clinical observations was recorded during the study at the high dose level (904 mg/kg/day) for both the male and female animals. It should be noted that most of the adverse clinical observations were related to the animals that either died or were found dead. Some of the more common clinical observations noted at the high dose level (1 000 mg/kg/day) in males included salivation, soft feces, breathing difficulties, discolored material around nose and mouth. During the premating, gestation, and lactation periods, similar clinical findings were observed in the female rats, but at a lower frequency. A low incidence of other clinical observations was noted that did not follow a dose response relationship and thus were not considered toxicologically meaningful.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality
At the high-dose level (904 mg/kg/day) two males (animal numbers 238 and 239) died on Study Days 49 and 57, respectively and four females (animal numbers 287,288,290, and 293) died on GD 16, LD1, Premating Day 4 and LD 3, respectively. The death of these animals was considered to be test article related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant changes in the mean body weight were found in any male treatment group during the course of the study. A decreased body weight gain in the high dose (904 mg/kg/day) males was statistically identified during the first two weeks of premating. A slight trend in decreased body weight gain at the end of the study period (weeks 8-9) was also noted at the male high dose level (904 mg/kg/day).

Mean female body weight values were comparable among the groups during the premating, gestation, and lactation periods. Body weight changes were significantly decreased in the high dose group (904 mg/kg/day) at the GD interval 14-20. The decrease in body weight changes was considered to be treatment related. A decrease in body weight change in the low dose group (60 mg/kg/day) during lactation was statistically identified. However, due to the lack of a clear dose-response relationship and its sporadic occurrence, it was not considered test article-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In male rats, food consumption was not affected at any of the dose levels examined. A statistically significant increase was noted for males in the high dose group (904 mg/kg/day) during the postmating interval (Weeks 7-8). This was not considered toxicologically meaningful, as the remaining food consumption values at that dose level and for the other dose groups were comparable. In female rats food consumption in the treated groups was not significantly reduced during the premating and gestation periods. Food consumption was significantly decreased in the low dose group (54 mg/kg/day) during lactation (78% of control value). This was consistent with the decrease in the body weight changes observed in the same dose group during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
At the high-dose level (904 mg/kg/day) two males (animal numbers 238 and 239) died on Study Days 49 and 57, respectively and four females (animal numbers 287,288,290, and 293) died on GD 16, LD1, Premating Day 4 and LD 3, respectively. The death of these animals was considered to be test article related.

Detailed Clinical Observations
Common clinical observations noted for the high dose (904 mg/kg/day) males (animal numbers 238 and 239) just prior to unscheduled deaths included activity decreased, brown material around mouth and soft feces. There were no common clinical observations noted for the high dose (904 mg/kg/day) females (animal numbers 287, 288, 290 and 293) just before unscheduled deaths. A greater incidence of adverse clinical observations was recorded during the study at the high dose level (904 mg/kg/day) for both the male and female animals. It should be noted that most of the adverse clinical observations were related to the animals that either died or were found dead. Some of the more common clinical observations noted at the high dose level (1 000 mg/kg/day) in males included salivation, soft feces, breathing difficulties, discolored material around nose and mouth. During the premating, gestation, and lactation periods, similar clinical findings were observed in the female rats, but at a lower frequency. A low incidence of other clinical observations was noted that did not follow a dose response relationship and thus were not considered toxicologically meaningful.

Body Weights and Body Weight Changes
No statistically significant changes in the mean body weight were found in any male treatment group during the course of the study. A decreased body weight gain in the high dose (904 mg/kg/day) males was statistically identified during the first two weeks of premating. A slight trend in decreased body weight gain at the end of the study period (weeks 8-9) was also noted at the male high dose level (904 mg/kg/day).

Mean female body weight values were comparable among the groups during the premating, gestation, and lactation periods. Body weight changes were significantly decreased in the high dose group (904 mg/kg/day) at the GD interval 14-20. The decrease in body weight changes was considered to be treatment related. A decrease in body weight change in the low dose group (60 mg/kg/day) during lactation was statistically identified. However, due to the lack of a clear dose-response relationship and its sporadic occurrence, it was not considered test article-related.

Food Consumption
In male rats, food consumption was not affected at any of the dose levels examined. A statistically significant increase was noted for males in the high dose group (904 mg/kg/day) during the postmating interval (Weeks 7-8). This was not considered toxicologically meaningful, as the remaining food consumption values at that dose level and for the other dose groups were comparable. In female rats food consumption in the treated groups was not significantly reduced during the premating and gestation periods. Food consumption was significantly decreased in the low dose group (54 mg/kg/day) during lactation (78% of control value). This was consistent with the decrease in the body weight changes observed in the same dose group during lactation.

Estrous Cyclicity
Mean cycle length and number of estrous cycles were comparable among the groups, including the control.

Postmortem Study Evaluations
Macroscopic Observations
No test article-related macroscopic changes were noted in rats of either sex that received DOWFAX C6L.

Brown discoloration of the liver was noted in one male (1 000 mg/kg/day) (animal number 238), that died on study (DOS), and this was considered to be incidental. Brown liver discoloration corresponded microscopically to minimal focal necrosis. This finding was not observed in any other animal at scheduled necropsy.

Other macroscopic changes noted were typical of those commonly seen in rats of the same strain and age, and were considered to be incidental.

Organ Weights
No test article-related organ weight changes were noted in rats of either sex. A statistically significant increase was noted in the relative liverbody weight ratio in females at the low dose level (54 mg/kg/day). As the relative liver/body weight ratio for females at the high dose levels were comparable to the control, this was considered incidental and not treatment related.

Microscopic Observations
No test article-related microscopic changes were noted in rats of either sex.

Minimal focal necrosis was noted in the liver of one DOS male (904 mg/kg/day), but this was not seen in rats of either sex at terminal necropsy, and was considered to be incidental.

Other microscopic changes noted were typical of those commonly seen in rats of the same strain and age or had a similar incidence in treated and control rats, and were considered to be incidental.

Reproductive Performance
Mating, fertility, and fecundity indices in male and female rats were not impacted by the test article. Copulatory interval (days) was increased slightly in the high dose group (904 mg/kg/day), but the value (3.8 days) was within the historical control range of 2 to 4.8 days.

Effect levels (P0)

Key result
Dose descriptor:
other: NOEL- maternal and developmental toxicity
Effect level:
181 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Parturition and Day 4 Litter Size Data
Gestation length and gestation index, number of pups born per litter, number of dead pups, stillborn index, and total implantation scars per litter were not affected. On LD 4, mean number of live pupsflitter was comparable among the groups.

Pup Sex Ratios
Pup sex ratio and mean number of live pups on LD 4 were not significantly different between the control and treatment groups.

Pup Survival Indices
The pup viability index (%) was slightly reduced in the high dose group (904 mg/kg/day), but it was not statistically different from control. The pup viability index in the high dose group (904 mg/kg/day) was 85.91%, as compared to the control values of 97.23%. This decrease may be correlated to a single litter at the high dose level (904 mg/kg/day) that gave birth to 15 pups, of which six were stillborn.

Pup Clinical Findings
In the pups, test article-related clinical findings were observed in the high dose group (904 mg/kg/day). These include decreased activity which was observed in ten pups on LD 0 and skin cold to touch, which was seen in seven pups on LD 0 and one pup on LD 0 and 4.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The pup viability index (%) was slightly reduced in the high dose group (904 mg/kg/day), but it was not statistically different from control. The pup viability index in the high dose group (904 mg/kg/day) was 85.91%, as compared to the control values of 97.23%. This decrease may be correlated to a single litter at the high dose level (904 mg/kg/day) that gave birth to 15 pups, of which six were stillborn.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup Body Weight
Pup body weight by sex and both sexes combined was significantly reduced in the high dose group (904 mg/kg/day) on LD 0. Similarly, on LD 4, male pup body weights and both sexes combined was significantly reduced in the high dose group (904mg/kg/day). However, female pup body weight on LD 4 was decreased, but not significantly.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

F1 Litter Data
Parturition and Day 4 Litter Size Data
Gestation length and gestation index, number of pups born per litter, number of dead pups, stillborn index, and total implantation scars per litter were not affected. On LD 4, mean number of live pupsflitter was comparable among the groups.

Pup Sex Ratios
Pup sex ratio and mean number of live pups on LD 4 were not significantly different between the control and treatment groups.

Pup Survival Indices
The pup viability index (%) was slightly reduced in the high dose group (904 mg/kg/day), but it was not statistically different from control. The pup viability index in the high dose group (904 mg/kg/day) was 85.91%, as compared to the control values of 97.23%. This decrease may be correlated to a single litter at the high dose level (904 mg/kg/day) that gave birth to 15 pups, of which six were stillborn.

Pup Clinical Findings
In the pups, test article-related clinical findings were observed in the high dose group (904 mg/kg/day). These include decreased activity which was observed in ten pups on LD 0 and skin cold to touch, which was seen in seven pups on LD 0 and one pup on LD 0 and 4.

Pup Body Weight
Pup body weight by sex and both sexes combined was significantly reduced in the high dose group (904 mg/kg/day) on LD 0. Similarly, on LD 4, male pup body weights and both sexes combined was significantly reduced in the high dose group (904 mg/kg/day). However, female pup body weight on LD 4 was decreased, but not significantly.

Pup Macroscopic
Macroscopic examination did not reveal any treatment-related macroscopic lesions in pups of either sex LD 4. All organs examined were within the normal limits, with the exception of six pups (four males and two females) in the control, one male pup in the low dose (54 mg/kg/day), and one male pup in the mid dose group (181 mg/kg/day) that had minor - findings.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Remarks on result:
not measured/tested

Results: F2 generation

General toxicity (F2)

Other effects:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The objective of this study was to generate limited information concerning the effects of the test article, DOWFAX C6L, on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Mortality and clinical signs of toxicity were observed in parental animals of the high dose group (1000 mg/kg/day). Body weight changes were also affected in the high dose group (904 mg/kg/day) during gestation. In pups, test article-related clinical signs and body weight effects were observed in the high dose group (904 mg/kg/day) and these findings observed in conjunction with maternal toxicity. Based on the results of this study, the No-Observed-Effect-Level (NOEL) for maternal and developmental toxicity was considered to be 181 mg/kg/day.
Executive summary:

This study was conducted for The Dow Chemical Company to generate limited information concerning the effects of the test article, DOWFAX C6L, on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Three treatment groups of 12 male and 12 female Sprague-Dawley [Crl: CD® (SD)] rats were administered the test article at dose levels of 54, 181, and 904 mg/kg/day of DOWFAX C6L (corresponding to 125, 417, and 2083 mg/kg/day bulk material dose levels, based on 43.4% purity) at a dose volume of 4 mL/kg. One additional group served as the control and received the vehicle, distilled water, at the same volume and dosing regimen as the treated groups. The vehicle control and test article were administered for at least 42 consecutive days to males (14 days premating, 14 days of mating, and 14 days after completion of the mating period after all females had delivered) and up to 54 days in females dependent on reproductive performance (14 days of premating, 14 days of mating [maximum], 22 days gestation and 4 days postpartum).

Observations included clinical signs, body weights, and food consumption during the premating, gestation, lactation, and postmating periods. After 2 weeks of vehicle or test article administration, the females were cohabited with males, one male to one female, from the same treatment group, for up to 14 days. Vaginal smears (lavage) were evaluated daily in females during the 14-day period to establish estrous cyclicity. Once mating was confirmed on Gestation Day 0 (GD 0), females were separated from the male for the remainder of gestation, and allowed to deliver and nurse litters until Lactation Day (LD) 4. Observations of the F1 (first generation offspring) pups included survival at birth and during lactation, individual pup body weights and sex, and clinical examinations on LD 0 and 4. Pups were euthanized and externally examined on LD 4. Complete necropsies were performed on all adult animals and protocol-designated organs and tissues were weighed and microscopically examined.

Two males and four females in the high dose group (904 mg/kg/day) died during the course of the study. The deaths did not appear to have been related to a mechanical dosing error, as no evidence of esophageal perforation was noted at necropsy. For the males, the deaths occurred on study days 49 and 57. For the females, the deaths occurred on premating day 4, GD 16, LD 1 and LD 3. The death of these animals was considered to be test article related.