Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Dec 2010 - 16 Oct 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP -guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EU Guidelines on Reproductive Toxicity Studies, in the Official Journal of the European Communities 91/414/EEC, February, 1995

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Health Canada, Guidelines on Reproduction Toxicity studies, Canada Gazette, Part II, Vol. 122 no. 2, 1988

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry, and Fisheries (JMAFF), 12 Nousan 8147 (Nov.24, 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

EC Number:

620-056-5

Cas Number:

874195-61-6

Molecular formula:

C14H13F3N4O5S

IUPAC Name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

Test animals

Species:

rat

Strain:

other: Wistar Han CRL:WI (HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. Portage, MI, USA
- Age at study initiation: (P-generation) 9 weeks
- Weight at randomisation: Males: 233.4–305.3 g; Females: 147.4–211.3 g
- Housing: Animals were housed individually (except during the mating phase) in suspended stainless steel cages, with deotized cage board in the bedding trays. During gestation and lactation, individual dams (and their litter) were housed in polycarbonate cages with corn-cob bedding.
A single Nylabone® was placed in the cage of each animal (to provide environmental enrichment). Adult males were given the Nylabone® for the duration of the study except for the cohousing phase. Adult females were given the Nylabone® during the premating phases only.
- Diet: Purina Mills Certified Rodent Diet 5002 in "meal" form; ad libitum
- Water: free and continuous access to tap water; ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.64-22.83
- Humidity (%): 47.69-60.15
- Air changes (per hr): 13.31
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test substance was mixed directly with the feed. Adjustments were not made for percentage purity of less than 100%. Treated diet was mixed at room temperature; aliquots of the chemical were taken from the original test batch and transferred to the mixing area. The control test diet was prepared in the same manner as the chemically-treated test diet, excluding the test substance. A sample of each batch of feed mixed was taken and retained in the freezer (average temperature -22±4 °C) until the study was complete and the analytical data deemed satisfactory. Replacement admixtures for each treatment group were prepared weekly and stored under freezer conditions (average temperature -22±4 °C) until presented to the animals the following week (or weeks).
The concentration of the test substance in the feed, for the females only, was adjusted during the lactation period (Days 0–21) by 50%. Samples from the first batch of adjusted feed for each dietary level were analyzed to measure the concentration. During the lactation phase a substantial increase in food consumption is observed in all dams which results in greatly increased intake of test substance (normal occurrence). A decrease in the dietary concentration of the test substance offset this increased food consumption, thereby maintaining an approximately constant test substance intake (mg/kg bw/day) throughout the study.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 consecutive days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: Females found to be inseminated were placed in a polycarbonate nesting cage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of AE 1887196 in the various test diets was analytically verified for batches intended for Weeks 1, 2, 3, and at monthly intervals thereafter.

Concentration Analysis: Mean analytical concentrations for each dietary group were 101, 506, and 2517 ppm, of which all dietary levels were 101% of the corresponding nominal concentrations of 100, 500, and 2500 ppm, respectively. During lactation, the concentration of the test substance in the food for the females was adjusted by 50%. Mean analytical concentrations for each dietary group during lactation were 49.5, 248, and 1249 ppm, ranging from 99–100% of the corresponding nominal concentrations of 50, 250, and 1250 ppm, respectively. The active ingredient of the test substance was not detected in the control diet. Mean overall recovery was 103% and ranged from 99–107% for rodent ration spiked with 50, 100, 250, 500, or 2500 ppm of AE 1887196.

Homogeneity Analysis: The mean concentrations of AE 1887196 in the food, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 25, 50, or 5000 ppm, were determined to be 25.1 ppm (range 24.4–25.8 ppm; %RSD = 1.9), 50.8 ppm (range 50.3–51.2 ppm; %RSD = 0.69), and 5012 ppm (range 4899–5165 ppm; %RSD = 1.5), respectively. Based on a %RSD to 10%, AE 1887196 was judged to be homogeneously distributed in the food over a concentration range of 25–5000 ppm.

Stability Analysis: Following 7 days of room temperature storage, the analytically determined concentration of the active ingredient of the test substance in the 25, 50, or 5000 ppm admixture was determined to be 20.6 ppm (23.7 ppm on Day 0), 43.7 ppm (49.2 ppm on Day 0), and 4803 ppm (4903 ppm on Day 0), respectively. Following 28 days of freezer storage, the analytically-determined concentration of the active ingredient of the test substance in the 25, 50, and 5000 ppm mixtures was determined to be 23.7 ppm (25.1 on Day 0), 49.2 ppm (50.8 on Day 0), and 4903 ppm (5012 on Day 0), respectively. AE 1887196 mixed in rodent ration was judged to be stable at room temperature for at least seven days and following freezer storage for a minimum of 28 days, over a concentration range of 25–5000 ppm.

Duration of treatment / exposure:

(P) Males: 10 weeks before mating, 14 days during mating
(P) Females: 10 weeks before mating, 14 days during mating, approximately 22 days during gestation, 21 days during lactation (weaning of the F1 offspring on Day 21).
F1-pups were maintained after weaning for approximately four to six weeks prior to initiation of the second generation.
(F1) Males: 10 weeks before mating, 14 days during mating
(F1) Females: 10 weeks before mating, 14 days during mating, approximately 22 days during gestation, 21 days during lactation

Frequency of treatment:

daily (7 days/week)

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

2 500 ppm (nominal)

No. of animals per sex per dose:

30 rats/sex/group
Number on animals on study: 240 (120/sex)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Dietary dose levels were selected after evaluation of the results of a previous 90-day toxicity study, where the NOAEL was 250 ppm of dietary AE 1887196 in both sexes (corresponding to 16.4 and 20.0 mg/kg bw/day in males and females, respectively). Significant toxicity (essentially consisting of histopathological changes in the liver and thyroid gland) was observed in animals from the 5000 ppm dietary group, which was the highest dose level tested (corresponding to 323 and 395 mg/kg bw/day in males and females, respectively).
A dose range-finding reproduction pilot study was also conducted at the test facility to help determine appropriate dietary levels. In the pilot study, the test substance was administered via the diet to male and female Wistar rats (10/sex/dietary level) at nominal concentrations of 100, 500, and 2500 ppm. Preliminary findings are as follows. For the P-generation, body weights were statistically (8–9%; Day 35 through Day 63) and non-statistically (7–8%; Day 70
through termination) decreased in 2500 ppm males. Relative liver weights were statistically increased 12% and 11% in 2500 ppm males and females, respectively. Additionally, absolute liver weights were non-statistically increased 10% in 2500 ppm females. For the F1-generation, Day 21 pup body weights were non-statistically decreased (8%, each) in 2500 ppm males, and males and females combined. Mean body weight change was statistically decreased (9%) in 2500 ppm males and females combined, and non-statistically decreased (9%) in 2500 ppm males on Day 4–21.
Based on these results, the dietary levels selected for the definitive reproduction toxicity study were 0, 100, 500, and 2500 ppm AE 1887196. This dietary range was intended to produce evidence of toxicity at the highest dietary concentration and no parental or reproductive effects at the lowest dietary concentration.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (a.m. and p.m.) during the workweek and once daily on weekends and holidays
- Cage side observations checked: mortality, moribundity, behavioral changes, signs of difficult or prolonged delivery, and overt toxicity by viewing the animal in the cage

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (a.m. and p.m.) during the workweek and once daily on weekends and holidays

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured once per week for both males and females during the 10-week premating period. During the mating period and until sacrifice, body weight for the males and unmated females was measured once per week. During gestation, dam body weight was measured on Days 0, 6, 13, and 20. During lactation, dam body weight was measured on Days 0, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured and fresh food provided once per week for both males and females during the 10-week premating period. During the mating period, fresh food was provided for both males and unmated females once each week without measuring food consumption. During gestation, fresh food was provided and food consumption measured on Days 0, 6, 13, and 20. During lactation, food consumption was measured on Days 0, 4, 7, 14, and 21. Fresh food was provided at least once per week.

Oestrous cyclicity (parental animals):

The estrous cycle (determined by examining daily vaginal smears) was characterized for all P and F1-generation females, over a three-week period prior to mating. Additionally, the estrous cycle stage was determined for all females just prior to termination.

Sperm parameters (parental animals):

For all P- and F1-generation males at termination, sperm was collected from one testis (left) and one epididymis (left) for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. An evaluation of the morphology and motility was performed on sperm sampled from the distal portion (closest to the urethra) of the vas deferens.
Sperm motility was conducted for all groups and morphology and sperm counts (testicular and epididymal) were conducted on the control and highest dietary groups of both generations.

Litter observations:

STANDARDISATION OF LITTERS
The size of each litter was adjusted on LD 4 to yield, as closely as possible, four males and four females per litter. When the number of male or female pups was less than four, a partial adjustment was made (e.g., three females and five males). No adjustment was made for litters of fewer than eight pups. Adjustments were made by random selection of the pups using software provided by SAS. Culled pups were sacrificed and discarded.

PARAMETERS EXAMINED
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Pups found dead or stillborn underwent a gross necropsy for possible defects and/or to determine the cause of death.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals (both P- and F1-generations): All surviving animals were sacrificed as soon as possible after the last litters were produced. F1-adult males were sacrificed after the beginning of the delivery phase for the F1-females.
- Maternal animals (both P- and F1-generations): All surviving animals were sacrificed following the weaning of their respective litters (LD 21).
Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed and necropsied after GD 24. Females that were never observed as being inseminated and/or with an internal vaginal plug and did not deliver at least 24 days after the completion of the mating phase were sacrificed and necropsied.

GROSS NECROPSY
The following organs were collected and weighed: brain, pituitary gland, liver, kidney, spleen, thyroid, thymus, adrenal, epididymis (right), ovary, prostate, seminal vesicle (with coagulating gland and fluid), testis (right), uterus (with oviduct and cervix), epididymis cauda (side not utilized for sperm)
- Maternal animals: the uterus was excised and the implantation sites, if present, were counted.
A quantitative evaluation of the ovarian follicles (preantral and antral) and corpora lutea were conducted on ten F1-dams (control and high-dose only) that delivered pups. Dams which died or did not deliver pups were not used in this randomization. For each dam, follicles in both ovaries were counted, using five step sections per ovary.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology was performed on the following tissues: pituitary gland, liver, kidney, spleen, thyroid, adrenal, epididymis (right), coagulating gland, ovary, oviduct, prostate, seminal vesicle (with coagulating gland and fluid), testis (right), uterus, cervix, lung, vagina, cervix, gross lesions

Processing of tissues and histopathological evaluations were initially conducted on the control and highest dietary groups, with one exception. The exception was that the reproductive organs were evaluated in any animal demonstrating reduced fertility (e.g., those who failed to mate, conceive, sire, or deliver healthy offspring), or where altered sperm motility was observed. When histopathological findings were attributed to treatment, the other dietary levels were evaluated beginning with the next highest dietary level until a NOAEL was established.

Postmortem examinations (offspring):

SACRIFICE
The F1- and F2-pups not culled on LD 4 were maintained with the dam until weaning on LD 21. On LD 21, a sufficient number of randomly selected F1-pups/sex/litter were maintained to produce the next generation (software provided by SAS for random selection).
F1-pups not selected to become parents of the next generation and all F2-pups were sacrificed, examined macroscopically and/or microscopically, and had organs weighed. One pup/sex/litter for each generation (as available), based on random selection utilizing software provided by SAS, had tissues collected and evaluated for any structural abnormalities or pathological changes, particularly as they may relate to the organs of the reproductive system.

GROSS NECROPSY
- The following organs were collected and weighed: Brain, Spleen, Thymus, Uterus

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology was performed on the following tissues: Epididymis, Coagulating Gland, Ovary, Oviduct, Prostate, Seminal Vesicle , Testis, Uterus, Vagina, Cervix, Gross Lesions

Statistics:

Data was analyzed using DATATOX, SAS, or TASC. Clinical observations for adults and pups were evaluated using Fisher’s Exact Test. Parametric data were analyzed using a univariate Analysis of Variance, and when significant differences were observed, a Dunnett's Test was performed. Nonparametric data were first analyzed by the Kruskal-Wallis Test and then subjected to Dunn's Test when significant differences were identified. Nonparametric dichotomous data (e.g., fertility and gestation indices) were initially analyzed by the Chi-Square Test and, when significance was observed between groups, then by the Fisher's Exact Test with the Bonferroni adjustment. Sperm parameters were analyzed using ANOVA (single factor) and the ovarian follicles and corpora lutea count data (mean data/animal values) were evaluated by the r-Test (two-sample assuming equal variance test) from Microsoft Excel® software programs. The frequency of gross lesions and other gross pathology data were examined visually and statistical analysis was deemed unnecessary. The organ and terminal body weight data for the adults were evaluated initially using Bartlett's Test to determine homogeneity of variance. An ANOVA was performed on homogeneous data followed by Dunnett's t-Test on parameters showing a significant effect by ANOVA. For nonhomogeneous data, a Kruskal-Wallis Analysis of Variance was performed followed by a pairwise Mann-Whitney U Test on parameters showing a significant overall effect. The pup terminal body weights and organ weights were evaluated using univariate ANOVA, and if significant differences were observed, a Dunnett’s Test was performed. Micropathology data for pups and adult animals were evaluated using the Chi-Square Test followed by a one-tailed Fisher’s Exact Test in cases of significant variation by the Chi-Square analysis. Differences between the control and test substance-treated groups were considered statistically significant when p < 0.05, p < 0.01, or p < 0.001.

Reproductive indices:

Mating Index (%) = # inseminated females (a) x 100 / # of females co-housed
Fertility Index (%) = # of pregnant females (b) x 100 / # of inseminated females
Gestation Index (%) = # of females with live pups x 100 / # of pregnant females
(a) Includes pregnant females not observed sperm positive or with an internal vaginal plug.
(b) Includes females which did not deliver, but had implantation sites.

Offspring viability indices:

Birth Index (%) = (total # of pups born/litter x 100) / (total # of implantation sites/litter)
Livebirth Index (%) = (# of live pups born/litter x 100) / (total # of pups/litter)
Viability Index (%) = (# of live pups/litter on Day 4 (pre-culling) x 100 / (# of live pups born/litter)
Lactation Index (%) = (# of live pups/litter on Day 21 x 100 / (# of live pups/litter on Day 4 (post-culling))
Gestation Length = Number of whole days from day in which insemination was observed in the vaginal smear [designated Day 0 of gestation (GD)] to Lactation Day (LD) 0 (delivery of pups and entry in computer system).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased incidence of urine stain at 2500 ppm in females of the P and F1 generation, but considered as a non-adverse effect

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

food consumption: incidental changes at 500 ppm in P-males; incidental changes at 2500, 500, 100 ppm in F1-females and at 2500 and 100 ppm in F1-males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

food consumption: incidental changes at 500 ppm in P-males; incidental changes at 2500, 500, 100 ppm in F1-females and at 2500 and 100 ppm in F1-males

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes were noted in livers of 2500 ppm females of P and F1 generation and in thyroid glands of 500 and 2500 ppm males and females of P- and F1-generation

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

statistical increase in median gestation length at the 2500 ppm F1-females but this is due to an increase in the number of individual F1-females that delivered on GD 23 or later

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
P-generation and F1-generation males: No test substance-related clinical observations were observed at any dietary level.
P-generation females: Test substance-related clinical observations were limited to an increased incidence of urine stain at the 2500 ppm dietary level. The increased incidence of urine stain was statistically significant during gestation (observed in 12 of 25 dams). Urine stain was seen in four dams during premating and five dams during lactation. Urine stain was considered non-adverse since there were no other observations or microscopic findings that could be correlated with this finding. No test substance-related clinical observations were observed at the 100 or 500 ppm dietary levels.
F1-generation females: Test substance-related clinical observations were limited to an increased incidence of urine stain at the 2500 ppm dietary level. The increased incidence of urine stain was statistically significant during gestation (observed in 7 of 28 dams). Urine stain was seen in five dams during premating and lactation. Urine stain was considered non-adverse since there were no other observations or microscopic findings that could be correlated with this finding. No test substance-related clinical observations were observed at the 100 or 500 ppm dietary levels.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
PREMATING
P-generation males: No test substance-related findings were observed on body weight, body weight gain or food consumption during the 10-week premating phase at any dietary level. There was one incidence of a statistical increase (5%) in food consumption (g/kg/day), compared to controls, for males during Week 7 at the 500 ppm dietary level, which is considered incidental and not related to the test substance since it was a single incidence and the incidence was small.
F1-generation males: No test substance-related findings were observed on body weight, body weight gain, or food consumption during the 10-week premating phase at any dietary level. At the 100 ppm dietary level, statistical changes in food consumption (g/kg/day), compared to controls, were seen in Weeks 8 and 9 (17% decreased and 6% increased, respectively). In addition, food consumption (g/animal/day) was statistically decreased, compared to controls, Week 8 (19%) in 100 ppm males and Weeks 2, 4, and 5 (6–8%) in 2500 ppm males. These transient, incidental changes are not thought to be related to the test substance since there were no corresponding decreases in body weight and there was no relationship to dose.
P-generation emales: No test substance-related findings were observed on body weight, body weight gain or in food consumption during the 10-week premating phase at any dietary level.
F1-generation females: No test substance-related findings were observed on body weight or body weight gain during the 10-week premating phase at any dietary level. At the 500 ppm dietary level, food consumption (g/kg/day) was statistically decreased Week 4 (4%) and Week 9 (5%), compared to controls. Food consumption (g/kg/day) was statistically decreased Weeks 3–5 (5–6%) and Weeks 8–9 (6–7%) at the 2500 ppm dietary level. Food consumption (g/animal/day) was also statistically decreased Week 4 (5%) at the 500 ppm dietary level and Weeks 3–6 (6–8%) and Weeks 8–9 (6–8%) at the 2500 ppm dietary level. These transient, incidental changes are not thought to be related to the test substance since there were no corresponding decreases in body weight and similar findings were not seen in the P-generation females.

GESTATION
P-generation: No test substance-related effects were observed on body weight or food consumption during gestation at any dietary level.
F1-generation: No test substance-related effects were observed on body weight or food consumption during gestation at any dietary level.

LACTATION
P-generation: No test substance-related effects were observed in body weight or food consumption during lactation at any dietary level. Food consumption (g/kg/day) was non statistically increased (7%) on Days 0–4 at the 100 and 500 ppm dietary levels but was not thought to be related to the test substance since this transient difference from control was small and there was no relationship to dose.
F1-generation: No test substance-related effects were observed in body weight or food consumption during lactation at any dietary level. Food consumption (g/kg/day) was statistically increased, compared to controls, on Days 0–4 at both the 500 and 2500 ppm dietary levels (22% and 16%, respectively). In addition, food consumption (g/animal/day) was statistically increased (22%) at the 500 ppm dietary level and non-statistically increased (15%) at the 2500 ppm dietary level on Days 0–4. These transient differences from control are not considered test substance-related since they were only seen on one occasion (Days 0–4), there was no relationship to dose and were more likely due to feed spillage, which is common among lactating females.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on food consumption, body weight, and dietary analyses results, the doses expressed as mean daily mg test substance/kg bbw during the pre-mating period (10 weeks for males and females) are presented in Table 1. Calculation for test substance intake is: Mean analytical concentration (ppm) specific for premating/1000 X mean weekly food consumption (g/kg/body weight/day) during premating (Tables 1 and 2).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no effects considered to be test-substance related on estrous cycle length or periodicity at any dietary level for either generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects considered to be test substance-related on any sperm parameter evaluated at any dietary level for either generation. Sperm motility (% motile and % progressive) and sperm counts (testis) were statistically increased in 2500 ppm F1-generation males. These increases, compared to control, are considered incidental and are due to a couple of control animals with lower values for these measurements.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
A slight statistical increase in median gestation length (22.5 days, compared to 22 days for controls) was seen at the 2500 ppm dietary level in the F1-females (Table 3). This is due to an increase in the number of individual F1-females that delivered on GD 23 or later (14 females in the 2500 ppm group versus 4 females in control group). Gestation length was not affected in the P-generation females at any dietary level.
Overall reproductive performance was not affected for any other parameter (e.g., mating, fertility, or gestation indices, days to insemination, or the number of implants) in either generation at any dietary level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
MALES
P-generation: There were no test substance-related effects on terminal body weights at any dietary level. A non-statistical increase in absolute and a statistical increase in relative liver weights (6% and 8%, respectively) were observed at the 2500 ppm dietary level. There were no other organ weight changes that were considered to be test substance-related at any dietary level.
F1-generation: There were no test substance-related effects on terminal body weights at any dietary level. Statistical increase (7%) in relative liver weights were observed at the 2500 ppm dietary level. Decreases in absolute and relative thymus weights (12% and 7%, respectively) were observed at the 2500 ppm dietary level. Decreases in absolute and relative thymus weights (7% and 6%, respectively) were also observed at the 500 ppm dietary level. The thymic weight changes were considered non-adverse since the changes were subtle, there was a lack of dose relationship in the relative thymic weights, and changes were likely due to subtle decrease in terminal body weights and/or stress. No decreases in thymus weights were observed at the 100 ppm dietary level, and there were no other organ weight changes that were considered to be test substance-related at any dietary level.

FEMALES
P-generation: There were no test substance-related effects on terminal body weights at any dietary level. A statistically significant increase in absolute and relative liver weights (12% and 10%, respectively) was observed at the 2500 ppm dietary level. There were no other organ weight changes that were considered to be test substance-related at any dietary level.
F1-generation: There were no test substance-related effects on terminal body weights at any dietary level. Statistical increase (7%) in relative liver weights were observed at the 2500 ppm dietary level. A decrease in absolute and relative thymus weights (10% and 8%, respectively) was observed at the 2500 ppm dietary level. The thymic weight changes were considered non adverse since the changes were subtle and changes were likely due to the subtle decrease in terminal body weights and/or stress. There were no other organ weight changes that were considered to be test substance-related at any dietary level.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross necropsy findings observed at any dietary level in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test substance-related micropathology changes were noted in
- livers of 2500 ppm females of P and F1 generation; and
- thyroid glands of 500 and 2500 ppm males and females of P- and F1-generation
In the P- and F1-generation females of the 2500 ppm dietary group, minimal centrilobular and/or midzonal hypertrophy of the liver was observed. The liver hypertrophy, coded as “Hypertrophy, hepatocellular, centrilobular and/or midzonal”, was characterized by enlarged hepatocytes primarily involving the cytoplasm of centrilobular areas and also occasionally involving the midzonal areas of the liver. The cytoplasmic appearance of hepatocytes varied from granular or intensively eosinophilic or pale. The liver hypertrophy change in the 2500 ppm dose level females correlated well with the statistically significant liver weight increases present at 2500 ppm dose level.
Minimal to moderate degree of colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) were noted in the thyroid glands of controls and test substance administered dose groups in P- and/or F1-generation adults. However, the incidence and severity of these findings were slightly higher at 500 and 2500 ppm dose groups (males and females) as compared to the controls of P- and F1-generation adults.
The colloid alteration consisted of pale, stippled, granular, or clumped colloid with variable staining characteristics. Hypertrophy of the follicular cells consisted of increase in the size of follicular epithelium with or without vacuolization. The test substance is a known hepatic microsomal enzyme inducer. The test substance could have induced hepatic microsomal enzymes and in turn might have caused the changes in thyroid glands. There is considerable evidence in the literature that the induction of hepatic microsomal enzymes not only alters the metabolism of xenobiotics but also alters the metabolism of various endogenous substances and thyroid function in rodents.
All other microscopic lesions observed in the P- and F1-generation adults were considered to be incidental and/or background seen in rats of this strain and age.

OVARIAN FOLLICLE COUNTS (F1 FEMALES)
There were no test substance-related effects observed on the mean primordial (preantral) follicles, antral follicles, or corpora luteal counts for the F1-females at any dietary level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 6.8 mg/kg bw/day for males and 8.3 mg/kg bw/day for females based on colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) noted in the thyroid glands of both P- and F1-generation adults

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 2 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to > 171.8 mg/kg bw/day highest dose level tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 2 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to > 171.8 mg/kg bw/day highest dose level tested

Results: P1 (second parental generation)

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systsemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 6.8 mg/kg bw/day for males and 8.3 mg/kg bw/day for females based on colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) noted in the thyroid glands

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 2 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to > 171.8 mg/kg bw/day; highest dose level tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to 41.3 mg/kg bw/day based on a slight increase in gestation length

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F2 pubs: a slight decrease in the livebirth and viability indices was observed at the 2500 ppm dietary level due to an increased incidence in the combination of stillborn, missing/cannibalized, and found dead pups

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
For the first generation, there were no test substance-related effects observed on the viability of the pups at any dietary level.
For the second generation, a slight decrease in the livebirth (historical control range 96.7–100) and viability (historical control range 94.3–100) indices was observed at the 2500 ppm dietary level due to an increased incidence in the combination of stillborn, missing/cannibalized, and found dead pups (Table 4). In the control group, the eight cannibalized pups and one found dead pup were from one female and the incidences in the pup viability for this group is within normal variation for this strain of rat. In the 2500 ppm dietary group, three dams lost their entire litter and one female lost half of her litter. Nearly all incidences of pups in this dietary group missing or found dead occurred by PND 1. Effects on viability were not observed at any other dietary level.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related clinical observations observed in either generation at any dietary level.

BODY WEIGHT (OFFSPRING)
F1-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. There were no test substance-related effects on pup body weight or body weight gain observed during the lactation period at any dietary level.
F2-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. There were no test substance-related effects on pup body weight or body weight gain observed during the lactation period at any dietary level.

SEXUAL MATURATION (OFFSPRING)
There were no test substance-related effects observed on either vaginal patency or balanopreputial separation for the pups from F1-generation at any dietary level. However, as the shift was noted in the number of days to passing sexual maturation criterion (longer time to passing than what is normally seen for the Wistar rat) in the test substance treated animals, anogenital distance was performed on the F2-pups. This shift is considered to be incidental to treatment with the test substance as it was observed at all dietary levels including control.

ANOGENITAL DISTANCE
There was no measureable difference on anogenital distance, compared to controls, at any dietary level for either males or females of the F2-generation.

ORGAN WEIGHTS (OFFSPRING)
There were no test substance-related changes in organ weights observed at any dietary level in either the F1- or F2-pups.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related gross necropsy findings observed at any dietary level in either the F1- or F2-pups.

HISTOPATHOLOGY (OFFSPRING)
There were no test substance-related microscopic findings for tissues examined at any dietary level in either the F1- or F2-pups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects noted up to and including 2500 ppm

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 41.3 mg/kg bw/day based on decreased viability index (missing/cannibalized and found dead pups) and decreased livebirth index (increased incidence of stillborn pups) of F2 pups

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Males: mean test substance intake during premating (in mg/kg bw/day)

	Male
	100 ppm in mg/kg bw/d*	500 ppm in mg/kg bw/d*	2500 ppm in mg/kg bw/d*
P	6.8	35.0	171.8
F1	6.3	32.4	158.9
Mean of both generations	6.6	33.7	165.4

*Individual values were based on the means for each particular phase

 

Table 2: Females: mean test substance intake during premating, gestation and lactation (in mg/kg bw/day)

Phase of study	Female
	100 ppm in mg/kg bw/d*	500 ppm in mg/kg bw/d*	2500 ppm in mg/kg bw/d*
P	8.3	40.2	200.5
F1	7.4	38.6	186.8
Mean of both generations	7.9	39.4	193.7
Gestation (P-gen) - Female	7.3	37.2	187.9
Gestation (F1-gen) - Female	7.0	36.6	179.1
Mean Gestation P and F1	7.2	36.9	183.5
Lactation (P-gen) - Female	8.3	41.3	203.9
Lactation (F1-gen) - Female	7.9	39.5	202.2
Mean Lactation P and F1	8.1	40.4	203.1

*Individual values were based on the means for each particular phase

                     

Table 3: Reproductive performance

Observation	Dose group (ppm)
	Control group	100 ppm	500 ppm	2500 ppm
P-Generation – F1-Offspring
Number Cohoused	30	30	30	30
Number Mated	28	29	27	29
Number of Animals Delivered	26	28	27	28
Number of Animals with Implants	27	28	27	28
Mating Index	93.3	96.7	90.0	96.7
Fertility Index	96.4	96.6	100.0	96.6
Gestation Index	96.3	96.4	100.0	100.0
Mean Number Days to Insemination (S.E.) Median	3.2 (0.56)       3.0	2.3 (0.23)       2.0	2.7 (0.56)       2.0	2.2 (0.23)       2.0
Mean Gestation Length (days) (S.E.) Median Gestation Length (days)	22.2 (0.11)     22.0	22.2 (0.12)     22.0	22.3 (0.12)     22.0	22.4 (0.13)     22.0
Total Number of Implantation Sites (Median)	302 (12.0)	319 (12.0)	322 (12.0)	331 (13.0)
F1-Generation – F2-Offspring
Number Cohoused	30	30	30	30
Number Mated	30	30	28	30
Number of Animals Delivered	30	30	27	28
Number of Animals with Implants	30	30	27	28
Mating Index	100.0	100.0	93.3	100.0
Fertility Index	100.0	100.0	96.4	93.3
Gestation Index	100.0	100.0	100.0	96.4
Mean Number Days to Insemination (S.E.) Median	2.3 (0.25)       2.0	2.4 (0.25)       2.0	2.7 (0.27)       3.0	2.6 (0.22)       3.0
Mean Gestation Length (days) (S.E.) Median Gestation Length (days)	22.1 (0.16)     22.0	22.2 (0.08)     22.0	22.1 (0.09)     22.0	22.5 (0.13)     22.5*
Total Number of Implantation Sites (Median)	332 (11.5)	330 (12.0)	318 (12.0)	310 (11.0)

* Statistically different from control, p ≤ 0.05

 

Table 4: Litter parameters

Observation	Dose group (ppm)
	Control group	100 ppm	500 ppm	2500 ppm
P-Generation
Total Number of Pups Born	295	286	313	317
Total Number Pups Missing/Litter	2/2	3/3	1/1	0/0
Total Number Pups Found Dead/Litter	5/3	0/0	2/2	1/1
Total Number Cannibalized/Litter	0/0	3/1	0/0	0/0
Number Stillborn	8	3	0	2
Sex Ratio Day 0 (% Male)	52.3	53.1	43.8	49.1
Mean Litter Size Day 0 Median	11.3   11.5	10.2   11.0	11.6   12.0	11.3   13.0
Birth Index	94.5	90.0	96.1	95.5
Live Birth Index	97.3	95.6	100.0	99.5
Viability Index	97.0	99.1	99.3	99.7
Lactation Index	99.5	100.0	99.5	100.0
F1-Generation
Total Number of Pups Born	318	314	302	288
Total Number Pups Missing/Litter	3/3	0/0	2/2	12/4
Total Number Pups Found Dead/Litter	3/3	0/0	3/3	3/3
Total Number Cannibalized/Litter	8/1	0/0	0/0	5/2
Number Stillborn	4	1	1	10
Sex Ratio Day 0 (% Male)	48.2	49.0	48.6	53.0
Mean Litter Size Day 0 Median	10.6   11.0	10.5   11.0	11.2   11.0	10.3   10.0
Birth Index	95.7	95.2	95.1	93.1
Live Birth Index	96.5	99.7	99.6	92.3
Viability Index	98.1	100.0	98.9	91.2
Lactation Index	99.6	100.0	99.1	99.5