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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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.

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

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.

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).

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: 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

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.

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

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

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

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

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

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

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

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.

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

Key result

Critical effects observed:

no

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

Reproductive effects observed:

not specified

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

 

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

41.3 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In accordance with OECD 416 (2001) and under GLP conditions, a two-generation reproductive toxicity study was performed in rats (M-445218-01-1). Triafamone was administered continuously in the food to rats (30 animals/dose level/sex) at dietary concentrations of 100, 500 and 2500 ppm. These doses corresponded to an achieved intake range of 6.3-6.8, 32.4-35.0, and 158.9-171.8 mg/kg bw/day in males during the premating phases and an achieved intake range of 7.0-8.3, 36.6-41.3 and 179.1-203.9 mg/kg bw/day in females during the premating, gestation and lactation phases. Body weight and food consumption determinations and detailed clinical examinations of each animal were conducted weekly throughout the study, as well as an evaluation of multiple reproductive parameters. In general, animals placed on study were subject to a postmortem examination, which included documenting and saving all gross lesions, weighing designated organs, and collecting representative tissue specimens for histopathological evaluation.

Effects attributed to exposure to Triafamone at the dietary dose level of 2500 ppm were as follows:

P-generation adults: Increased incidence of urine stain in dams. 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 in five dams during lactation. Urine stain was not considered to be adverse since there were no other observations or microscopic findings that could be correlated with this finding. An increase in absolute and relative liver weights was noted in males (6% and 8%, respectively) and females (12% and 10%, respectively). Minimal centrilobular hypertrophy of the liver in females was found, which correlated with the statistically significant increase in liver weights. In addition, findings in the thyroid glands consisting of colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) were observed.

F1-offspring: No test substance-related findings were observed.

F1-generation adults: Increased incidence of urine stain in dams. The increased incidence of urine stain was statistically significant during gestation (observed in 7 of 28 dams). Urine stain was seen in five dams, each 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. In male and female rats, a statistically significant increase in relative liver weights (7%, each) was noted. Colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) of the thyroid glands were found. Furthermore, a decrease in absolute and relative thymus weights (12% and 7%, respectively) in males (non-statistically decreased) and (10% and 8%, respectively) in females (statistically decreased) were observed. Thymic weight changes were considered non-adverse since the changes were subtle and there was a lack of dose-relationship in the relative thymic weights in males.

F2-offspring: Slight decreases in the live birth and viability indices were noted which are due to an increased incidence in the combination of stillborn, missing/cannibalized, and found dead pups.

Reproductive performance (P and F1): Slight statistical increase in median gestation length in F1-adults was found.

At the dietary dose level of 500 ppm colloid alteration (multifocal to diffuse) and follicular hypertrophy (multifocal to diffuse) of the thyroid glands were observed in the P- and F1-generation adults. In addition, in the F1-generation adults a non-statistical decrease in absolute and relative thymus weights (7% and 6%, respectively) was found in males. Thymic weight changes were considered non-adverse since the changes were subtle and there was a lack of dose-relationship in the relative thymic weights. No test substance-related findings were observed in F1- and F2 offsprings. Triafamone had no effect on the reproductive performance of the P- and F1- generation.

At the dietary dose level of 100 ppm, no test substance-related findings were observed neither in the P- or F1-generation nor in F1- or F2 -offsprings. The reproductive performance of the P- and F1-generation was not affected by Triafamone.

In conclusion, the parental systemic NOAEL is 100 ppm in both males and females (corresponding to 6.8 mg/kg bw/day and 8.3 mg/kg bw/day, respectively) 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. The reproductive NOAEL is greater than 2500 ppm for males and 500 ppm for females (> 171.8 mg/kg bw/day in males; 41.3 mg/kg bw/day in females) based on a slight but statistically significant increase in gestation length. The offspring NOAEL is 500 ppm (41.3 mg/kg bw/day) based on decreased viability (missing/cannibalized and found dead pups) and decreased livebirth index (increased incidence of stillborn pups).

Short description of key information:
2-generation toxicity study (OECD 416), rat:
NOAEL (fertility): > 171.8 mg/kg bw/day for males and 41.3 mg/kg bw/day for females
NOAEL (systemic): 6.8 mg/kg bw/day for males and 8.3 mg/kg bw/day for females
NOAEL (offspring): 41.3 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
The reliable GLP compliant OECD Guideline study was chosen.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414), rat: NOAEL (developmental) = 100 mg/kg bw/day; NOAEL (maternal) 20 mg/kg bw/day
Prenatal developmental toxicity (OECD 414), rabbit: NOAEL (developmental) >= 75 mg/kg bw/day; NOAEL (maternal) >= 75 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Aug 2011 - 19 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japenese Ministry of Agriculture, Forestry and Fisheries, notification 12 Nousan N°8147 (November, 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: New Zealand White Crl:KBL (NZW) rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Châtillon-sur-Chalaronne, France
- Age at study initiation: 18±1 weeks old
- Weight at study initiation: between 2.96 and 3.89 kg
- Housing: Individual housing of pregnant females in suspended stainless steel wire mesh cages.
- Diet: 110 C-10 pelleted animal diet from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) ad libitum.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 4 or 5 days prior to commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod: 16 h light/8 hrs dark ( 5am-9pm)

IN-LIFE DATES: From: 2011-08-04 To: 2012-04-19

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test item was suspended (w/w) in an aqueous solution of 0.5% methylcellulose 400 and stored at approximately 5 °C (±3 °C). Test formulations were prepared 6 times (F1 to F6) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in 0.5% aqueous methylcellulose was demonstrated in study SA 08145 at concentrations of 0.5 and 250 g/L for up to 29 days under similar conditions to those of the current study. Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (3.75 and 18.75 g/L). The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration (7.5 g/L) of the first formulation (F1) and all concentrations of the remaining formulations (F2 to F6) were checked. Data were recorded and analyzed using Empower 2 (Build 2154).
Results: Homogeneity and concentration analysis: 95 to 100% of nominal concentrations, which is within the in-house target range of 90 to 110% of nominal concentration. Therefore dose preparations were considered acceptable for use on this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Time-mated female rabbits were received from the supplier on GD 1 or GD 2. Nulliparous females were mated with stock males of the same strain and same supplier. The day of mating was designated as gestation Day 0 (GD 0).

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

daily

Duration of test:

On GD 29, all surviving females were sacrificed (34 days).

Remarks:

Doses / Concentrations:
15, 30, 75 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

animals assigned: 23
animals pregnant at start of treatment: 22, 22, 20, 21 (in the control group, in the 15, 30 and 75 mg/kg bw/d group, respectively)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses was selected based on the severity of maternal toxic effects obtained in a range-finding study (SA 08261), where groups of 8 presumed-pregnant rabbits were administered the test item by gavage at 0, 25, 75 or 200 mg/kg bw/day, from GD 6-28 inclusive. A dose level of 200 mg/kg bw/day resulted in marked maternal toxicity as evidenced by clinical signs, body weight losses, reduced food consumption and increased liver weight (+21%; p < 0.01). At 75 mg/kg bw/day, there was a slight maternal toxicity as evidenced by a slight reduction of 22% in body weight gain between GD 6-29 (not statistically significant). Mean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was slightly more pronounced than in the controls (-0.27 kg compared to -0.21 kg, not statistically significant) and was slightly outside the range of in-house HCD (-0.08 to -0.25 kg). At necropsy, mean liver weight was unaffected by the treatment. At the macroscopic examination, no treatment-related findings were noted. At 25 mg/kg bw/day, no treatment-related maternal findings were noted.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All cages were checked for dead or moribund animals twice daily, once in the morning and again in the afternoon (except at weekends and public holidays when checking was carried out once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. All animals were examined daily from GD 2-29.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD: 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION: Yes
- Full feeder weights were measured on GD: 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28
- Empty feeder weights were measured on GD: 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29
From these records the mean daily consumption was calculated. Food spillage was also noted.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29
- Organs examined: visceral organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes
- Individual weights of live fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group.
Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses.
Statistical analyses were performed on the following parameters using SAS programs (Version 9.2).
- Maternal endpoints: body weight changes calculated according to interval periods; calculated corrected body weight change (body weight data measured on different days throughout gestation were not statistically analyzed; only descriptive statistics are presented); average food consumption calculated according to interval periods, liver weight
- Litter based endpoints: number of corpora lutea; number of implantation sites; number of resorptions (early, late); pre- and post-implantation loss percentages; fetal body weight (combinded sexes and per sex)
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.
- Fetal endpoints: fetal sex; fetal death status
If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.
For fetal sex (male vs. female fetuses) and fetal death status (live vs. dead fetuses) endpoints, control group and each exposed group were compared using the Chi-square test for fetal sex parameter, using the Fisher Exact test (2-sided) for fetal death status parameter. Death status was analyzed both using the fetus as the statistical unit and using the litter as the statistical unit. Group means were compared at the 5% and 1% levels of significance.
In addition, statistical analysis was conducted on selected visceral and skeletal fetal observations.

Indices:

Pre-implantation loss
Post-implantation loss
Number of live fetuses per litter
Number of dead fetuses per litter

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There was no treatment-related mortality throughout the study.
One female in the control group was found dead on GD 7. At the macroscopic examination, the left lobes of the lungs were found collapsed. Thus, the death was attributed to a gavage error.
One animal treated at 75 mg/kg bw/day AE 1887196 aborted on GD 28 after having lost 250 g between GD 20-26 and 330 g between GD 26-28 (early necropsy). Food consumption was reduced between GD 20-26. There were no macroscopic findings at necropsy. In isolation and as there was no severe maternal toxicity noted in the other animals of this dose group, this abortion was not attributed to the treatment.

Clinical observation
At 75 mg/kg bw/day, 12/21 animals had few feces (compared to 6/23 in the controls) on several days.
The other clinical signs were those commonly encountered in pregnant rabbits or were observed with no dose-relationship.

Body weight
Up to the highest dose of 75 mg/kg bw/day, there were no treatment-related effects on mean maternal body weights, body weight gains and maternal corrected body weight change. One animal treated at 15 mg/kg bw/day had total litter resorptions (no live fetuses).

Food consumption
At 75 mg/kg bw/day, the mean food consumption of all pregnant females with live fetuses was slightly reduced by 13% between GD 6-8 (p < 0.05) and by approximately 10 % between GD 8-14 compared to the controls (not statistically significant). Thereafter, mean food consumption was unaffected by treatment. This effect was considered to be treatment-related, though it remained within the range of in-house Historical Control Data (HCD). Up to the dose of 30 mg/kg bw/day, mean food consumption was unaffected by treatment.

Gross pathology and organ weights
The few macroscopic findings observed occurred in isolation and/or with no doserelationship and were thus considered to be incidental.
At 75 mg/kg bw/day, the mean liver weight of pregnant females was increased by 8% when compared to the concurrent control group (not statistically significant). Individual values of liver weight from animals treated at 75 mg/kg bw/day ranged between 73-141 g compared to 73-117 g in the control group. There was no other change in mean liver weights at any dose level.

Histopathology
No microscopic changes were considered to be treatment-related

Cesarean section data
Pregnancy rate was unaffected by treatment. The pregnancy rate was 91% in the 30 and 75 mg/kg bw/day dose groups, 96% in the 15 mg/kg bw/day dose group and 100% in control group. Litter parameters including the number of live fetuses, the number of implant sites per dam, the percentages of pre and post implantation losses, the number of early and late resorptions, the fetal death status, the percentage of male fetuses, and the fetal body weight for combined and separate sexes were unaffected by treatment.
One animal treated at 15 mg/kg bw/day had a total litter loss (resorptions). This finding was considered incidental and not treatment-related.

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External observations (Table 3)
There were no treatment-related malformations or variations noted at the fetal external examination. The few variations observed were considered to be incidental, as they occurred in isolation and were well within the range of in-house HCD.

Visceral observations (Table 4)
There were no treatment-related malformations or variations noted at the fetal visceral examination. The visceral malformations observed in a total of 10 fetuses were distributed between the different doses groups including the control (1, 2, 5, 2 malformed fetuses at 0, 15, 30 and 75 mg/kg bw/day groups, respectively).
One fetus from the high dose group, 3 fetuses from the mid dose group, 2 fetuses from the low dose group and one fetus from the controls had cardiac malformations. One fetus from the high dose group and one from the medium dose group had retina fold unilateral. One fetus from the mid dose group had an absence of kidney. Since these findings were observed with no dose-effect relationship and as they were well within the in-house HCD, they were considered to have occurred spontaneously.
The incidences of the visceral variation “Thymic remnant present (uni/bi)” and “Ureter retrocaval (uni)” were higher at 75 mg/kg bw/day than in control group, at both litter and fetal levels. However, since the incidences were well within or at the border of the in-house HCD and since the differences from controls were not statistically significant; these findings were considered not to be treatment-related.
Other variations observed did not occur in a dose-related manner, were within the in-house HCD or occurred as isolated findings and were considered incidental.

Skeletal observations (Table 5)
There were no treatment-related malformations or variations noted at the fetal skeletal examination. Skeletal malformations were observed in a total of 8 fetuses from all dose groups including the control.
One fetus from each treated group had thoracic malformations. Two fetuses in two different litters from the high dose group had lumbar malformations. With the exception of hemivertebrae, each finding on these fetuses was however observed within the range of the in-house HCD. There were 2 cases of 1 lumbar hemivertebra (1.1% fetuses, 10% litters) compared to 1 case (0.5% fetuses, 4.2% litters) in the HCD. Therefore, and in the absence of any other treatment-related malformations in the skeletal axis, these malformations were considered to be incidental.
The incidences of the skeletal variations “Extra ossification point(s) (uni/bi) on 7th cervical vertebra” and “Insertion point(s) (uni/bi) of pelvic girdle on 2nd sacral vertebra” were higher in all treated groups than in the control group, at the litter and/or fetal level. However, as the values were not statistically different from controls, did not occur in a dose-related manner and were well within the in-house HCD, these findings were considered not to be treatment-related.
The incidence of the skeletal variation “13th thoracic rib (bi)” was higher at 30 and 75 mg/kg bw/day than in the control group, at both litter and fetal levels, and statistically significant (p < 0.01) only at the fetal level at the high dose group. However, the incidences were well within the in-house HCD, this finding was therefore considered not to be treatment-related.
Other variations observed did not occur in a dose-related manner, were within the in-house HCD or occurred as isolated findings and were considered incidental.

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Mean (±SD) Maternal Body Weight Gain (kg) of all pregnant females

Interval	Dose level of AE 1887196 in mg/kg bw/day
	0	15	30	75
Number of pregnant females at start of treatment	22	22(21)	21	20
Pretreatment, GD 3-6:	0.04 ± 0.060	0.05 ± 0.083 (0.05 ± 0.085)	0.04 ± 0.050	0.04 ± 0.058
Treatment, GD 6-8:	0.03 ± 0.034	0.03 ± 0.036 (0.03 ± 0.037)	0.01 ± 0.072	0.02 ± 0.035
Treatment, GD 8-10:	0.04 ± 0.033	0.04 ± 0.034 (0.04 ± 0.035)	0.04 ± 0.026	0.03 ± 0.023
Treatment, GD 10-14:	0.07 ± 0.047	0.09 ± 0.060 (0.09 ± 0.061)	0.08 ± 0.051	0.09 ± 0.041
Treatment, GD 14-18:	0.07 ± 0.051	0.04 ± 0.098 (0.05 ± 0.089)	0.08 ± 0.052	0.05 ±0.065
Treatment, GD 18-22:	0.05 ± 0.044	0.05 ± 0.041 (0.04 ± 0.037)	0.07 ± 0.040	0.04 ± 0.046
Treatment, GD 22-26:	0.05 ± 0.074	0.05 ± 0.061 (0.04 ± 0.059)	0.07 ± 0.046	0.05 ± 0.084
Treatment, GD 26-29:	0.06 ± 0.038	0.08 ± 0.044 (0.08 ± 0.045)	0.08 ± 0.045	0.06 ± 0.051
Treatment, GD 6-29:	0.37 ± 0.129	0.36 ± 0.148 (0.36 ± 0.151)	0.43 ± 0.113	0.36 ± 0.151
Corrected BW gain	-0.17 ± 0.121	-0.16 ± 0.154 (-0.16 ± 0.154)	-0.11 ± 0.158	-0.15 ± 0.147

SD: standard deviation

One animal treated at 15 mg/kg bw/day had total litter loss. For this reason, the figures in parentheses are the means calculated once the data for this female are excluded.

 

Table 2: Cesarean Section Observations

Observation	Dose Level of AE 1887196 (mg/kg bw/day)
	0	15	30	75
Maternal data:
No. Animals assigned	23	23	23	23
No. Animals pregnant	23	22	21	21
Pregnancy rate, %	100	96	91	91
No. Animals non-pregnant	0	1	2	2
Uterine data at scheduled sacrifice:
Total No. corpora lutea (c)	256	253	242	226
Total No. Implantations (c)	229	217	203	194
Total No. Litters (c)	22	22	21	20
Total No. live fetuses (c)	207	196	190	178
Total No. dead fetuses	6	9	4	8
Total No. early resorptions (c)   Total No. late resorptions (c)   Litters with total resorptions (c,d)	13     3     0	12 (7)     0     1	8     1     0	6     2     0
Mean fetal weight, combined sexes [g]	39.36 ± 4.17	38.87 ± 4.17	41.37 ± 2.86	37.84 ± 3.30
Sex ratio, % male	44.0 ± 21.6	47.2 ± 17.2	49.4 ± 19.3	54.0 ± 20.8
Preimplantation loss per litter, %	10.55 ± 13.07	13.46 ± 15.49 (14.10 ±15.58)	15.65 ± 15.17	14.16 ± 10.51
Postimplantation loss per litter, %	9.2 ± 8.8	10.8 ± 21.4 (6.6 ± 8.1)	5.9 ± 8.0	9.1 ± 16.0

(c) statistical analysis was not conducted on this endpoints

(d) Also includes litters with dead fetuses only or dead fetuses and resorptions

The figures in parentheses are the means calculated once the data for the animal having total litter loss are excluded.

 

Table 3: External examinations

Dose level [mg/kg bw/day]	0	15	30	75	Historical control range	0	15	30	75	Historical control range
Observations	Number of litters examined					Number of fetuses examined
	22	21	21	20		207	196	190	178
	Number of litters affected (Percentage of litters affected)					Number of fetuses affected (Percentage of fetuses affected)
Malformations Omphalocele	0 (0.0)	0 (0.0)	0 (0.0)	1 (5.0)	(0.0-4.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.6)	(0.0-0.5)

 

 

Table 4: Visceral examinations

Dose level [mg/kg bw/day]	0	15		30		75	HCR	0	15	30	75	HCR
Observations	Number of litters examined							Number of fetuses examined
	22	21		21		20		207	196	190	178
								Number of heads examined
								97	92	90	85
	Number of litters affected (Percentage of litters affected)							Number of fetuses affected (Percentage of fetuses affected)
Variations
#Thymic remnant present (uni/bi)	11 (50.0)	7 (33.3)	6 (28.6)		12 (60.0)		(20.8 - 84.2)	25 (12.1)	10 (5.1)	8 (4.2)	25 (14.0)	(3.8 - 23.9)
#Ureter (uni): retrocaval	2 (9.1)	0 (0.0)	1 (4.8)		2 (10.0)		(0.0 - 18.2)	3 (1.4)	0 (0.0)	1 (0.5)	5 (2.8)	(0.0 - 2.3)

# : Statistical analysis was conducted on this observation

HCR: historical control range

 

Table 5: Summary of skeletal malformations

Dose level [mg/kg bw/day]	0	15				30		75		0	15		30		75
	Number of litters examined									Number of fetuses examined
	22		21		21				20	207		196		190		178
	Number of litters examined									Number of heads examined
	22		21		21				20	97	92		90		85
Malformations	Number of litters affected									Number of fetuses affected
One thoracic vertebra : hemivertebra. One rib (uni) : absent	1	0		0			0			1	0		0		0
Two ribs (uni) : fused. Two thoracic centrum : hemicentrum and fused	0	1		1			0			0	1		1		0
One lumbar vertebra : hemivertebra. One lumbar hemicentrum and one lumbar centrum : fused	0	0		0			2			0	0		0		2
Caudal and all sacral vertebrae : fused. Caudal vertebrae : absent	1	0		0			0			1	0		0		0
All caudal vertebrae (except 1st): fused	0	0		1			0			0	0		1		0

 

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

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

According to OECD 414 (2001) and under GLP conditions, two studies were performed to assess the potential effects of Triafamone on the pre-natal development in rats and in rabbits.

Groups of 23 sperm-positive female rats were exposed to Triafamone by oral gavage from gestation day (GD) 6 to 20 (M-428307-01-1). The doses given were 20, 100 and 600 mg/kg bw/day in suspension in aqueous solution of 0.5% methylcellulose 400. Clinical observations, maternal body weights and food consumption were recorded during the treatment period. At scheduled sacrifice, on GD 21, a macroscopic examination of the visceral organs was performed, the gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). In addition, the liver was weighed at scheduled sacrifice for all pregnant females and histological examinations were performed on the liver in all groups. Live fetuses were removed from the uterus, counted, weighed, sexed and examined externally.

Pregnancy rate was unaffected by treatment. There was no treatment-related mortality throughout the study. Litter parameters, including the number of live fetuses, the number of implant sites per dam, the percentages of pre and post implantation losses, the numbers of early and late resorptions, the fetal death status, the fetal body weight and the percentage of male fetuses, were unaffected by treatment. There were no treatment-related malformations or external findings observed at the fetal examination.

At 600 mg/kg bw/day: Two animals (out of 23) had soiled anogenital region for several days. Between GD 6-8, the mean maternal body weight gain of all pregnant females was reduced by 67% (not statistically significant) and the mean food consumption was reduced by 9% (p 0.05), compared to the control group. Thereafter the body weight parameters and food consumption were unaffected by treatment. At necropsy, 7/23 animals had enlarged liver and prominent lobulation and the mean liver weight of pregnant females was increased by 17% (p < 0.01), when compared to the concurrent control group. Microscopically, minimum to slight centrilobular hepatocellular hypertrophy was observed in the liver of 22/23 animals.

At the visceral fetal examination, the incidence of the variation “Thymic remnant present (uni/bi)” was higher than in the control group and was outside the in-house historical control data (HCD) at both litter and fetal levels. At the skeletal fetal examination, the incidences of the variations “Interparietal: split”, “Frontal: split”, “Supraoccipital: incomplete ossification”, “5th and/or 6th sternebrae: incomplete ossification” and “13th rib (uni/bi) short/costal cartilage absent” were higher than in the control group at both litter and fetal levels and were outside the in-house HCD.

At 100 mg/kg bw/day: There was no treatment-related effect on in-life parameters of the dams. At necropsy, 4/23 dams had enlarged livers and 1/23 animals had prominent lobulation. Microscopically, minimal centrilobular hepatocellular hypertrophy was observed in the liver of 9/23 animals.

There was no treatment-related effect on litter or fetal parameters.

At 20 mg/kg bw/day: No treatment-related maternal findings were noted. There were no treatment-related effects on litter or fetal parameters.

In conclusion, Triafamone resulted in maternal toxicity, as evidenced by effects on body weight, food consumption and histological changes in the liver. Increased incidences of a few visceral and skeletal fetal variations were observed. Overall, Triafamone did not show evidence of a teratogenic potential in the rat up to 600 mg/kg bw/day, which was the highest dose level tested. A dose level of 20 mg/kg bw/day was considered to be a NOAEL in terms of maternal toxicity, whilst a dose level of 100 mg/kg bw/day was considered to be a NOAEL for developmental toxicity.

Developmental toxicity was also examined in rabbits (M-431539-03-1). Groups of 23 time-mated pregnant female New Zealand White rabbits were exposed to Triafamone by oral gavage from gestation day (GD) 6-28. The selected doses were 15, 30 and 75 mg/kg bw/day suspended in aqueous solution of 0.5% methylcellulose 400.

Clinical observations, maternal body weights and food consumption were recorded during the treatment period. At scheduled sacrifice, on GD 21, a macroscopic examination of the visceral organs was performed, the gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). In addition, the liver was weighed at scheduled sacrifice for all pregnant females and histological examinations were performed on the liver in all groups. Live fetuses were removed from the uterus, counted, weighed, sexed and examined externally.

At scheduled sacrifice, on GD 29, a macroscopic examination was performed, the gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). In addition, the liver was weighed at scheduled sacrifice from all pregnant females. Histological examination was performed on the liver in the control and high dose groups. Live fetuses were removed from the uterus, counted, weighed and examined externally.

Up to the highest dose of 75 mg/kg bw/day, there were no treatment-related effects on mortality, mean maternal body weights, body weight gains, maternal corrected body weight change. There were no treatment-related macroscopic and microscopic changes on the liver. Pregnancy rate was unaffected by treatment: 91% in the 30 and 75 mg/kg bw/day dose groups, 96% in the 15 mg/kg bw/day dose group and 100% in control group.

Litter parameters including the number of live fetuses, the number of implant sites per dam, the percentages of pre and post implantation losses, the number of early and late resorptions, the fetal death status, the percentage of male fetuses, and the fetal body weight for combined and separate sexes were unaffected by treatment. There were no treatment-related malformations or variations noted at the fetal external, visceral and skeletal examinations.

Overall, Triafamone did not show evidence of teratogenic potential in the rabbit up to 75 mg/kg bw/day, which was the highest dose level tested. The NOAEL for both maternal and developmental toxicity was considered to be equivalent to 75 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
The selected study is the most adequate and reliable study with the lowest dose descriptor.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.