Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Principles of method if other than guideline:
The study is a two generation study also designed to assess subchronic exposure of the test material.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD (SD) IGS BR strain
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited. Animals were inspected and acclimatised for fifteen days during which time their health status was assessed. At the start of the experiment males weighed 180 to 255 g and the females weighted 145 to 196 g and were approximately six to eight weeks old.

Animals were housed in groups of four in cages meeting criteria. During the mating phase, animals were transferred to similar cages on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually, during gestation and lactation.

Animals were allowed fee access to food and water. The rate of air exchanges was at least 15 air changes per hour with low intensity flourescent lighting to allow for 12 hours light/dark cycle. The temperature and relative humidity controls were set to achieve target values of 21 + or - 2 degrees C and 55 + or - 15%, respectively. Deviations from relative humidity targets were considered not to affect the purpose or integrity of the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material was incorporated into the diet by mixing with a small amount of basal laboratory diet for 19 minutes at a constant speed. The pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further 30 minutes at a constant speed.
Details on mating procedure:
F0 and F1 animals were paired, following subsequent maturation periods, on a 1 male: 1 female basis within each does group, for a period of up to 21 days. Cage trayliners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Each pregnant female was observed at 0830, 1230 and 1630 hours and around the period of expected parturition. On Weekends and Holidays, observations were carried out at approx 0830 and 1230 hours. The following was recorded for each female: date of mating; date and time of observed start of parturition; date and time of observed completion of parturition; and duration of gestation.

On completion of parturition the number of live and dead offspring were recorded. The date and time of Day post partum litter observations were standardised. For each litter the following was recorded: number of pups born; number of pups alive recorded daily and reported on Day 1, 4, 7, 14, 21 post partum. On days 1 and 21 the sex of the individual offspring were recorded; clinical condition of pups from birth to weaning; individual pup and litter weights on Day 1, 4, 7, 14 and 21 post partum; and necropsy findings.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the dietary admixtures was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.
Frequency of treatment:
The test material was administered daily by dietary admixture. Control animals were treated in an identical manner with untreated laboratory diet.
Details on study schedule:
Pretreatment ophthalmoscopic and behavioural assessments were performed on the ten randomly F0 selected males and females from control and treatment groups.
Groups of 28 F0- male and 28 F0 female animals were treated at the appropriate dose level throughout the treatment period.
The ten selected F0 males and ten F0 females from each treatments and control group, were observed for signs of functional/behavioral toxicity once weekly.
A vaginal smear was prepared daily for 21 days prior to pairing for all F0 females.
During week 10, the ten F0 selected animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli, grip strength and motor activity. The ten selected F0 males and F0 females were also subjects to ophthalmoscopic examination and blood sampling for haematological and blood chemical analysis. The animals were not fasted prior to sampling.
On day 74, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of 21 days.
Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
Pregnant F0 females were allowed to give birth and maintain their offspring on the appropriate diet until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight, clinical observations, developmental landmarks and reflexological signs were also performed during this period.
At Day 21 post partum, where ever possible, one male and one female offspring from the F0 mating phase were selected to form the next generation (F1). Surviving adult F0 females and unselected offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 females and selected organs were weighed for one unselected male offspring and one unselected female offspring.
Following completion of the F0 female gestation and lactation phases, the F0 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
Selected F1 males and F1 females were treated at the appropriate dose level for ten weeks during which they were evaluated for evidence of sexual maturation.
A vaginal smear was prepared daily for 21 days prior to paring for all F1 females.
All F1 animals were paired on a 1 male:1 female basis within each dose group for a max of 21-days.
Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
Pregnant F1 females were allowed to give birth and maintain their offspring on the appropriate diet until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight, ano-genital distance, clinical observations, developmental landmarks and reflexological signs were also performed during this period.
At Day 21 post partum, all surviving F1 females and offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 females and selected organs were weighed for one male and one female offspring.
Following completion of the F1 female gestation and lactation phases, the F1 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
Remarks:
Doses / Concentrations:
3000 ppm (250 mg/kg)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6000 ppm (500 mg/kg)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
12000 ppm (1000 mg/kg)
Basis:
nominal in diet
No. of animals per sex per dose:
F0 generation: 28 males/28 females
F1 generation: 24 males/24 females
Control animals:
yes, plain diet
Details on study design:
The test material was administered by dietary ad mixture to three groups each of twenty-eight male and female F0 Generation Sprague-Dawley Crl:CD (SD) IGS BR strain rats, at dietary concentrations of 3000, 6000, and 12000 ppm (equivalent to a mean achieved dosage of 250, 500 and 1000 mg/kg/day, respectively).

A further group of 28 male and female F0 Generation was exposed to basal laboratory diet to serve as control.Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. During Week 10, extensive functional observations and ophthalmoscopy examinations were performed on test selected animals of either sex from each dose group, together with haematology and blood chemistry assessment.After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum, followed by the termination of all F0 male dose groups. The offspring selected for the F1 generation were dosed for at least 10 weeks and the paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving F1 females and their offspring were killed, followed by termination of all F1 male dose groups.Oestrous cycle assessment was performed daily for three weeks prior to mating. Observations for positive evidence of mating were recorded together with the start and completion of parturition. During the maturation phase of the F1 generation offspring, males and females were evaluated for sexual maturation. The ano-genital distance was recorded for all F2 generation offspring on Day post partum. During the lactation phases daily clinical observations were performed on all surviving offspring, together with litter size. Litter weight, individual offspring weights and landmark developmental signs were also recorded on specific days post partum.All animals at termination were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Positive control:
Not applicable.
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded for F0 males on Day 0 and then weekly for F0 and F1 males until termination. Fo and F1 females were weighed weekly until mating was evident. Bodyweights for females showing evidence of mating were recorded on Days 1, 4, 7, 14 and 21 post coitum. Females with live litters were weighed on Days 1, 4, 7, 14 and 21 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes

Oestrous cyclicity (parental animals):
Oestrous Cycle: Each of the following stages were evaluated and classified as follows:
Di-oestrus (D) - Predominately leucocytes present although some epithelial and cornified cells can be seen.
Pro-oestrus(P) - Predominately epithelial cells, usually in significant numbers.
Early Oestrus (E1) - Predominately cornified cells, usually seen as small groups or isolated cells.
Late Oestrus (E2) - Predominately cornified cells usually seen as clumps of cells.
Met-oestrus (M) - Large number of leucocytes with discrete clumps of cornified cells.
The oestrous cycles are classified according to the following criteria:
Regular oestrous - The pattern of daily stages of oestrous show a four to five day cycle that is generally repeated over 21 days.
Extended oestrous - The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous - Where the predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle- An irregular length of oestrous cycle is observed over the 21 day evaluation period.
Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.
Sperm parameters (parental animals):
Sperm Analysis: Sperm motility characteristics were recorded for individual sperm analysis:
Concentration (millions/ml): Based on total number of motile and non-motile cells identified.
Motility: Is the ratio of recorded motile cells to the total concentration.
Progressive Velocity (VSL): Is the straight line distance between the beginning and end of the track divided by time elapsed.
Trackspeed (VSL): Is the total distance between each CB (Centre of Brightness of the sperm head) position for a given cell during image acquisition.
Path Velocity (VAP): Is the smoothed average position of the CB and gives an average cell path velocity.
Straightness (Str): Measures the departure of a cell path from a straight line.
Linearity (LIN): Measures the departure of a cell track from a straight line.
Amplitude of a lateral head displacement (ALH): Corresponds to the mean width of the head oscillation as the cell swims.
Beat cross frequency (BCF): Is determined by measuring the frequency with which the cell track crosses the cell path in either direction.

The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approx - 20C. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. A minimum of 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
A sample of semen was preserved in formalin and then stained with eosin. A sub-sample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed manually.
The cauda epididymis was separated from the body of the epididumis, and then weighed. The cauda epididymis was then frozen at approximately -20 deg C. At an appropriate later data the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Sample of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, clinical condition, weight gain
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the F0 female gestation and lactation phases.
- Maternal animals: At Day 21 post partum.

GROSS NECROPSY
- All adult animals including those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs, removed from the ten selected F0 males and F0 females from each treatment group that were killed at the end of the study, were dissected free from fat and weighed before fixation. In addition, the reproductive organs were weighed from all F0 animals:
Adrenals, Brain, Epididymides, Heart, Kidnenys, Liver, Spleen Testes, Thymus, Thyroid gland, Ovaries, Pituitary, Prostate, Seminal vesicles (with coagulating gland), Uterus (with cervix).

Samples of the following tissues were preserved from the ten selected F0 males and females from each dose group, in buffered 10% formalin.
Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Eyes (preserved in Davidson's fluid), Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi (lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative)), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Oesophagus, Pancreas, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder.

The following tissues were also removed from the remaining animals:
Adrenals, Coagulating gland, Right epididymis (preserved un Bounis fluid and then in 70% IMS after 48 hours), Ovaries, Pituitary, Prostate, Seminal vesicles. Right testes (preserved in Bouins fluid and then in 70% after 48 hours), Uterus/Cervix/Oviducts, Vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All adult animals including those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following organs, removed from the ten selected F0 males and F0 females from each treatment group that were killed at the end of the study, were dissected free from fat and weighed before fixation. In addition, the reproductive organs were weighed from all F0 animals:
Epididymides, Testes, Ovaries, Pituitary, Prostate, Seminal vesicles (with coagulating gland), Uterus (with cervix).

Samples of the following tissues were preserved from all F1 males and F1 females from each dose group, in buffered 10% formalin:
Adrenals, Bone & bone marrow (femur including stifle joint), Coagulating gland, Right Epididymide (preserved in Bouins fluid and then in 70% after 48 hours), Kidneys, Mesenteric lymph nodes, Ovaries, Pituitary, Prostate, Seminal vesicles, Right teste (preserved in Bouins fluid and then in 70% after 48 hours), Thymus, Uterus/Cervix/Oviducts, Vagina.
Statistics:
Data were processsed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.

Non-parametric methods were also used to analyse implantation loss, offspring sex ration and developmental landmarks and reflexological responses.

The haematology variable basophils was not analysed since consistently greater than30% of the data were recorded as the same value.

Histopathological data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes: 1) Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater; 2) Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
Please see 'Any other information on materials and methods' for details.

Offspring viability indices:
Gestation and Parturition Data: The following parameters were calculated for individual data during the gestation and parturition period of the F0 and F1 generation: Gestation length and parturition index.

Lactation Data: Live Birth and Viability Indices were calculated for each group from group mean data.
Sex ratio (% males) were calculated based on group mean values from each litter value on Day 1 and 21. Implantation losses (%): Group mean percentile pre-implantation and post implantation loss were calculated.

Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observable signs of toxicity were detected in test of control animals throughout the study period.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic abnormalities were detected at terminal kill.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no treatment related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
A statistically significant reduction was evident in spermatid count for 12000 ppm males, however in the absence of any histopathological correlates the intergroup difference was considered of no toxicological significance.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment related effects on mating performance or fertility.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female treated with 12000 ppm was killed on humane grounds on Day 40 due to a damaged tail tip. This death was a result of physical injury and unrelated to test material toxicity.

One male treated with 3000 ppm was found dead on Day 11 and showed no clinical signs prior to death. The observed histopathology findings did not indicate any obvious cause of this death and while the aetiology of this is uncertain, there were no similar mortalities at this or the higher dose levels in either generation and in isolation, it was considered not to be toxicologically significant. There were no further unscheduled deaths.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males treated with 1200 ppm showed a statistically significant reduction in bodyweight gain compared to concurrent control animals during the first 8 weeks of treatment. Thereafter, weekly gains were frequently lower than controls, although differences were not as marked and failed to attain statistical significance. Females from this treatment group were also adversely affected. Slightly lower bodyweight gains were detected during the first four weeks of treatment compared to control animals with differences attaining statistical significance during Week 2. Subsequent bodyweight gains for the remaining pre-pairing phase were essentially similar to controls.

No such toxicological significant effects were detected in animals of either sex treated with 6000 or 3000 ppm.

Males treated with 6000 ppm showed a reduction in bodyweight gain during Week 8 only. In isolation this intergroup difference is considered of minimal toxicological importance.

Females treated with 3000 ppm showed an increase in bodyweight gain during Week 4 only. There was no dose-related response for this finding and in any event increases in bodyweight gains are unlikely to be indicative of test material toxicity.

Gestations bodyweight:
Females treated with 12000 ppm showed a reduction in bodyweight gain during the first week of gestation, although subsequent bodyweight gain during week two was similar to control animals. During the third week of gestation bodyweight gain was again notably lower than controls, with differences attaining statistical significance.

No such effects were detected in females treated with 6000 or 3000 ppm.

Lactation bodyweight:
Bodyweight gain during the first two weeks of lactation was notably lower than concurrent control animals for females treated with 12000 ppm, however, statistical significance was only obtained during Week 1.

No such effects were detected in females treated with 6000 or 3000 ppm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The distribution of pre-coital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing. The distribution of animals with pre-coital intervals in excess of four days showed no treatment related trends. One female in the 3000 ppm treatment group and two females in the 12000 ppm dose group failed to achieve pregnancy. In the absence of a convincing dose related response, these were considered low incidence normal findings observed for mated rats of the stain employed and considered unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Females treated with 12000 ppm showed statistically significant reductions in thymus, ovary and uterus weight both absolute and relative to bodyweight. The effect on ovary, uterus and thymus (absolute) weight extended to the 6000 ppm treatment group. Females treated with 12000 ppm also showed an increase in relative kidney weight.

No such treatment effects were detected in males treated with 12000 ppm or animals of either sex treated with 3000 ppm.

Animals of either sex treated with 12000 ppm showed a reduction in absolute adrenal, liver, pituitary and spleen weight while males from this treatment showed a reduction in absolute kidney weight. There were no histological correlates and in the absence of a similar reduction in the weight of these organs when expressed relative to terminal bodyweight, the intergroup differences were considered attributable to the bodyweight effect seen in this study and not a direct effect of treatment. Animals of either sex treated with 12000 ppm showed an increase in relative testis, epididymis and caudal weight while females showed increases in relative brain and liver weight. The effect on epididymides extended to 6000 ppm males. In the absence of any histopathological correlates these findings were considered to be without toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Isolated incidents of red lungs, small testes, epididymides or seminal vesicles, blue testes and fluid filled kidneys were detected for a number of treated males. A number of females from control and treated groups showed isolated incidents of enlarged/red lungs, dark adrenals, hard/dark liver, small/thin or encased spleen and a mass on the upper chest. Such findings are consistent with normally expected low incidence findings in laboratory maintained rats of the strain and aged used and are of no toxicological importance. One male treated with 3000 ppm showed numerous red lesions along the length of the gastro-intestinal tract, a severely enlarged liver and spleen, pale lungs, pancreas, pituitary and stomach, small prostate, red/enlarged mesenteric lymph nodes and a nodular appearance on the thymus. Histopathologically these organs were shown to have malignant lymphomas. This finding is a low incidence finding in laboratory maintained rats of the stain and age used and is considered unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Kidneys:
Basophilia of renal tubules was observed in relation to treatment for females treated with 12000 ppm (P <0.001). Males were not similarly affected. Accumulations of yellow pigment were observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.01 for males and P <0.05 for females). The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin.

Mesenteric lymph nodes:
Sinus histiocytosis was observed as a treatment related effect in females treated with 12000 ppm (P <0.01) or 6000 ppm (P <0.05) dose levels. Male rats were not similarly affected.

Thymus:
A greater incidence of higher grades of severity lymphoic atrophy of the thymus was seen for females treated with 12000 ppm (P<0.05).

Bone Marrow:
Generally higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen in relation to treatment for females only treated with 12000 ppm. Although this condition did not attain statistical significance the overall picture suggested a probable if marginal effect of treatment for higher dose females only.

Ovaries:
Fine cytoplasmic vacuolation of interstitial cells were observed with lower incidence among animals treated with 12000 ppm (P <0.001) or 6000 ppm (P <0.05) of the test material. The significance of this change is uncertain. A greater incidence of higher grades of severity of yellow pigment accumulation was also observed as an effect of treatment for females treated with 12000 ppm (P <0.01). The pigment was demonstrated to be haemosiderin by Perl's staining technique.

Dose descriptor:
NOEL
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive: Reproductive screening: litter size
Dose descriptor:
NOEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic effects Body weight Food consumption Haematology Organ weights Histopathology: Kidneys Mesenteric lymph nodes Thymus Bone marrow Ovaries
Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical findings were observed.
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic abnormalities were selected at terminal kill.
Histopathological findings:
effects observed, treatment-related
VIABILITY (OFFSPRING)
In total there were 28, 27, 28 and 25 females at 0 (control), 3000, 6000 and 12000 ppm, respectively, who gave birth to a live litter and successfully reared young to weaning (Day 21 of age) and have been included in the following assessment of litter responses.

The mean numbers of corpora lutea observed for treated F0 females were considered to be acceptable and did not indicate any adverse effect of dietary exposure at 3000, 6000 or 12000 ppm. Although values for treated groups were slightly lower than control animals, attaining statistical significance at both 3000 and 12000 ppm, there was no clear dose related response and these differences were considered to represent normal biological variation rather than an effect of maternal exposure to the test material. Subsequent pre and post implantation losses for treated animals were essentially similar to controls however the resultant mean number of implantations and litter size at birth observed for 12000 ppm females were lower than concurrent control animals. Following birth, live birth index for 12000 ppm females was lower than controls, suggesting increase mortality among the F1 offspring prior to Day 1 of age; however subsequent offspring viability throughout lactation was similar to controls.

No such effects were detected in 6000 or 3000 ppm females. Sex ratio at birth for live offspring at Day 1 of age and at weaning (Day 21) was similar in all treatment groups and did not indicate any selective effects for either sex.

BODY WEIGHT (OFFSPRING)
Mean offspring bodyweights on Day 1 of age were unaffected by maternal exposure in females treated with 12000 ppm. Subsequent bodyweight gain of the offspring to weaning was progressively lower than concurrent control animals, with differences being particularly marked between Days 14 and 21 of lactation. Mean offspring bodyweights at this treatment group were significantly lower than control animals from Days 14 of age and this, combined with the slightly lower litter size, resulted in statistically differences in mean litter weight from Day 7 onwards.

No such effects were detected in females treated with 6000 or 3000 ppm.

Inter-group differences in offspring maturation (as assessed by the onset and completion of pinna unfolding, incisor eruption and eye opening) and reflexological assessments (percentage successful at surface righting, mid-air righting and pupil and startle reflex) did not indicate any adverse effects of dietary exposure at 3000, 6000 or 12000 ppm. These findings suggest that the impaired growth observed for offspring at 12000 ppm did not affect the development of the offspring.

SEXUAL MATURATION (OFFSPRING)
Animals of either sex treated with 12000 ppm showed a statistically significant increase in age at completion of sexual maturation together witha reduction in bodyweight (males only) at sexual maturation compared to control animals.

No such treatment-related effects were detected in animals of either sex treated with 6000 or 3000 ppm.

Males treated with 6000 ppm showed a statistically significant reduction in bodyweight at sexual maturation. In the absence of any associated effect on the age at sexual maturation the intergroup difference was considered to be of no toxicological importance.

ORGAN WEIGHTS (OFFSPRING)
Animals of either sex treated with 12000 ppm showed a statistically significant increase in kidney weight relative to bodyweight. The effect on relative kidney weight extended to 6000 and 3000 ppm males. A statistically significant reduction in absolute uterus weight was also detected at 12000 ppm.

The remaining statistically significant intergroup differences in the adrenals, brain, heart, kidneys, liver, prostrate, pituitary, reproductive organs, spleen, thymus and thyroid detected throughout the treatment groups were not associated with any histological correlates and were considered attributable to the bodyweight effect seen in this study and not a direct consequence of treatment.

HISTOPATHOLOGY (OFFSPRING)
Kidneys:
Basophilia of renal tubules was observed in relation to treatment for females treated with 12000 ppm (P <0.001). Male rats were not similarly affected. Accumulations of yellow pigment were observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.001), and for males treated with 6000 ppm (P <0.05) and at 3000 ppm (P <0.05), although there was no dose response and the effect must be regarded as being of marginal toxicological significance for these two lower dose levels. The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin.

Mesenteric lymph node:
A greater incidence of accumulations of yellow pigment was observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.001 for males and P <0.05 for females). The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin. For females only, sinus histiocytosis was also observed as a treatment-related effect at 12000 ppm (P <0.01) and at 6000 ppm (P <0.05).

Thymus:
A greater incidence and generally higher grades of severity of lymphoid atrophy were seen for females treated with 12000 ppm (P <0.001), 6000 ppm (P <0.01), or at 3000 ppm (P <0.05). Male rats were not similarly affected.

Bone marrow:
Generally higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen in relation to treatment for females only treated with 12000 ppm (P <0.001). An opposite effect was observed for males at this dose level and reached statistical significance but this was probably an anomaly.

Ovaries:
Fine cytoplasmic vacuolation of interstitial cells was observed with a lower incidence among females treated with 12000 ppm (P <0.001) or at 6000 ppm (P <0.01).

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Dose descriptor:
NOEL
Generation:
F1
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity: Offspring growth and development Sexual Development A NOEL for systemic toxicity was not achieved in the F1 generation Histopathology: Kidney Mesenteric lymph node Thymus Bone marrow Ovaries
Reproductive effects observed:
not specified

No clinically observable signs of toxicity were detected in treatment groups from either the F0 adults or F1 adults throughout the study period. Bodyweight development, dietary intake and food utilisation were however adversely affect in 12000 ppm adult animals in both the F0 and F1 generation, resulting in a deterioration in physical condition of the animals. Haematological investigations of selected F0 adult animals revealed a normocytic, normochromic anaemia. This was probably associated with the bone marrow changes seen microscopically. Changes were identified as higher grades of severity of adipose infiltration of the marrow, indicating hypoplasia for adult females treated with 12000 ppm from either generation. Absolute and relative thymus weight was reduced in adult females from either generation at this dose level and microscopic examination of thymus sections revealed changes identified as a greater incidence of higher grades of severity of lymphoid atrophy. Sinus histocytosis was evident microscopically in the in the mesenteric lymph nodes for 12000 ppm adult females from either generation together with accumulations of yellow pigment also being observed in 12000 ppm adult females from the F1 generation only. Such effects in the thymus and mesenteric lymph nodes may possibly be associated with the reduced physical conditions seen in these animals, however the latter condition seen in 12000 ppm F1 adult females in the mesenteric lymph nodes may simply represent the accumulation of the coloured test material or coloured metabolite(s) and as such is of minimal toxicological significance.

Relative kidney weights were elevated for the F0 adult females treated with 12000 ppm and for F1 adult animals of either sex treated with 12000 ppm. Microscopic examination of kidney sections revealed basophilia of renal tubules in F0 and F1 adult females at this dose level. Further renal microscopic changes were evident in adult animals of either sex from both the F0 and F1 generations and were identified as accumulations of yellow pigment.

Microscopic changes were also identified in the ovaries. A lower incidence of fine cytoplasmic vacuolation of interstitial cells was observed for both F0 adult and F1 adult females treated with 12000 ppm and a greater incidence of higher grades of severity of yellow pigment accumulations (identified as haemosiderin) for F0 adult females at this dose level. Organ weight data supported these findings with reductions in absolute and relative ovary weight for adult females from the parental generation at this dose level. A reduction in absolute and relative uterus weight was also evident in these animals.

Toxicologically significant effects extended to the 6000 ppm dose group. F0 adult males showed a reduction in bodyweight gain and food consumption during maturation albeit to a lesser extent. F1 adult animals however showed a similar reduction in bodyweight gain and food consumption as observed in the 12000 ppm dose group.

A reduction in ovary, uterus and thymus weight was evident in F0 adult females together with the microscopic changes of sinus histocytosis in the mesenteric lymph nodes and a lower incidence of fine cytoplasmic vacuolation of intestinal cells in the ovaries. F1 adult females showed a reduction in absolute ovary weight together with microscopic changes of sinus histocytosis in the mesenteric lymph nodes and a lower incidence of fine cytoplasmic vacuolation of interstitial cells in the ovaries, while F1 adult males showed an increase in relative kidney weight together with microscopic renal changes of accumulation of yellow pigment.

Results for the functional observation are reported separately under Specific Investigations.

Changes detected in 3000 ppm were confined to the F1 adult animals. Adult males showed statistically significant increase in relative kidney weight together with microscopic changes of accumulations of yellow pigment in the kidneys while adult females showed a reduction in absolute ovary weight and a greater incidence and higher grades of severity of lymphoid atrophy in the thymus.

No such effects were detected in the F0 generation animals with 3000 ppm.

There were no treatment-related effects on reproductive performance in animals of either sex in treatment groups from either generation.

Offspring:

F0 adult females treated with 12000 ppm showed a reduction in live litter size at birth. A reduction in live birth index was also evident for F0 adult females together with a reduction in corpora lutea. There were no effect on intra-uterine embryonic deaths however, the reduced corpora lutea count may have resulted in the reduced live litter size at birth. F1 adult females treated with 12000 ppm showed a reduction in corpora lutea subsequently resulting in a lower number of implantations and litter size at birth. The toxicological relevance of these findings however is dubious given the absence of a true dose related response for corpora lutea and the deterioration in the physical condition of the animals. It should be noted that ovarian oocyte counts for selected F1 adult females was not affected by treatment at this dose level. Mean offspring bodyweights on Day 1 of age were unaffected by maternal exposure at 12000 ppm in either generation. However, litter weight was notably lower due to the aforementioned litter size at this dietary level. Subsequent bodyweight gain in either generation to weaning was progressively lower than concurrent controls, with differences being particularly marked from Day 7 of age. This may also have been a consequence of the offspring beginning to feed on the test diet rather than a developmental effect.

Selected F1 offspring treated with 12000 ppm showed an increase in age at completion of sexual maturation together with a reduction in bodyweight at sexual maturation. This did not effect the oestrous cycles of females prior to mating or the subsequent mating performance of these animals, and was most probably a consequence of the reduced offspring size.

Unselected F1 offspring and F2 offspring treated with 12000 ppm showed a reduction in absolute brain, spleen and thymus weight with the effect on brain weight extending to the 6000 ppm dose group.

No such toxicologically significant effects were detected in F0 or F1 generation animals of either sex treated with 6000 or 3000 ppm.

Conclusions:
Following OECD TG 416 the oral administration of Molyvan 855 in Sprague-Dawleyrats by dietary administration at a maximum dose level of 12000 ppm (600 mg/kg) for two successive generations resulted in adult toxicity in the F0 generation at 12000 (1000 mg/kg) and 6000 ppm (500 mg/kg) and in all treatment groups for the F1 generation.

The 'No Observed Effect Level' (NOEL) and No Observed Adverse Effect Level (NOAEL) for reproductive toxicity of Molyvan 855 for both generations was considered to be 6000 ppm (500 mg/kg).
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Additional information

Toxicity to Reproduction:

Key study

In an OECD TG 416 study conducted under GLP conditions, the oral administration of Molyvan 855 in Sprague-Dawley rats by dietary administration at a maximum dose level of 12000 ppm (1000 mg/kg) for two successibe generations resulted in adult toxicity in the F0 generation at 12000 (1000 mg/kg) and 6000 ppm (500 mg/kg) and in all treatment groups for the F1 generation (SPL 2006). The 'No Observed Effect Level' (NOEL)of Molyvan 855 for reproductive toxicity for both generations was considered to be 6000 ppm (500 mg/kg).


Short description of key information:
A dietary two generation reproduction study in the rat (OECD 416), with evaluation of subchronic toxicity, was conducted to evaluate the effects on fertility.

Justification for selection of Effect on fertility via oral route:
Well conducted study in accordance with OECD Guideline 416 and GLP.

Effects on developmental toxicity

Description of key information
A dietary two generation reproduction study in the rat (OECD 416), with evaluation of subchronic toxicity, was conducted to evaluate the effects on fertility.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline under GLP conditions.
Qualifier:
according to
Guideline:
other: OECD 416 "Two Generation Reproduction Toxicity Study"
Deviations:
no
Principles of method if other than guideline:
The study is a two generation study also designed to assess developmental toxicity of the test material.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Kent.

- Age at study initiation: Six to eight weeks old.

- Weight at study initiation: Makes: 180 - 255kg. Females: 145 - 196kg.

- Fasting period before study: Not applicable.

- Housing: Housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper.

- Diet: Ad libitum

- Water: Ad libitum

- Acclimation period: The animals were acclimatised for fifteen days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 deg C

- Humidity (%): 55 +/- 15%

- Air changes (per hr): At least fifteen air changes per hour.

- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet: Daily
- Mixing appropriate amounts with: Ground diet of Rodent PMI 5002 (Certified) diet.
- Storage temperature of food: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the dietary admixtures was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.
Details on mating procedure:
F0 and F1 animals were paired, following subsequent maturation periods, on a 1 male: 1 female basis within each does group, for a period of up to 21 days. Cage trayliners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Each pregnant female was observed at 0830, 1230 and 1630 hours and around the period of expected parturition. On Weekends and Holidays, observations were carried out at approx 0830 and 1230 hours. The following was recorded for each female: date of mating; date and time of observed start of parturition; date and time of observed completion of parturition; and duration of gestation.

On completion of parturition the number of live and dead offspring were recorded. The date and time of Day post partum litter observations were standardised. For each litter the following was recorded: number of pups born; number of pups alive recorded daily and reported on Day 1, 4, 7, 14, 21 post partum. On days 1 and 21 the sex of the individual offspring were recorded; clinical condition of pups from birth to weaning; individual pup and litter weights on Day 1, 4, 7, 14 and 21 post partum; and necropsy findings.



Duration of test:
The test material was administered daily by dietary admixture. Control animals were treated in an identical manner with untreated laboratory diet.
Remarks:
Doses / Concentrations:
12000 ppm (1000 mg/kg)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm (250 mg/kg)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6000 ppm (500 mg/kg)
Basis:
nominal in diet
No. of animals per sex per dose:
F0 generation: 28 males/28 females
F1 generation: 24 males/24 females
Control animals:
yes, plain diet
Details on study design:
The test material was administered by dietary ad mixture to three groups each of twenty-eight male and female F0 Generation Sprague-Dawley Crl:CD (SD) IGS BR strain rats, at dietary concentrations of 3000, 6000, and 12000 ppm (equivalent to a mean achieved dosage of 250, 500 and 1000 mg/kg/day, respectively). A further group of 28 male and female F0 Generation was exposed to basal laboratory diet to serve as control.

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. During Week 10, extensive functional observations and ophthalmoscopic examinations were performed on test selected animals of either sex from each dose group, together with haematology and blood chemistry assessment.

After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum, followed by the termination of all F0 male dose groups. The offspring selected for the F1 generation were dosed for at least 10 weeks and the paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving F1 females and their offspring were killed, followed by termination of all F1 male dose groups.

Oestrous cycle assessment was performed daily for three weeks prior to mating. Observations for positive evidence of mating were recorded together with the start and completion of parturition. During the maturation phase of the F1 generation offspring, males and females were evaluated for sexual maturation. The ano-genital distance was recorded for all F2 generation offspring on Day post partum. During the lactation phases daily clinical observations were performed on all surviving offspring, together with litter size. Litter weight, individual offspring weights and landmark developmental signs were also recorded on specific days post partum.

All animals at termination were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporation Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.

Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.

The haematology variable basophils was not analysed since consistently greater than30% of the data were recorded as the same value.

Histopathological data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes: 1) Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater; 2) Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Indices:
Gestation and Parturition Data: The following parameters were calculated for individual data during the gestation and parturition period of the F0 and F1 generation: Gestation length and parturition index.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female treated with 12000 ppm was killed on humane grounds on Day 40 due to a damaged tail tip. This death was a result of physical injury and unrelated to test material toxicity.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Females treated with 12000 ppm showed statistically significant reductions in thymus, ovary and uterus weight both absolute and relative to bodyweight. The effect on ovary, uterus and thymus (absolute) weight extended to the 6000 ppm treatment group. Females treated with 12000 ppm also showed an increase in relative kidney weight.

No such treatment effects were detected in males treated with 12000 ppm or animals of either sex treated with 3000 ppm.

Animals of either sex treated with 12000 ppm showed a reduction in absolute adrenal, liver, pituitary and spleen weight. There were no histological correlates and in the absence of a similar reduction in the weight of these organs when expressed relative to terminal bodyweight, the intergroup differences were considered attributable to the bodyweight effect seen in this study and not a direct effect of treatment. Females treated with 12000 ppm showed an increase in relative brain and liver weight.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A number of females from control and treated groups showed isolated incidents of enlarged/red lungs, dark adrenals, hard/dark liver, small/thin or encased spleen and a mass on the upper chest. Such findings are consistent with normally expected low incidence findings in laboratory maintained rats of the strain and aged used and are of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Kidneys:
Basophilia of renal tubules was observed in relation to treatment for females treated with 12000 ppm (P <0.001). Males were not similarly affected. Accumulations of yellow pigment were observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.01 for males and P <0.05 for females). The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin.

Mesenteric lymph nodes:
Sinus histiocytosis was observed as a treatment related effect in females treated with 12000 ppm (P <0.01) or 6000 ppm (P <0.05) dose levels. Male rats were not similarly affected.

Thymus:
A greater incidence of higher grades of severity lymphoic atrophy of the thymus was seen for females treated with 12000 ppm (P<0.05).

Bone Marrow:
Generally higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen in relation to treatment for females only treated with 12000 ppm. Although this condition did not attain statistical significance the overall picture suggested a probable if marginal effect of treatment for higher dose females only.

Ovaries:
Fine cytoplasmic vacuolation of interstitial cells were observed with lower incidence among animals treated with 12000 ppm (P <0.001) or 6000 ppm (P <0.05) of the test material. The significance of this change is uncertain. A greater incidence of higher grades of severity of yellow pigment accumulation was also observed as an effect of treatment for females treated with 12000 ppm (P <0.01). The pigment was demonstrated to be haemosiderin by Perl's staining technique.



BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)


No such toxicological significant effects were detected in animals of either sex treated with 6000 or 3000 ppm.

Females treated with 3000 ppm showed an increase in bodyweight gain during Week 4 only. There was no dose-related response for this finding and in any event increases in bodyweight gains are unlikely to be indicative of test material toxicity.

Gestations bodyweight:
Females treated with 12000 ppm showed a reduction in bodyweight gain during the first week of gestation, although subsequent bodyweight gain during week two was similar to control animals. During the third week of gestation bodyweight gain was again notably lower than controls, with differences attaining statistical significance.

No such effects were detected in females treated with 6000 or 3000 ppm.

Lactation bodyweight:
Bodyweight gain during the first two weeks of lactation was notably lower than concurrent control animals for females treated with 12000 ppm, however, statistical significance was only obtained during Week 1.

No such effects were detected in females treated with 6000 or 3000 ppm.

Dose descriptor:
NOEL
Effect level:
6 000 ppm (nominal)
Based on:
test mat.
Remarks:
(500 mg/kg)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
3 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
In total there were 28, 27, 28 and 25 females at 0 (control), 3000, 6000 and 12000 ppm, respectively, who gave birth to a live litter and successfully reared young to weaning (Day 21 of age) and have been included in the following assessment of litter responses.

The mean numbers of corpora lutea observed for treated F0 females were considered to be acceptable and did not indicate any adverse effect of dietary exposure at 3000, 6000 or 12000 ppm. Although values for treated groups were slightly lower than control animals, attaining statistical significance at both 3000 and 12000 ppm, there was no clear dose related response and these differences were considered to represent normal biological variation rather than an effect of maternal exposure to the test material. Subsequent pre and post implantation losses for treated animals were essentially similar to controls however the resultant mean number of implantations and litter size at birth observed for 12000 ppm females were lower than concurrent control animals. Following birth, live birth index for 12000 ppm females was lower than controls, suggesting increase mortality among the F1 offspring prior to Day 1 of age; however subsequent offspring viability throughout lactation was similar to controls.

No such effects were detected in 6000 or 3000 ppm females. Sex ratio at birth for live offspring at Day 1 of age and at weaning (Day 21) was similar in all treatment groups and did not indicate any selective effects for either sex.

BODY WEIGHT (OFFSPRING)
Mean offspring bodyweights on Day 1 of age were unaffected by maternal exposure in females treated with 12000 ppm. Subsequent bodyweight gain of the offspring to weaning was progressively lower than concurrent control animals, with differences being particularly marked between Days 14 and 21 of lactation. Mean offspring bodyweights at this treatment group were significantly lower than control animals from Days 14 of age and this, combined with the slightly lower litter size, resulted in statistically differences in mean litter weight from Day 7 onwards.

No such effects were detected in females treated with 6000 or 3000 ppm.

Inter-group differences in offspring maturation (as assessed by the onset and completion of pinna unfolding, incisor eruption and eye opening) and reflexological assessments (percentage successful at surface righting, mid-air righting and pupil and startle reflex) did not indicate any adverse effects of dietary exposure at 3000, 6000 or 12000 ppm. These findings suggest that the impaired growth observed for offspring at 12000 ppm did not affect the development of the offspring.

SEXUAL MATURATION (OFFSPRING)
Animals of either sex treated with 12000 ppm showed a statistically significant increase in age at completion of sexual maturation together witha reduction in bodyweight (males only) at sexual maturation compared to control animals.

No such treatment-related effects were detected in animals of either sex treated with 6000 or 3000 ppm.

Males treated with 6000 ppm showed a statistically significant reduction in bodyweight at sexual maturation. In the absence of any associated effect on the age at sexual maturation the intergroup difference was considered to be of no toxicological importance.

ORGAN WEIGHTS (OFFSPRING)
Animals of either sex treated with 12000 ppm showed a statistically significant increase in kidney weight relative to bodyweight. The effect on relative kidney weight extended to 6000 and 3000 ppm males. A statistically significant reduction in absolute uterus weight was also detected at 12000 ppm.

The remaining statistically significant intergroup differences in the adrenals, brain, heart, kidneys, liver, prostrate, pituitary, reproductive organs, spleen, thymus and thyroid detected throughout the treatment groups were not associated with any histological correlates and were considered attributable to the bodyweight effect seen in this study and not a direct consequence of treatment.

HISTOPATHOLOGY (OFFSPRING)
Kidneys:
Basophilia of renal tubules was observed in relation to treatment for females treated with 12000 ppm (P <0.001). Male rats were not similarly affected. Accumulations of yellow pigment were observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.001), and for males treated with 6000 ppm (P <0.05) and at 3000 ppm (P <0.05), although there was no dose response and the effect must be regarded as being of marginal toxicological significance for these two lower dose levels. The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin.

Mesenteric lymph node:
A greater incidence of accumulations of yellow pigment was observed as a consequence of treatment for animals of either sex treated with 12000 ppm (P <0.001 for males and P <0.05 for females). The pigment was not demonstrated to be Perl's positive, thus was probably not haemosiderin. For females only, sinus histiocytosis was also observed as a treatment-related effect at 12000 ppm (P <0.01) and at 6000 ppm (P <0.05).

Thymus:
A greater incidence and generally higher grades of severity of lymphoid atrophy were seen for females treated with 12000 ppm (P <0.001), 6000 ppm (P <0.01), or at 3000 ppm (P <0.05). Male rats were not similarly affected.

Bone marrow:
Generally higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen in relation to treatment for females only treated with 12000 ppm (P <0.001). An opposite effect was observed for males at this dose level and reached statistical significance but this was probably an anomaly.

Ovaries:
Fine cytoplasmic vacuolation of interstitial cells was observed with a lower incidence among females treated with 12000 ppm (P <0.001) or at 6000 ppm (P <0.01).

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
Abnormalities:
not specified
Developmental effects observed:
not specified

No clinically observable signs of toxicity were detected in treatment groups from either the F0 adults or F1 adults throughout the study period. Bodyweight development, dietary intake and food utilisation were however adversely affect in 12000 ppm adult animals in both the F0 and F1 generation, resulting in a deterioration in physical condition of the animals. Haematological investigations of selected F0 adult animals revealed a normocytic, normochromic anaemia. This was probably associated with the bone marrow changes seen microscopically. Changes were identified as higher grades of severity of adipose infiltration of the marrow, indicating hypoplasia for adult females treated with 12000 ppm from either generation. Absolute and relative thymus weight was reduced in adult females from either generation at this dose level and microscopic examination of thymus sections revealed changes identified as a greater incidence of higher grades of severity of lymphoid atrophy. Sinus histocytosis was evident microscopically in the in the mesenteric lymph nodes for 12000 ppm adult females from either generation together with accumulations of yellow pigment also being observed in 12000 ppm adult females from the F1 generation only. Such effects in the thymus and mesenteric lymph nodes may possibly be associated with the reduced physical conditions seen in these animals, however the latter condition seen in 12000 ppm F1 adult females in the mesenteric lymph nodes may simply represent the accumulation of the coloured test material or coloured metabolite(s) and as such is of minimal toxicological significance.

Relative kidney weights were elevated for the F0 adult females treated with 12000 ppm and for F1 adult animals of either sex treated with 12000 ppm. Microscopic examination of kidney sections revealed basophilia of renal tubules in F0 and F1 adult females at this dose level. Further renal microscopic changes were evident in adult animals of either sex from both the F0 and F1 generations and were identified as accumulations of yellow pigment.

Microscopic changes were also identified in the ovaries. A lower incidence of fine cytoplasmic vacuolation of interstitial cells was observed for both F0 adult and F1 adult females treated with 12000 ppm and a greater incidence of higher grades of severity of yellow pigment accumulations (identified as haemosiderin) for F0 adult females at this dose level. Organ weight data supported these findings with reductions in absolute and relative ovary weight for adult females from the parental generation at this dose level. A reduction in absolute and relative uterus weight was also evident in these animals.

Toxicologically significant effects extended to the 6000 ppm dose group. F0 adult males showed a reduction in bodyweight gain and food consumption during maturation albeit to a lesser extent. F1 adult animals however showed a similar reduction in bodyweight gain and food consumption as observed in the 12000 ppm dose group.

A reduction in ovary, uterus and thymus weight was evident in F0 adult females together with the microscopic changes of sinus histocytosis in the mesenteric lymph nodes and a lower incidence of fine cytoplasmic vacuolation of intestinal cells in the ovaries. F1 adult females showed a reduction in absolute ovary weight together with microscopic changes of sinus histocytosis in the mesenteric lymph nodes and a lower incidence of fine cytoplasmic vacuolation of interstitial cells in the ovaries, while F1 adult males showed an increase in relative kidney weight together with microscopic renal changes of accumulation of yellow pigment.

Changes detected in 3000 ppm were confined to the F1 adult animals. Adult males showed statistically significant increase in relative kidney weight together with microscopic changes of accumulations of yellow pigment in the kidneys while adult females showed a reduction in absolute ovary weight and a greater incidence and higher grades of severity of lymphoid atrophy in the thymus.

No such effects were detected in the F0 generation animals with 3000 ppm.

There were no treatment-related effects on reproductive performance in animals of either sex in treatment groups from either generation.

Offspring:

F0 adult females treated with 12000 ppm showed a reduction in live litter size at birth. A reduction in live birth index was also evident for F0 adult females together with a reduction in corpora lutea. There were no effect on intra-uterine embryonic deaths however, the reduced corpora lutea count may have resulted in the reduced live litter size at birth. F1 adult females treated with 12000 ppm showed a reduction in corpora lutea subsequently resulting in a lower number of implantations and litter size at birth. The toxicological relevance of these findings however is dubious given the absence of a true dose related response for corpora lutea and the deterioration in the physical condition of the animals. It should be noted that ovarian oocyte counts for selected F1 adult females was not affected by treatment at this dose level. Mean offspring bodyweights on Day 1 of age were unaffected by maternal exposure at 12000 ppm in either generation. However, litter weight was notably lower due to the aforementioned litter size at this dietary level. Subsequent bodyweight gain in either generation to weaning was progressively lower than concurrent controls, with differences being particularly marked from Day 7 of age. This may also have been a consequence of the offspring beginning to feed on the test diet rather than a developmental effect.

Selected F1 offspring treated with 12000 ppm showed an increase in age at completion of sexual maturation together with a reduction in bodyweight at sexual maturation. This did not effect the oestrous cycles of females prior to mating or the subsequent mating performance of these animals, and was most probably a consequence of the reduced offspring size.

Unselected F1 offspring and F2 offspring treated with 12000 ppm showed a reduction in absolute brain, spleen and thymus weight with the effect on brain weight extending to the 6000 ppm dose group.

No such toxicologically significant effects were detected in F0 or F1 generation animals of either sex treated with 6000 or 3000 ppm.

Conclusions:
The 'No Observed Effect Level' (NOEL) and 'No Observed Adverse Effect Level' (NOAEL) of Molyvan 855 for developmental toxicity for both generations was considered to be 6000 ppm.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Additional information

Developmental toxicity:

Key study

The 'No Observed Effect Level' (NOEL) of Molyvan 855 for developmental toxicity for both generations was considered to be 6000 ppm (500 mg/kg ) (SPL 2006).


Justification for selection of Effect on developmental toxicity: via oral route:
Well conducted study in accordance with OECD Guideline 416 and GLP.

Justification for classification or non-classification

Reproductive toxicity

Based on Regulation No 1272/2008, Molyvan 855 is not classified for reproductive toxicity.

Developmental toxicity

Based on Regulation No 1272/2008, Molyvan 855 is not classified for developmental toxicity.