Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2011

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Molyvan 855 (OD-855)
Dark brown paste
Date recvd: 27 February 2004
Storage conditions: room temperature in the Dark

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the sponsor.

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.