Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
This second multi-generation reproductive toxicity study was conducted to meet the data requirements of a non-EU, non-chemicals legislation. It has been selected as key study due to the additional investigations conducted in this second study.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jul 2002 to 17 Feb 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
1998
GLP compliance:
yes
Limit test:
no
Justification for study design:
- Premating exposure duration for parental (P0) animals : 10 weeks
- Basis for dose level selection : The dose levels were selected in relation to the results of the previous study.
- Route of administration : oral (feed)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Tif:RAIf (F2 generation i.e. first offspring from F1 hybrids)
Details on species / strain selection:
The Tif:RAIf strain of rat was used for a previous reproduction study and therefore selected for use in this study. The oral (dietary) route of administration was selected as this represents is a possible route of exposure for humans and other mammalian species.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: at least 5 weeks (P)
- Weight at study initiation: 124-194 g males (P),113-167 g females (P)
- Housing: Two/cage/sex except during mating (1 male with 1 female) and females individually during pregnancy and lactation
- Diet: CT1 diet with or without the test material, ad libitum
- Water: ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 to 70%
- Air changes: At least 15 / hour.
- Photoperiod: 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The experimental diets were prepared in 40 kg or 60 batches using one or two 1kg premixes prepared by triturating the appropriate amount of test material with milled diet. The premixes were then added to the required amount of control diet and mixed thoroughly.
Details on mating procedure:
One male was housed with one female from the same group for a maximum period of 14 days (brother/sister matings were avoided). Daily vaginal smears were examined for confirmation of mating. Females were separated from the males following the detection of a sperm-positive vaginal smear.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples from all dietary levels (including controls) were taken at intervals throughout the study and analysed quantitatively for the test material. The homogeneity of the test material in CT1 diet was determined for each batch size by analysing samples from the 20 and 2500 ppm dietary levels. The chemical stability of the test material in diet was also determined. All analysis was by High Performance Liquid Chromatography.
- Concentration analysis results: The mean achieved concentrations of the test material in diet were within 12% of the nominal concentrations and the overall concentration was within 4% of nominal.
- Homogeneity results: The homogeneity of the test material in diet at concentrations of 20 ppm and 2500 ppm was satisfactory and within 4% of the overall mean.
- Stability results: The reanalysis of the test material in diet preparations at concentrations of 20 ppm and 2500 ppm stored at room temperature was acceptable for 13 and 24 days respectively.
Frequency of treatment:
The test material was fed in the diet continuously throughout the study.
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
20 ppm
Remarks:
Dietary concentration; see Table 1 in “Any other information on materials and methods incl. tables” for mean dose received in mg/kg bw/day
Dose / conc.:
50 ppm
Remarks:
Dietary concentration; see Table 1 in “Any other information on materials and methods incl. tables” for mean dose received in mg/kg bw/day
Dose / conc.:
1 000 ppm
Remarks:
Dietary concentration; see Table 1 in “Any other information on materials and methods incl. tables” for mean dose received in mg/kg bw/day
Dose / conc.:
2 500 ppm
Remarks:
Dietary concentration; see Table 1 in “Any other information on materials and methods incl. tables” for mean dose received in mg/kg bw/day
No. of animals per sex per dose:
- test group: 26 animals per sex per dose
- satelite P1 group: 14 males, to generate histological data on testis only
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected in relation to the results of the previous study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS & DETAILED CLINICAL OBSERVATIONS
P0, P1, Satellite P1 were examined daily and significant changes recorded. Detailed observations were recorded at the same time as body weight.

BODY WEIGHT
- Males - P0, P1 & Satellite P1: body weights recorded weekly and at termination. F1: also on the day of preputial separation.
- Females - P0 & P1: body weights recorded weekly prior to mating, on gestation days 1, 8 15 and 22, on days 1, 5, 8, 15 and 22 post partum and at termination. P1: also on the day of vaginal opening.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Males - P0, P1 & Satellite P1: weekly except during the mating period.
- Females - P0 & P1: weekly during the pre-mating, gestation and lactation periods.

DEVELOPMENTAL LANDMARKS
P1 males and females: the day on which preputial separation occurred in males and vaginal opening occurred in females was recorded.
Oestrous cyclicity (parental animals):
P0 & P1: For 25 days prior to the mating period, daily vaginal smears were examined for cyclicity. The stage of oestrus was also determined on the day of termination.
Sperm parameters (parental animals):
- Sperm analysis: All males at scheduled termination excluding satellite P1 males.
- Sperm count, motility and velocity: sperm obtained from the right cauda epididymis were analysed using a Computer-Assisted Sperm Analyser for total and static count and straight line, curvilinear and average path velocity. Percentage motility and motile count were calculated. A sample of minced cauda epididymis was stained with IDENT, a DNA specific stain and the number of sperm in the sample counted.
- Sperm morphology: samples of minced cauda epididymis were stained using a multicoloured single step stain and the morphology of the sperm evaluated.
- Homogenisation resistant testicular sperm: The right testis from control and high dose males was used for analysis of homogenisation resistant testicular spermatid head counts.
Litter observations:
PARAMETERS EXAMINED
The sex, body weight and clinical condition of each pup was recorded on completion of parturition (day 1) and on days 5, 8, 15 and 22 post partum. Litters were examined daily for dead, unhealthy or abnormal pups.

SELECTION OF PUPS
On day 22 post partum, 26 males and 26 females per group were selected from the F1A litters to become the next generation of parents (P1). Fourteen males per group were also selected to generate histological data on the testis only.
Postmortem examinations (parental animals):
TERMINATION
- All surviving rats and those requiring euthanasia were killed by exsanguination under terminal anaesthesia. Female rats were similarly killed if they were observed to have gestation or parturition difficulties, if they failed to litter by nominal day 25 of gestation or had total litter loss (although some P0 females were retained and not terminated as scheduled, for possible remating due to the unexpected number of whole litter losses – no remating was ultimately required).
- The P0 & P1 females were killed on day 22 post partum together with any unselected pups. The P0 & P1 males including satellite P1 males were killed when they were approximately 33 weeks of age (for parity with the previous study). All rats were killed by exsanguination under terminal anaesthesia

MACROSOPIC EXAMINATION
All rats (excluding satellite P1 males) were given an examination of all cranial, thoracic and abdominal organs and structures. For females, the uterus was examined for the presence and number of implantation sites.

ORGAN WEIGHTS
- The following organs from P0 and P1 rats (excluding satellite P1 males) were weighed at scheduled termination: adrenal glands, pituitary gland, brain, prostate gland, left epididymis (including cauda), seminal vesicle (including prostate, coagulating gland and fluids), right epididymis (including cauda), right cauda epididymis, spleen, kidneys, liver, left testis, right testis, ovaries, uterus (with oviducts and cervix)
- Paired organs were weighed together unless stated otherwise. A total weight was calculated for paired organs weighed separately.

TISSUE SUBMISSION
- The following tissues from all P0 and P1 rats (excluding satellite P1 males) were taken and preserved in fixative: abnormal tissue, pituitary gland, adrenal gland, prostate gland, cervix, seminal vesicle (including coagulating gland), kidney (target organ), left testis, left epididymis, efferent ductules, mammary gland (females only), uterus with oviducts, ovary, vagina
- Only the testes and epididymides from the satellite P1 males were taken and preserved.

TISSUE PROCESSION AND EXAMINATION
All submitted tissues were processed, except the efferent ductules, and H&E sections prepared. The left testis, left epididymis and kidneys were examined by light microscopy for P0 and P1 animals. Both testes and epididymides were examined for all satellite P1 males. Other processed tissues from the control and high dose animals only were examined. The reproductive organs from animals with suspected infertility were examined. A quantitative evaluation of primordial follicles was conducted using the left ovary from 10 control and 10 high dose P1 females.

HISTOLOGICAL EXAMINATION OF THE TESTES
For each testis, four transverse sections were examined and the number of tubular cross sections showing germ cell loss was recorded for each section separately. For each affected tubular cross-section, the degree of germ cell loss was described as either complete or partial/complete. The distribution of affected tubules within the testis cross-section was described as either focal or multifocal/diffuse. This resulted in four different morphological descriptions of germ cell loss. Every section showing germ cell loss was given one of 5 severity grades. The overall severity grade of germ cell loss for the whole testis or pair of testes was determined.
Postmortem examinations (offspring):
TERMINATION
All pups found dead or requiring euthanasia prior to day 22 post partum were given a macroscopic examination. On day 22 post partum, up to three male and three female F1A and F2A pups per litter were killed by exsanguination under terminal anaesthesia. Remaining pups were killed and discarded without examination.

MACROSCOPIC EXAMINATION
All pups of more than 18 days of age were given an examination of the cranial, thoracic and abdominal organs and structures.

ORGAN WEIGHTS
The following organs were recorded from one male and one female pup per litter (selected from the 3/sex/litter at scheduled termination): brain, spleen, thymus

TISSUE SUBMISSION
Abnormal tissues were taken from pups more than 18 days of age given a macroscopic examination and preserved in fixative. These tissues were not processed or examined histologically.
Statistics:
Analysis of variance, analysis of covariance, double arcsine transformation of Freeman and Tukey, Fisher's Exact Test. For more details see "Any other information on materials and methods incl. tables" section.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on the incidence of mortality or on the clinical condition of the P0 parent animals
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no effect of the test substance on the incidence of mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There was an effect of the test substance on body weight with lower body weights being recorded for the P0 males given 2500 ppm in comparison with the control group. This effect was accompanied by a reduction in food consumption and food utilisation during weeks 1-4. The lower body weights of the P0 males given 1000 ppm that were not statistically significantly different from the control body weights (with one exception at week 4), were not associated with any reduction in food consumption and were therefore considered to be of no toxicological significance.
- There was no effect of 1000 or 2500 ppm test substance on the body weight of the P0 females during the pre-mating period, gestation or post partum.
- There was no effect of 20 or 50 ppm test substance on the body weight of the P0 animals.
- See Table 1 in "Any other information on results incl. tables" for more information.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There was an effect of test substance on body weight with lower body weights being recorded for the P0 males given 2500 ppm in comparison with the control group. This effect was accompanied by a reduction in food consumption and food utilisation during weeks 1-4.
- There was no effect of 1000 or 2500 ppm test substance on the food consumption of the P0 females during the pre-mating period, gestation or post partum.
- There was no effect of 20 or 50 ppm test substance on the food consumption of the P0 animals.
- For the females with litters, food consumption was statistically significantly lower in the 2500 ppm group in comparison with the control group for week 3 post partum for the P0 females only. However, at this time, pups are also consuming the diet.
- See Table 2 and 3 in "Any other information on results incl. tables" for more information.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histological change was seen in the kidneys of the P0 males given 1000 or 2500 ppm. The histological change was consistent with α-2µ globulin nephropathy seen previously in rats treated with the test substance. This effect is a phenomenon specific to the male rat and of no relevance to human risk assessment. There was evidence to show that the treatment-related lesion was bilateral. The change was considered to be minimal, affecting only small segments of very few tubules with no effect on reproductive function or spermatogenesis and thus was considered not to be adverse.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- There was no effect of the test substance on oestrus cyclicity.




Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- There was no effect of treatment on sperm motility.
- For the P0 males, there was no effect on the number of sperm in the right cauda epididymis.
- For the P0 males, there was no effect of the test substance on straight line, curvilinear or average path velocities. In context with the historical range of values, the mean velocities were considered to be within normal variability.
Reproductive performance:
no effects observed
Description (incidence and severity):
- There was no effect of the test substance on pre-coital interval, the duration of gestation or on the success of mating.
- For the P0 females, the proportion and percentage of successful matings were lower in the 20 and 2500 ppm groups in comparison with the control group but were not statistically significantly different.
- In the absence of a relationship with dose, the variation in the proportion of successful matings was considered incidental to treatment with the test substance.
- Uterine implantations: There was no effect of the test substance on the number of uterine implantation sites or on post-implantation loss for either the P0 females.
- See Table 4 in "Any other information on results incl. tables" for more information.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
155.6 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Remarks on result:
other: Value is based on highest dose (2500 ppm)
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
158.9 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Value is based on highest dose (2500 ppm)
Remarks:
equivalent to the dose of parental animals P0 during gestation
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
61.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Value is based on medium dose (1000 ppm)
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
158.9 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Remarks on result:
other: Value is based on highest dose (2500 ppm)
Remarks:
equivalent to the dose of parental animals P0 during gestation

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
61.7 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on the clinical condition of the P1 parent animals or the P1 satellite males.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no effect of he test substance on the incidence of mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There was no effect of 1000 or 2500 ppm test substance on the body weight of the P1 males or on the body weight of the P1 females during the pre-mating period, gestation or post partum.
- There was no effect of 20 or 50 ppm test substance on the body weight of the P1 animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There was no effect of 1000 or 2500 ppm test substance on the food consumption of the P1 males or on the food consumption of the P1 females during the pre-mating period, gestation or post partum.
- There was no effect of 20 or 50 ppm test substance on the food consumption of the P1 animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- There was a possible treatment-related increase in epididymal weight in P1 males given 1000 ppm or 2500 ppm.
- Other changes in organ weights were considered unlikely to be of toxicological significance as they were not consistently seen in both sexes, or were not dose-related or were not accompanied by histological change or were consistent with historical control values.
- See Table 5 in "Any other information on results incl. tables" for more information.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Treatment-related histological change was seen in the kidneys of the P1 males given 1000 or 2500 ppm. The histological change was consistent with α-2µ globulin nephropathy seen previously in rats treated with the test substance. This effect is a phenomenon specific to the male rat and of no relevance to human risk assessment.
- An increased incidence of a very minor testicular tubular lesion (minimal germ cell loss/disorganisation +/- Sertoli cell vacuolation) was confined to P1 males given 2500 ppm. There was evidence to show that the treatment-related lesion was bilateral. The change was considered to be minimal, affecting only small segments of very few tubules with no effect on reproductive function or spermatogenesis and thus was considered not to be adverse.
- See Table 6 in "Any other information on results incl. tables" for more information.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on oestrus cyclicity.

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- There was no effect of treatment on sperm motility.
- For the P1 males given 2500 ppm, the total number of sperm and the number of sperm per gram of right cauda epididymis were statistically significantly higher than in the control group. In context with the historical range of values, the values for the 2500 ppm group were seen to be slightly higher. Histological examination of the epididymis and morphological examination of the sperm did not reveal any treatment-related abnormalities. Although possibly treatment-related, the higher number of sperm was considered not to be an adverse finding. The number of sperm in the right testis was significantly lower in the P1 males given 50, 1000 or 2000 ppm but there was no evidence of a dose-related effect and the values were within or just below the historical control range. The minimal histological effect of treatment on the testis was considered not to have any potential impact on sperm number.
- For the P1 males given 2500 ppm, the straight line, curvilinear and average path velocities were statistically significantly lower in comparison with the control group. In context with the historical range of values, the mean velocities were considered to be within normal variability.
- See Table 7 and 8 in "Any other information on results incl. tables" for more information.
Reproductive performance:
no effects observed
Description (incidence and severity):
- Reproductive performance of P1 parental animals was not affected by the administration of the test substance.
- There was no effect of the test substance on the pre-coital interval, the duration of gestation or on the success of mating.
- There was no effect on the proportion of successful matings for the P1 females.
- Uterine implantations: There was no effect of the test substance on the number of uterine implantation sites or on post-implantation loss for either the F1 females.
- Oocyte count: The numbers of small follicles in the ovaries of P1 females receiving 2500 ppm test substance were comparable to the numbers observed in control females.
- See Table 4 in "Any other information on results incl. tables" for more information.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
191.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Remarks on result:
other: Value is based on highest dose (2500 ppm)
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
181.8 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: Value based on highest dose (2500 ppm)
Remarks:
equivalent to the dose of parental animals P1 during gestation
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
181.8 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Remarks on result:
other: Value based on highest dose (2500 ppm)
Remarks:
equivalent to the dose of parental animals P1 during gestation
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
191.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: Value based on highest dose (2500 ppm)

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
74.8 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on clinical condition.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Up to 5 whole litter losses occurred in each group of the P0 generations. Four whole litter losses occurred in the P0 control group. The incidence of litters affected was not dose-related and was considered to be incidental to treatment.
- There was no effect of the test substance on the proportion or percentage of pups live born or on the proportion of litters with all pups born alive, on mean litter size on days 1, 5, 8, 15 and 22 and no effect on pup survival, for either the F1A litters. Intergroup variation in pup survival was related to the number of whole litter losses, which were considered not to be treatment-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- There was no effect of the test substance on body weight.
- On days 15 and 22, total litter weight of the F1A pups was statistically significantly lower in the 2500 ppm group in comparison with the control group due to a slightly smaller litter size in the combined with a slightly lower pup body weight (rather than a slightly higher pup body weight typically associated with smaller litters). It was therefore concluded that the slightly lower total litter weight was due to the direct consumption of diet containing 2500 ppm by the pups, rather than a developmental effect.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
- There was no effect of the test substance on pup sex distribution.
- For the F1 animals, there was no effect on the mean day of age when preputial separation or vaginal opening occurred.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related findings on brain, spleen or thymus weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on clinical condition.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Up to 5 whole litter losses occurred in each group of the P1 generations. No litter losses occurred in the P1 control group. The incidence of litters affected was not dose-related and was considered to be incidental to treatment.
- There was no effect of the test substance on the proportion or percentage of pups live born or on the proportion of litters with all pups born alive, on mean litter size on days 1, 5, 8, 15 and 22 and no effect on pup survival, for either the F2A litters. Intergroup variation in pup survival was related to the number of whole litter losses, which were considered not to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There was no effect of the test substance on body weight.
- There were no statistically significant differences in total litter weight for the F2A pups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There was no effect of the test substance on pup sex distribution.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related findings on brain, spleen or thymus weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Intergroup comparison of P0 male body weights (g) (selected timepoints; adjusted mean values)

 

Dietary Concentration of test substance (ppm)

week

0

20

50

1000

2500

2

219.6

221.6

221.1

217.9

215.9*

4

297.5

302.3

301.5

290.4*

282.8**

10

399.6

408.0

405.4

391.9

374.5**

20

478.5

490.6

484.1

471.7

450.0**

28

514.6

523.0

520.6

504.3

479.9**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 2: Intergroup comparison of P0 male food consumption and food utilisation (selected timepoints)

Food consumption (g/rat/day)

Dietary Concentration of test substance (ppm)

week

0

20

50

1000

2500

1

22.6

21.8

22.8

21.6

20.7**

2

25.1

24.5

25.0

23.9

23.4**

7

25.5

25.3

26.4

25.5

24.2*

10

24.4

24.3

24.8

23.7

23.4

26

22.6

21.7

22.5

21.6

21.6

Overall food utilization (g growth/100g food)

 

 

 

 

 

 (weeks 1-4)

(weeks 1-10)

23.11

14.02

23.88

14.35

23.39

14.01

22.25

13.53

21.61**

13.26*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 3: Intergroup comparison of P0 female food consumption (g/rat/day)post partum

 

Dietary Concentration of test substance (ppm)

week

0

20

50

1000

2500

1

28.2

24.7

26.4

28.5

27.1

3

77.1

70.4

73.0

75.7

70.6*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

Table 4: Outcome of mating of each generation

P0 Generation

Dose level of test substance(ppm)

0

20

50

1000

2500

Number of pairings

26

26

26

26

26

Number of successful matings

21

18

24

20

18

Number of females which littered but no live pups were found ^

1

3

0

4

1

Number of non pregnant females

2

5

1

1

5

Number of females which failed to litter with uterine implantation sites

1

0

1

0

1

Died due to difficult parturition

1

0

0

1

1

P1 Generation

 

P1 Pairings

26

26

26

26

26

Number of successful matings

23

23

24

21

26

Number of females which littered but no live pups were found^

0

0

0

3

0

Number of non pregnant females

1

3

1

1

0

Number of females which failed to litter with uterine implantation sites

2

0

1

1

0

Died due to difficult parturition

0

0

0

0

0

^ Classified as whole litter losses

Table 5: Organ weight (adjusted for final body weight) - males

 

Dietary Concentration of test substance (ppm)

Organ

0

20

50

1000

2500

P0

 

 

 

 

 

Kidney

3.00

3.03

3.01

2.95

3.11*

     Epididymis

1.638

1.545

1.619

1.582

1.674

P1

 

 

 

 

 

Kidney

2.83

2.88

2.85

2.81

2.85

     Epididymis

1.584

1.617

1.615

1.656*

1.668*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 6: Incidence of P1 Males with Testicular Tubules showing Germ Cell Loss/Disorganisation +/- Sertoli Cell Vacuolation

 

Dose level of the test substance (ppm)

 

0

20

50

1000

2500

Total number of P1 Males (main study + satellites)

40 (26+14)

40 (26+14)

40 (26+14)

40 (26+14)

40 (26+14)

Main study: uni/bilateral status unknown

3/26

1/26

1/26

3/26

15/26

Satellites: unilateral

1/14

4/14

2/14

3/14

0/14

Satellites: bilateral

1/14

0/14

0/14

1/14

5/14

Total incidence

5/40

5/40

3/40

7/40

20/40

 

Table 7: Sperm number (millions) – P1 parent males

Observation

Dietary Concentration of test substance (ppm)

0

20

50

1000

2500

No. sperm per g right cauda

505

523

532

546

601*

No. sperm per g right testis

52

52

42**

36**

43**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 8: Sperm velocity (µm/s) – P1 parent males

Observation

Dietary Concentration of test substance (ppm)

0

20

50

1000

2500

Straight line velocity

71.6

71.8

72.3

71.3

67.6*

Curvilinear velocity

305.0

297.2

302.4

297.7

289.6**

Average path velocity

123.9

122.1

123.1

120.4

116.2**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Applicant's summary and conclusion

Conclusions:
The fertility and reproductive performance of two generations of parental animals (P0 and P1) was not affected by the administration of the test substance and there was no effect on the survival or development of the pups born to the parental animals. The no observed effect level for reproductive effects was 2500 ppm of the test substance. Minimal histological change in the testis of the P1 males given 2500 ppm was considered not to represent an adverse effect of treatment. Possible treatment-related increases in epididymal weight in P1 males given 1000 or 2500 ppm, and in epididymal sperm number in P1 males given 2500 ppm, were also considered not to be adverse. The test substance at 1000 and 2500 ppm in diet was associated with histological change in the kidney of the P0 and P1 males. The absolute no effect level in this study, based on pathological change in the kidney was 50 ppm of the test substance.
Executive summary:

In a GLP compliant two generation reproduction study, performed in accordance with OECD 416, the test material was administered to groups of 26 male and 26 female (P0 parents) Tif:RAIf rats in diet containing 0 (control), 20, 50, 1000 or 2500 ppm of the test material. After 10 weeks, the animals were mated and allowed to rear the ensuing F1A litters to weaning. The breeding programme was repeated with P1 parents selected from the F1A pups to produce the F2A litters, after a 10 week pre-mating period. The test substance was fed in the diet continuously throughout the study. In addition, 14 males per group in the F1 generation were selected from the remaining pups and retained to generate histological data on the testis only (and referred to as Satellite males). The growth of both parental generations, reproductive function, mating behaviour, conception, gestation, parturition, lactation and weaning and the growth and development of the pups were determined. There was no effect of the test substance on the clinical condition of the P0 or P1 animals. Lower body weights in P0 males were associated with 2500 ppm and accompanied by a reduction in food consumption and, food utilisation between weeks 1-4. There was no effect of the test substance on oestrus cyclicity, pre-coital interval, the duration of gestation or on the success of mating. Also, there was no effect of the test substance on the proportion or percentage of pups born live or on the proportion of litters with all pups born alive for either the F1A or F2A litter. Mean litter size and pup body weight on days 1, 5 and 8 were unaffected by treatment. However, on days 15 and 22, there was evidence for an effect on body weight due to the direct consumption of diet containing 2500 ppm by the F1A pups. A similar difference was not evident in F2A pups. The incidence of whole litter losses was unrelated to treatment and there was no effect on pup survival due to the test substance. The mean day of age when preputial separation or vaginal opening occurred was not affected by treatment. Histological change was seen in the kidney of the P0 and P1 males given 1000 ppm or 2500 ppm with an increase in kidney weight in the P0 males given 2500 ppm. The primary change was increased hyaline droplet accumulation within the proximal tubular epithelium and in the tubular lumen. This effect is generally recognised to be a phenomenon specific to the male rat and of no relevance to human risk assessment. An increased incidence of a very minor testicular tubular lesion (minimal germ cell loss/disorganisation +/- Sertoli cell vacuolation) was confined to P1 males given 2500 ppm. There was evidence to show that the treatment-related lesion was bilateral. The change was considered to be minimal, affecting only small segments of very few tubules with no effect on reproductive function or spermatogenesis and thus was considered not to be adverse. There was a possible treatment-related increase in epididymal weight in P1 males given 1000 ppm or 2500 ppm and an increase in epididymal sperm number in P1 males given 2500 ppm; both were considered not to be adverse. The fertility and reproductive performance of two generations of parental animals (P0 and P1) was not affected by the administration of the test substance and there was no effect on the survival or development of the pups born to the parental animals. The no observed effect level for reproductive effects was 2500 ppm of the test substance. Minimal histological change in the testis of the P1 males given 2500 ppm was considered not to represent an adverse effect of treatment. Possible treatment-related increases in epididymal weight in P1 males given 1000 or 2500 ppm, and in epididymal sperm number in P1 males given 2500 ppm, were also considered not to be adverse. The test substance at 1000 and 2500 ppm in diet was associated with histological change in the kidney of the P0 and P1 males. The absolute no effect level in this study, based on pathological change in the kidney was 50 ppm of the test substance.