Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two generation study (OECD 416): NOAEL = 2500 ppm (based on highest dose); no effect on fertility and reproductive performance of two generations of parental animals (P0 and P1)

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Link to relevant study records
Reference
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
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)
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
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
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.
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.
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: 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.
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
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
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: 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.
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)
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
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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)

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
155.6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant, OECD 453 study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 416 - rat

In a GLP compliant two generation reproduction study (Moxon, 2004), 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, 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.

In a second study (Doubovetzky 1998), the substance was administered orally to two successive generations (F0 and F1) of male and female Tif:RAIf (Sprague Dawley derived) rats by admixture in the diet at nominal concentrations of 0, 10, 30, 1000 and 2500 ppm. F0 animals (30/sex/group) were treated for 10 weeks pre-mating and throughout the production of 2 litters until necropsy. Litters were culled to 4 pups/sex, where possible, on day 4 post partum. The F1 generation was selected from the first litters of the F0 generation and these animals were similarly treated for 10 weeks pre-mating and throughout the production of 2 litters until necropsy. Clinical signs, body weights, food consumption, mating, gestation and parturition parameters, pup survival and developmental/behavioural landmarks were recorded. A gross necropsy examination was performed on all pups not selected for mating. All parental animals were necropsied after weaning of the second litters and subjected to macroscopic examination and histopathology of the sex and target organs. Sperm analysis (motility, morphology and spermatid counts) was performed on 15 males/group of the F0 and F1parental generations. However, the results of the sperm analysis were considered to be flawed and an additional study was undertaken to study the effects of CGA293343 technical on sperm cell parameters. In this additional study, groups of 30 virgin male rats of the same specification as the main study were fed CGA293343 technical in the diet for 10 weeks at nominal concentrations of 0, 10, 30, 1000 or 2500 ppm. Body weight, food consumption and clinical signs were recorded. After 10 weeks, the animals were killed and necropsied, the testes, epididymides, prostate, and seminal vesicles (with coagulating glands) were weighed, and epididymis sperm cells and/or testis spermatids were evaluated for number, motility, and morphology. Changes in procedure from the first study were implemented to reduce and standardise the time for sperm collection, to refine the technique for opening the cauda epididymis, and to randomise the order in which sperm evaluations were performed to minimise inter-day bias.

There were no treatment-related clinical signs or deaths. Reductions in body weight and food consumption were confined to the F0 males given 2500 ppm. For both generations and all matings, there were no treatment-related effects on the number of animals mating, the number of females becoming pregnant, mean pre-coital time or the mean duration of gestation. For the first F0 mating only, mean litter size was slightly reduced in the high dose group at birth and on day 4 post partum (prior to culling). There were no treatment-related effects on pup survival (postnatal loss), sex ratios or clinical signs. Pup body weight gain was reduced during lactation at 2500 ppm. For F1 pups, pup body weight gain was also reduced at 1000 ppm.

There were no treatment-related macroscopic findings or changes in organ weights.  Increased incidences of minimal to marked hyaline change (eosinophilic droplets in the cytoplasm of renal tubular epithelium and occasionally within the tubular lumen) in renal tubules were present in male F0 and F1 animals treated with 1000 ppm and 2500 ppm and in one F1 female treated with 2500 ppm. Minimal to marked renal tubular cast was found in F1 males at 1000 ppm and in males of both generations at 2500 ppm in slightly increased incidences. No treatment- related findings were detected in the reproductive system of either sex or, in any of the sperm characteristics.

At 2500 ppm, reduced food consumption and retarded body weight gain were observed in F0 male rats only. Reduced body weight gain of pups was observed at both matings of each generation at 2500 ppm. Increased incidences of minimal to moderate hyaline change in renal tubules were observed in F0 and F1 males and one F1 female at 2500 ppm; renal tubular casts graded minimal to slight were of slightly increased incidences in the males. At 1000 ppm, reduced body weight gain of pups during lactation was observed. Increased incidences of minimal to moderate hyaline change in renal tubules were observed in F1 males.

Female gonadal function, mating behaviour, conception, parturition, lactation and weaning were not adversely affected by the administration of CGA293343 technical at any dose level, in either the F0 or F1 generation. Male gonadal function, mating behaviour and outcome and sperm characteristics were not adversely affected by the administration of CGA293343 technical at any dose level.

Pathology Working Group Peer Review (PWGPR)

A Pathology Working Group Review was performed to evaluate atrophic lesions of the testes in F1 generation male rats from a dietary two-generation reproduction study of the test substance (Doubovetzky 1998). Parental (P generation) animals in the original study were administered the test substance in the diet at dose levels of 10, 30, 1000 and 2500 ppm. The PWG considered the increased incidence of tubular atrophy in F1 animals at the 1000 and 2500 ppm groups to be associated with the dietary administration of the test substance. Based upon the historical control data, findings at the 30 ppm level were considered equivocal while the 10 ppm dose group represented the "No Effect Level". (Seely J, 2000).

OECD 416 - rat - Historical control

Two additional studies were conducted (Moxon 2004b and 2004c) for the purpose of generating contemporaneous historical control data to aid interpretation of a two generation study conducted in the Tif:RAif rat (Moxon 2004a; described above). In each study, groups of 26 male and 26 female (FO parents) Tif:RAif rats were fed control diet throughout the study. After 10 weeks, the animals were mated and allowed to rear the ensuing F1A litters to weaning. The breeding programme was repeated with F1 parents selected from the F1A pups to produce the F2A litters, after a 10 week pre-mating period.

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.

Effects on developmental toxicity

Description of key information

Developmental toxicity rabbit (OECD 414): NOAEL for maternal toxicity = 15 mg/kg bw/day; NOAEL for developmental toxicity = 50 mg/kg bw/day; no evidence of teratogenicity

Developmental toxicity rat (OECD 414): NOAEL for maternal toxicity = 30 mg/kg bw/day; NOAEL for developmental toxicity = 200 mg/kg bw/day; no evidence of teratogenicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 Aug 1995 to 14 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
1984
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
1987
Qualifier:
according to
Guideline:
other: Japanese MAFF 59 Nohsan No. 4200
Version / remarks:
1985
Qualifier:
according to
Guideline:
other: European Communities Commission Directive 87/302/EEC, OJ No. L133/24
Version / remarks:
1988
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
other: Russian Chbb:HM
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: at least 3 months
- Weight at study initiation: 2424-3668 g
- Fasting period before study: not specified
- Housing: Individually in battery cages with steel slatted floors
- Diet: Pelleted certified standard feed (Nafag No. 814), ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 7 days prior to insemination

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 2°C
- Humidity: 50 ± 20%
- Air changes: Approximately 16/hour
- Photoperiod: 12 hours light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %(w/w) aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered in an aqueous solution of carboxymethylcellulose (0.5% w/w). Suspensions at the appropriate concentrations were freshly prepared every day using a high-speed homogeniser. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Verification analyses of the actual test material concentrations, stability and homogeneity in the prepared suspensions were performed on two dates during the administration. All samples were analysed by HPLC.
- Concentration analysis results: The mean concentrations of the test substance in suspensions were 98.3%, 97.2 %, 99.0% and 101.4 % of the nominal concentrations (1.25, 3.75, 12.5 and 37.5 mg/mL respectively).
- Homogeneity results: The homogeneity of the test substance in suspension was considered to be in an acceptable range (-2% to +2%).
- Stability results: The test substance was found to be stable in suspension for the period of dosing.
Details on mating procedure:
- Impregnation procedure: artificial insemination with diluted semen from bucks of the same strain
- The day of insemination was designated day 0 of pregnancy
Duration of treatment / exposure:
day 7 to day 19 of gestation
Frequency of treatment:
once daily
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
low
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low-mid
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
high-mid
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high
No. of animals per sex per dose:
19
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of the results of a dose range finding study.
- Justification species selection: The rabbit is one of the species recommended for teratology studies required by regulatory authorities. Background data for this strain were available at the test facility.
- Justification route selection: Administration by gavage was selected as the oral route is a possible route for human exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS
Animals were examined twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS
- Time schedule for examinations: daily

BODY WEIGHT
- Time schedule for examinations: daily

FOOD CONSUMPTION
Food consumption was recorded on days 4, 7, 12, 16, 20, 24 and 29.

POST-MORTEM EXAMINATIONS
- The animals were killed on day 29 by intravenous injection of a fast-acting barbiturate anaesthetic and foetuses were removed by hysterectomy.
- The following were recorded: Macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
The foetuses were numbered, sexed (on the basis of anogenital distance), externally examined and weighed. They were then killed and processed for visceral or skeletal examination. Foetal heads were assigned to either visceral or skeletal examination at an approximate 1:1 ratio within each litter, independent of sex and starting with skeletal. In the case of gross external anomaly or malformation, foetuses were allocated to one technique depending on the type and incidence of finding.
Statistics:
Analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-test in case of a significant result in the ANOVA. Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher’s Exact test in case of a significant result in the Chi-Square test. Non-parametric data (e.g. mean percent affected foetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One high dose group animal (150 mg/kg bw/day) presented with vaginal bloody discharge on day 19. A second high dose animal had vaginal bloody discharge on days 18 & 19. Bloody discharge in the perineal area was noticed for a third high dose animal on day 22 and was terminated on the same day, due to severe weight loss. Bloody discharge in the perineal area was detected in a total of 13/19 dams in the high dose group between days 14 and 23. These observations were considered treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One high dose group animal (150 mg/kg bw/day) presented with vaginal bloody discharge on day 19 and was found dead on day 20. A second high dose animal had vaginal bloody discharge on days 18 & 19 and was killed moribund on day 19. A third high dose animal was terminated due to severe weight loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The absolute mean body weight of high dose group dams (150 mg/kg bw/day) was slightly reduced from gestation day 16 to day 29 as compared to controls but without statistical significance. A marked body weight loss was noted between days 7 and 12 and during the whole treatment period. A reduced weight gain resulted from day 7 to 29 (not statistically significant). The reduction was attributed to treatment. A similar non-significant reduction was noted at 50 mg/kg bw/day for days 7-19.
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):
Mean maternal food consumption was significantly and dose-dependently reduced in the 50 and 150 mg/kg bw/day groups during the treatment period (days 7 to 12 and days 12 to 16) and from day 16 to day 20 in the 150 mg/kg bw/day group. A significant, compensatory increase in food consumption in the high dose group was recorded in the post-treatment period.
See Table 2 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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose females found dead or killed prior to day 29, haemorrhagic contents of the uterus were observed and considered treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Pre-implantation loss and the number of implantation sites were comparable for all groups. There was a higher post-implantation loss at the high dose due to an increase in early resorptions. Increased numbers of post-implantation losses in 3 high dose dams resulted from total resorption and were considered treatment-related.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Number of total resorption at term at highest doses are increased.
See Table 3 in "Any other information on results incl. tables" for more information.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Mean number of early resorptions was increased at the highest dose.
See Table 3 in "Any other information on results incl. tables" for more information.
Dead fetuses:
no effects observed
Description (incidence and severity):
Values of mean number of live foetuses remains almost the same with the dose range.
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Increased "number pregnant, premature deaths" at the highest dose.
See Table 3 in "Any other information on results incl. tables" for more information.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
pre and post implantation loss
early or late resorptions
necropsy findings
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
vagina
Description (incidence and severity):
bloody vaginal discharge in most animals in high dose group, haemorrhagic contents of the uterus found in three animals found dead or killed before scheduled termination.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the highest dose, mean foetal body weights were significantly lower than controls.
See Table 3 in "Any other information on results incl. tables" for more information.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
See Table 3 in "Any other information on results incl. tables" for more information.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related malformations, anomalies or variations
See Table 4 in "Any other information on results incl. tables" for more information.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related malformations. Significantly increased foetal (but not litter) incidence of fused sternebrae 3 and 4 in the high dose group was correlated with reductions in mean foetal body weight and considered related to maternal toxicity due to treatment. Other skeletal anomalies were unaffected by treatment. The significantly increased foetal (but not litter) incidence of absent ossification of the medial phalanx of anterior digit 5 in four high dose foetuses may indicate slight maternal toxicity. Other skeletal variations were considered unrelated to treatment.
See Table 4 in "Any other information on results incl. tables" for more information.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related malformations, anomalies or variations
See Table 4 in "Any other information on results incl. tables" for more information.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced mean body weight in high dose group
Description (incidence and severity):
Mean foetal body weights were significantly lower than controls in high dose groups. No teratogenicity observed.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Intergroup comparison of body weight gain(g) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

15

50

150

7-12

-26

-23

-17

-30

-105**

12-16

57

69

73

47

38

16-20

21

-2

-17**

3

-6

7-19

51

61

40

17

-67**

7-29

114

144

167

134

38

Net change

-112

-159

-150

-160

-172

** Statistically significant difference from control group mean, p<0.01

Net change = carcass weight minus day 7 body weight

 

Table 2: Intergroup comparison of food consumption (g/animal/day) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

15

50

150

7-12

88.7

92.4

88.5

69.2**

21.4**

12-16

86.7

91.2

79.1

64.2**

38.5**

16-20

96.3

95.9

88.5

80.6

56.8**

20-24

96.3

99.2

93.1

100.9

125.1*

24-29

93.4

96.8

100.3

103.3

121.6*

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 3: Ceasarean section observations

Observation

Dose level (mg/kg bw/day)

0 (control)

5

15

50

150

Number of females inseminated

19

19

19

19

19

Number not pregnant

4

0

0

0

1

Number pregnant, premature deaths

0

0

0

0

3

Number pregnant at term

15

19

19

19

15

Number with total resorption at term

0

0

0

1

3

Number with live foetuses at term

15

19

19

18

12

Gravid uterus weight (g)

226

303

316

294

210

Mean number of corpora lutea

6.7

7.2

7.2

6.9

7.1

Mean number of implantation sites

4.7

5.4

5.9

5.4

5.4

% Pre-implantation loss

32.6

23.1

17.5

23.7

25.1

Mean number live foetuses

3.7

5.1

5.4

4.6

3.0

Mean number early resorptions

1.0

0.3

0.5

0.6

2.4

Mean number late resorptions

0.0

0.0

0.1

0.2

0.0

% post implantation loss

21.0

6.3

9.9

16.3

45.6

Total number viable foetuses - males

31

48

46

46

22

Total number viable foetuses - females

24

49

56

40

23

% Males

56.4

49.5

45.1

53.5

48.9

Mean foetal body weight (g)

44.0

41.5

41.7

42.1

37.5**

Mean male foetal body weight (g)

44.4

41.7

43.0

42.2

38.8**

Mean female foetal body weight (g)

41.8

40.9

40.8

41.1

36.6*

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 4: Incidence of selected foetal skeletal anomalies and variations (% foetuses affected per litter)

Observation

Dose level (mg/kg bw/day)

0 (control)

5

15

50

150

Fused sternebra 3 and sternebra 4

0/55

(0.0)

0/97

(0.0)

2/102

(1.8)

1/88

(0.8)

5/45*

(14.6)

Anterior digit 5, medial phalanx, absent ossification

0/55

(0.0)

1/97

(1.3)

0/102

(0.0)

0/88

(0.0)

4/45*

(5.9)

* Statistically significant difference from control group mean, p<0.05

Conclusions:
- Maternal toxicity occurred at 150 mg/kg bw/day (3 premature deaths, bloody discharge, reduced food consumption, body weight and body weight gain) resulting in foetal toxicity (increased post-implantation loss, reduced foetal weight and increased incidence of two skeletal observations).
- Maternal toxicity also occurred at 50 mg/kg bw/day (reduced food consumption) but there was no foetal toxicity.
- On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 15 mg/kg bw/day and for developmental toxicity was 50 mg/kg bw/day.
- There was no evidence of teratogenicity.
Executive summary:

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rabbits. The test material was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 5, 15, 50 and 150 mg/kg body weight to 19 inseminated Russian Chbb:HM rabbits per group from day 7 to 19 of pregnancy inclusive, using a dose volume of 4 mL/kg body weight. Dams were killed on day 29 of pregnancy, just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent examination. In the 150 mg/kg bw/day, there were a number of treatment-related findings. Bloody discharge in the perineal area or from the vagina was detected in 15 dams. Three of those dams were found dead, killed in moribund condition or terminated prematurely due to severe weight loss and bloody discharge. Mean body weights were reduced from day 16 to 29 and there was a mean body weight loss during the treatment period (days 7-19). A slight non-significant mean body weight gain reduction occurred in the 50 mg/kg bw/day dose group. Food consumption was dose-dependently reduced during treatment in the 150 and 50 mg/kg bw/day groups. A compensatory increase in food consumption was noticed during post-treatment in the high dose group.Increased treatment-related post implantation losses resulted from total resorption in 3 dams of the high dose group. At necropsy, haemorrhagic contents were noted in the uteri of the 3 dams not surviving to scheduled termination. Foetal weights in the 150 mg/kg bw/day group were significantly reduced compared to controls and this finding was attributed to maternal toxicity. The incidence and type of external, visceral and skeletal findings was generally not affected by the treatment but a few isolated skeletal findings may indicate a treatment-related delay in ossification. Maternal toxicity occurred at 150 mg/kg bw/day (3 premature deaths, bloody discharge, reduced food consumption, body weight and body weight gain) resulting in foetal toxicity (increased post-implantation loss, reduced foetal weight and increased incidence of two skeletal observations). Maternal toxicity also occurred at 50 mg/kg bw/day (reduced food consumption) but there was no foetal toxicity. On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 15 mg/kg bw/day and for developmental toxicity was 50 mg/kg bw/day. There was no evidence of teratogenicity.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 Jul 1995 to 24 Oct 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
1984
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Version / remarks:
1987
Qualifier:
according to
Guideline:
other: Japanese MAFF 59 Nohsan No. 4200
Version / remarks:
1985
Qualifier:
according to
Guideline:
other: European Communities Commission Directive 87/302/EEC, OJ No. L133/24
Version / remarks:
1988
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Tif:RAIf (SPF), hybrids of RII/1 x RII/2
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: at least 8 weeks
- Weight at study initiation: 177-217 g
- Fasting period before study: not specified
- Housing: Individually in Macrolon cages with wire mesh tops
- Diet: Pelleted certified standard feed (Nafag No. 890), ad libitum
- Water: Tab water, ad libitum
- Acclimation period: at least 7 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 50 ± 20%
- Air changes: Approximately 16/hour
- Photoperiod: 12 hours light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % (w/w) aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test material was administered as a suspension in the vehicle.
- Suspensions at the appropriate concentrations were freshly prepared every day using a high-speed homogeniser.
- Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.
- Dosage volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Verification analyses of the actual test material concentrations, stability and homogeneity in the prepared suspensions were performed on two dates during the administration. All samples were analysed by HPLC.
- Concentration analysis results: The mean concentrations of the test substance in suspensions were 98.1%, 94.7 %, 95.0% and 99.5 % of the nominal concentrations (0.5, 3, 20 and 75 mg/mL respectively).
- Homogeneity results: The homogeneity of the test substance in suspension was considered to be in an acceptable range (-4% to +5%).
- Stability results: the test substance was found to be stable in suspension for the period of dosing.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: nulliparous females were mated overnight with males of the same stock and proven fertility
- M/F ratio per cage: 3 females to 1 male
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: presumed pregnant females were removed from the mating cages and the procedure repeated for remaining females until sufficient mated dams were produced.
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 to 15 of gestation
Frequency of treatment:
once daily
Duration of test:
21 days
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low-mid dose
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
High-mid dose
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected on the basis of the results of a dose range finding study.
- Species selection rationale: The rat is one of the species recommended for teratology studies required by regulatory authorities. Background data for this strain were available at the test facility.
- Route of exposure rationale: administration by gavage was selected as the oral route is a possible route for human exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS
Animals were examined twice daily for mortality

DETAILED CLINICAL OBSERVATIONS
Time schedule for examinations: daily

BODY WEIGHTS
Time schedule for examinations: daily

FOOD CONSUMPTION
Food consumption was recorded on days 6, 11, 16 and 21 p.c.

POST-MORTEM EXAMINATIONS
- The animals were killed on day 21 by carbon dioxide inhalation, and foetuses were removed by hysterectomy
- Macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the genitals was performed

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- The foetuses were numbered, sexed (on the basis of anogenital distance), externally examined and weighed. They were then killed and processed for visceral or skeletal examination.
- Foetuses were assigned to either visceral or skeletal evaluation at an approximate 1: 1 ratio within each litter, independent of sex (starting with skeletal). In the case of gross external anomaly or malformation, foetuses were allocated to one technique depending on the type and incidence of finding.
Statistics:
Analysis of continuous data (e.g. body weight, feed consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-test in case of a significant result in the ANOVA. Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher’s Exact test in case of a significant result in the Chi-Square test. Non-parametric data (e.g. mean percent affected foetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One high dose group animal (750 mg/kg bw/day) showed regurgitation of substance, hypoactive behaviour and piloerection on day 7 and was found on day 9 to have hunched posture, weight loss and piloerection. It was killed for humane reasons. At this highest dose, 17 dams had transient hypoactive behaviour and piloerection one hour after treatment start and lasting up to 4 days in a few animals. Regurgitation of substance was recorded for two other high dose animals. There were no other treatment-related findings.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal was killed for humane reasons, after showing regurgitation of substance, hypoactive behaviour and piloerection on day 7 and was found on day 9 to have hunched posture, weight loss and piloerection.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, body weight was significantly lower than controls from one day after the first dose until termination. Mean maternal body weight gain was significantly reduced in the 200 and 750 mg/kg bw/day groups from days 6-11 and days 6-16 and in the 750 mg/kg bw/day group from days 6-21. The net weight change from day 6 was significantly reduced in the 200 and 750 mg/kg bw/day groups compared to controls.
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):
Mean maternal food consumption was transiently reduced in the 200 and 750 mg/kg bw/day groups from day 6-11 p.c. compared to controls. Reduced food consumption lasted from day 6 p.c. up to day 16 in the 750 mg/kg bw/day group. A slight compensation after treatment was noted in both groups.
See Table 2 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:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the high dose dam killed on day 9 p.c. for welfare reasons, the oesophagus was found to be filled with bedding material. There were no other remarkable observations
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre & post-implantation losses, live litter size and sex ratios were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Values were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean foetal body weight significantly lower in the top dose group compared to controls, due to the maternal toxicity observed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Values were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Values were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Values were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Values were similar in all groups, see Table 3 in "Any other information on results incl. tables" section
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related malformations, anomalies or variations.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related malformations. Skeletal anomalies regarded as a consequence of maternal toxicity due to 750 mg/kg bw/day included an increased incidence of asymmetrically shaped sternebra 6 and irregular ossification of the occipital bone. The skeletal variations considered to be treatment-related included an increased incidence of poor ossification of sternebra 5, absent ossification of metatarsal 1, shortened rib 13, absent ossification of the proximal phalanx of anterior digits 2 & 5, poor or absent ossification of the distal or proximal phalanx of posterior digits 1 – 5.
See Table 4 in "Any other information on results incl. tables" for more information.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformation was limited to diaphragmatic hernia in one foetus in the high dose group. Occasional anomalies were considered not to be treatment-related due to scattered occurrence, lack of dose-relationship and historical control data. Variations included enlarged thymus in 7, 6, 4 and 5 foetuses from the 5, 30, 200 and 750 mg/kg/day groups respectively. Accessory lobulets on the liver were observed in 1, 3, 1, 2 and 3 foetuses in the 0, 5, 30, 200 and 750 mg/kg/day groups respectively. These variations were also considered not to be treatment-related for the same reasons as the anomalies.
See Table 5 in "Any other information on results incl. tables" for more information.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
changes in litter size and weights
skeletal malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Intergroup comparison of body weight gain(g) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

30

200

750

6-11

25.6

25.2

25.4

18.5**

-1.0**

6-16

63.4

62.0

63.8

53.4**

33.9**

6-21

135.8

133.8

134.6

128.5

110.3**

Net change

36.8

40.1

36.3

25.9*

20.0**

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Net change = carcass weight minus day 6 body weight

Table 2: Intergroup comparison of food consumption(g/animal/day) – selected time points

 

Dose level (mg/kg bw/day)

Days

0 (control)

5

30

200

750

6-11

23.4

24.0

23.4

19.9**

10.8**

11-16

25.6

25.7

25.7

23.9

21.1**

16-21

25.9

27.3

25.9

26.3

28.8**

** Statistically significant difference from control group mean, p<0.01

 

Table 3: Caesarean section observations

Observation

Dose level (mg/kg bw/day)

0 (control)

5

30

200

750

Number of females mated

24

24

24

24

24

Number pregnant at term

22

23

23

22

22

Gravid uterus weight (g)

99.0

93.7

98.3

102.6

90.2

Mean number of corpora lutea

15.5

14.7

15.0

16.0

15.4

Mean number of implantation sites

14.5

13.5

14.4

14.9

14.3

% Pre-implantation loss

7.1

8.3

3.5

6.7

9.3

Mean number live foetuses

14.0

13.1

14.0

14.6

13.9

Mean number early resorptions

0.5

0.4

0.4

0.3

0.4

Mean number late resorptions

0.0

0.0

0.0

0.0

0.0

% post implantation loss

3.3

3.1

2.9

2.2

2.8

Total number viable foetuses - males

158

130

155

172

144

Total number viable foetuses - females

151

172

167

149

161

% Males

51.1

43.0

48.1

53.6

47.2

Mean foetal body weight (g)

5.3

5.3

5.2

5.2

4.8**

Mean male foetal body weight (g)

5.4

5.4

5.3

5.3

4.9**

Mean female foetal body weight (g)

5.1

5.2

5.1

5.1

4.7**

** Statistically significant difference from control group mean, p<0.01

 

Table 4: Selected foetal skeletal anomalies and variations (% foetuses affected per litter)

Observation

Dose level (mg/kg bw/day)

0 (control)

5

30

200

750

Asymmetrically shaped sternebra 6

2.5

2.0

1.3

1.7

6.8

Irregular ossification occipital bone

1.9

0.0

1.2

1.1

7.8

Poor ossification sternebra 5

0.0

0.0

0.5

0.0

5.7

Absent ossification metatarsal 1

9.6

20.8

14.8

9.6

35.7

Shortened rib 13

8.3

2.9

2.6

13.6

16.5

Absent ossification, proximal phalanx anterior digit 2

5.3

8.8

4.5

3.7

17.0

Absent ossification, proximal phalanx anterior digit 5

9.9

13.3

6.9

8.9

25.9

Poor ossification, distal phalanx posterior digit 1

1.3

0.6

2.7

1.2

6.0

Absent ossification, proximal phalanx posterior digit 2

38.1

42.9

41.0

40.7

78.0

Poor ossification, distal phalanx posterior digit 2

1.3

0.6

2.7

1.8

6.2

Absent ossification, proximal phalanx posterior digit 3

28.7

36.5

30.2

33.6

64.0

Poor ossification, distal phalanx posterior digit 3

1.3

0.6

2.7

1.2

6.8

Absent ossification, proximal phalanx posterior digit 4

28.9

34.8

32.7

38.6

64.3

Poor ossification, distal phalanx posterior digit 4

1.3

0.6

2.7

1.2

6.8

Absent ossification, proximal phalanx posterior digit 5

57.6

54.7

62.7

55.3

92.2

No statistically significant differences from control group mean

 

 

Table 5: Selected foetal visceral variations (% foetuses affected per litter)

Observation

Dose level (mg/kg bw/day)

0 (control)

5

30

200

750

Enlarged thymus

0.00

4.87

4.00

2.54

3.35

Liver accessory lobulet

0.65

1.96

0.62

1.22

1.96

No statistically significant differences from control group mean

 

Conclusions:
- Maternal toxicity (reduced food consumption and body weight gain) was seen at 200 and 750 mg/kg bw/day.
- At the highest dose level only, an increased incidence of foetal skeletal anomalies and variations and reduced foetal weight were consistent with delayed development resulting from or secondary to, significant maternal toxicity.
- On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day and for developmental toxicity was 200 mg/kg bw/day.
- There was no evidence of teratogenicity.
Executive summary:

In a GLP compliant teratogenicity study, performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rats. The test substance was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 5, 30, 200 and 750 mg/kg body weight to 24 mated Tif:RAIf rats per group from day 6 to 15 of pregnancy inclusive, using a dose volume of 10 mL/kg body weight. Dams were killed on day 21 p.c., just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent examination. The report amendment describes an additional procedure designed to obtain more information on the prenatal development of the thymus. The thymus weight of foetuses previously used for visceral examination was recorded.  At the highest dose of 750 mg/kg bw/day, transient hypoactive behaviour was recorded for 17/24 dams which lasted for 1 hour to 4 days after the first dose administration. Regurgitation of substance was observed in 2 dams. One dam was killed on day 9 following marked weight loss. Body weight of the high dose dams was significantly reduced from day 6 through to termination. Body weight gain at 200 and 750 mg/kg bw/day was reduced between days 6 and 16. Maternal food consumption was depressed at 750 mg/kg bw/day from days 6 to 16 and at 200 mg/kg bw/day from days 6 to 11. Mean carcass net weight change from day 6 was reduced at 200 and 750 mg/kg bw/day. Necropsy examination revealed no significant findings in surviving animals. For all groups, there was no evidence of an adverse effect on any of the reproductive parameters (pre-implantation loss, implantation sites, post-implantation loss, live litter size and sex ratios). At 750 mg/kg bw/day, mean foetal body weights were significantly lower than those of the controls. There were no foetal external or visceral observations that were considered to be related to treatment. At skeletal examination, there was an increased incidence of asymmetrically shaped sternebra; irregular, poor or absent ossification of cranial bones, sternebra, metatarsals and phalanges; shortened ribs at 750 mg/kg bw/day. These findings were considered to represent a treatment-related delay of foetal development (ossification) resulting from or secondary to significant maternal toxicity. No specific effects on prenatal development of the thymus were detected. Maternal toxicity (reduced food consumption and body weight gain) was seen at 200 and 750 mg/kg bw/day. At the highest dose level only, an increased incidence of foetal skeletal anomalies and variations and reduced foetal weight were consistent with delayed development resulting from or secondary to, significant maternal toxicity. On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day and for developmental toxicity was 200 mg/kg bw/day. There was no evidence of teratogenicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
GLP compliant, OECD 414 study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 - rat

In a GLP compliant teratogenicity study (Winkler, 1996), performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rats. The test substance was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 5, 30, 200 and 750 mg/kg body weight to 24 mated Tif:RAIf rats per group from day 6 to 15 of pregnancy inclusive, using a dose volume of 10 mL/kg body weight. Dams were killed on day 21 p.c., just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent examination. The report amendment describes an additional procedure designed to obtain more information on the prenatal development of the thymus. The thymus weight of foetuses previously used for visceral examination was recorded. At the highest dose of 750 mg/kg bw/day, transient hypoactive behaviour was recorded for 17/24 dams which lasted for 1 hour to 4 days after the first dose administration. Regurgitation of substance was observed in 2 dams. One dam was killed on day 9 following marked weight loss. Body weight of the high dose dams was significantly reduced from day 6 through to termination. Body weight gain at 200 and 750 mg/kg bw/day was reduced between days 6 and 16. Maternal food consumption was depressed at 750 mg/kg bw/day from days 6 to 16 and at 200 mg/kg bw/day from days 6 to 11. Mean carcass net weight change from day 6 was reduced at 200 and 750 mg/kg bw/day. Necropsy examination revealed no significant findings in surviving animals. For all groups, there was no evidence of an adverse effect on any of the reproductive parameters (pre-implantation loss, implantation sites, post-implantation loss, live litter size and sex ratios). At 750 mg/kg bw/day, mean foetal body weights were significantly lower than those of the controls. There were no foetal external or visceral observations that were considered to be related to treatment. At skeletal examination, there was an increased incidence of asymmetrically shaped sternebra; irregular, poor or absent ossification of cranial bones, sternebra, metatarsals and phalanges; shortened ribs at 750 mg/kg bw/day. These findings were considered to represent a treatment-related delay of foetal development (ossification) resulting from or secondary to significant maternal toxicity. No specific effects on prenatal development of the thymus were detected. Maternal toxicity (reduced food consumption and body weight gain) was seen at 200 and 750 mg/kg bw/day. At the highest dose level only, an increased incidence of foetal skeletal anomalies and variations and reduced foetal weight were consistent with delayed development resulting from or secondary to, significant maternal toxicity. On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 30 mg/kg bw/day and for developmental toxicity was 200 mg/kg bw/day. There was no evidence of teratogenicity.

OECD 414 - rabbit

In a GLP compliant teratogenicity study (Winkler, 1996), performed in accordance with OECD 414, the test substance was tested for its embryonic, foetotoxic, and teratogenic potential in rabbits. The test material was administered by gavage in an aqueous solution of carboxymethylcellulose (0.5% w/w) at daily doses of 0, 5, 15, 50 and 150 mg/kg body weight to 19 inseminated Russian Chbb:HM rabbits per group from day 7 to 19 of pregnancy inclusive, using a dose volume of 4 mL/kg body weight. Dams were killed on day 29 of pregnancy, just prior to the expected delivery and foetuses were removed by Caesarean section for subsequent examination. In the 150 mg/kg bw/day, there were a number of treatment-related findings. Bloody discharge in the perineal area or from the vagina was detected in 15 dams. Three of those dams were found dead, killed in moribund condition or terminated prematurely due to severe weight loss and bloody discharge. Mean body weights were reduced from day 16 to 29 and there was a mean body weight loss during the treatment period (days 7-19). A slight non-significant mean body weight gain reduction occurred in the 50 mg/kg bw/day dose group. Food consumption was dose-dependently reduced during treatment in the 150 and 50 mg/kg bw/day groups. A compensatory increase in food consumption was noticed during post-treatment in the high dose group. Increased treatment-related post implantation losses resulted from total resorption in 3 dams of the high dose group. At necropsy, haemorrhagic contents were noted in the uteri of the 3 dams not surviving to scheduled termination. Foetal weights in the 150 mg/kg bw/day group were significantly reduced compared to controls and this finding was attributed to maternal toxicity. The incidence and type of external, visceral and skeletal findings was generally not affected by the treatment but a few isolated skeletal findings may indicate a treatment-related delay in ossification. Maternal toxicity occurred at 150 mg/kg bw/day (3 premature deaths, bloody discharge, reduced food consumption, body weight and body weight gain) resulting in foetal toxicity (increased post-implantation loss, reduced foetal weight and increased incidence of two skeletal observations). Maternal toxicity also occurred at 50 mg/kg bw/day (reduced food consumption) but there was no foetal toxicity. On the basis of these results, the no observed effect level (NOAEL) for maternal toxicity was 15 mg/kg bw/day and for developmental toxicity was 50 mg/kg bw/day. There was no evidence of teratogenicity.

Justification for classification or non-classification

Based on the available information, classification for reproductive toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.