Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version 27 July 1995
Deviations:
no
GLP compliance:
yes
Remarks:
Research Toxicology Center S.p.A., Pomezia, Italy
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary
Specific details on test material used for the study:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Analytical purity: 95%
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Test animals

Species:
rat
Strain:
other: HsdBrl: wistar Han rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: (P) ♂: 183 - 197 g; ♀: 155 - 164 g
- Housing: in a limited access rodent facility
- Pre-mating period: 5 animals per sex per cage
- Mating period: one male to one female
- Post mating: animals were housed individually
- Diet: commercially available laboratory powdered rodent diet (4 RF 21, Mucedola R.r.l., Italy)
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 2011-03-24 to 2011-05-21

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diet were prepared according to the stability of the test substance in the diet.
- Mixing appropriate amounts with: commercially available laboratory powdered rodent diet (4 RF 21, Mucedola R.r.l., Italy)
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: monogamous
- Length of cohabitation: until mating
- Proof of pregnancy: vaginal plug or sperm in vaginal smear were referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged: in individual clear polycarbonate cages with stainless mesh lid and floor
- Any other deviations from standard protocol: - for females from day 14 to day 20 post coitum and from day 0 to day 4 post partum the achieved dosage was calculated at 6 and 4 days interval, respectively and not weekly as indicated in the study protocol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis were performed to confirm that the proposed formulation procedure was acceptable and that the homogeneity and stability of the formulation were satisfactory. Samples of the formulations prepared in week 1 and last week of the study were analysed to check the homogeneity and concentration. Chemical analysis were carried out by the Analytical Chemistry Department at RTC. The software used for this activity was the Empower® Pro build No. 2154.
Duration of treatment / exposure:
♂: 7 days a week for 2 consecutive weeks prior to pairing and thereafter until day of necropsy (total of 30-31 days of treatment).
♀: 7 days a week for 2 consecutive weeks prior to pairing and thereafter during mating, gestation and post partum until day 4 post partum.
Frequency of treatment:
daily, 7 days a week
Details on study schedule:
- Age at mating of the mated animals in the study: 10 - 11 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
Dose / conc.:
15 000 ppm (nominal)
Dose / conc.:
123 mg/kg bw/day
Remarks:
♂. mean dose value as calculated from the reported body weight and standard food intake values
Dose / conc.:
420 mg/kg bw/day
Remarks:
♂: mean dose value as calculated from the reported body weight and standard food intake values
Dose / conc.:
1 271 mg/kg bw/day
Remarks:
♂: mean dose value as calculated from the reported body weight and standard food intake values
Dose / conc.:
151 mg/kg bw/day
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values
Dose / conc.:
537 mg/kg bw/day
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values
Dose / conc.:
1 496 mg/kg bw/day
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose levels were selected in consultation with the sponsor.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on day of allocation to the treatment groups. ♂: additionally on day that treatment commenced, weekly thereafter and just prior to necropsy. ♀: weekly from the first day of treatment to mating, on days 0, 7, 14, and 20 post coitum and on days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption calculated as ppm food/kg body weight/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
The distribution of the oestrus cycle was determined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
Yes, for size and external abnormalities. Possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after the mating of all females (30 - 31 days of treatment).
- Maternal animals: All surviving animals, females with live pups were killed on day 4 post partum, females with no birth 25 days after positive identification of mating were killed shortly after, one female with litter loss was killed on day1 post partum.

GROSS NECROPSY
- Gross necropsy consisted of external examination of surfaces and orifices and requisite organs with changes were weighed and tissue samples preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: external abnormalities, sex confirmation by gonadal inspection
Statistics:
For continous variables significance of differences amongst group means were assessed by Dunnett's test or a modified t-test depending on homogeneity of data. Non-continous variables were statistically analyzed by means of Kruskal-Wallis Test and intergroup differences between the control and treated groups assessed by a non-parametric version of Williams test. Statistical analyzes of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The mean values and standard deviations were calculated from actual values in the computer without rounding off.
Reproductive indices:
♂:
Copulatory Index (%) = no. of animals mated x 100/No. of animals paired
Fertility Index (%) = no. of males which induced pregnancy x 100/No. of males paired

♀:
Copulatory Index (%) = no. of animals mated x 100/No. of animals paired
Fertility Index (%) = no. of pregnant females x 100/No. of females paired

♂ and ♀:
Copulatory Interval = Mean number of days between pairing and mating
Offspring viability indices:
Pre-birth loss (%): (No. of visible implantations - total litter size) x 100/No. of visible implantations

Pup loss at birth (%): (Total litter size - live litter size) x 100/Total litter size

Cumulative pup loss on day 4 post partum (%): (Total litter size at birth - live litter size at day 4) x 100/Total litter size at birth

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Yellow staining of the fur was evident in all treated males and in mid and high dose females during entire treatment period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A slight statistically significant reduction in body weight was noted in the high dose females on gestation day 20. This change was not considered to be of toxicological relevance since it was related to the presence of one female with total litter loss and the smal size of few litters.
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Minimal degenerative changes were noted in the seminoferous epithelium of the testes of one male of the high dose group. However, regular layering of the different cell populations in the germinal epithelium in each stage leads to consider such effect as spontaneous pathology.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The distribution of the oestrus cycle and reproductive parameters were comparable between all animals mated.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the control group, 2 in the low dose group, one in the mid-dose group and one in the high dose group were not pregnant. Unilateral implantation was present in one mid-dose and one high-dose female. One mid-dose female had unilateral total resorption and one high-dose female had total litter loss.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested
Dose descriptor:
NOAEL
Effect level:
1 271 mg/kg bw/day
Based on:
other: mean dose value as calculated from the reported body weight and standard food intake values
Sex:
male
Basis for effect level:
other: NOAEL corresponds to the highest achieved dose level
Dose descriptor:
NOAEL
Effect level:
1 496 mg/kg bw/day
Based on:
other: mean dose value as calculated from the reported body weight and standard food intake values
Sex:
female
Basis for effect level:
other: NOAEL corresponds to the highest achieved dose level

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Sexual maturation:
no effects observed
Gross pathological findings:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
> 15 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
mortality

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 2: Implantation, pre-birth loss and gestation length - Group mean data

 Group    Corpora Lutea  Implantations  Total Litter Size  Pre-birth loss (%)  Gestation length (days)
 1 Mean 13.22 13.00 11.89 8.70 22.0
  Std  1.56  1.58 1.83 7.40 0.71
  9 9 9 9 9
2 Mean 11.50  11.00 9.38 16.95  22.0
   Std  3.25  3.96  3.54  11.9  0.53
   N  8  8  8  8  8
 3  Mean  12.25  12.00  11.25  7.29  22.00
   Std 3.15   3.07  3.15  6.23  0.00
 N  8  8  8  8  8
 4  Mean  12.50  11.00  9.50  14.90  22.0
   Std  2.83  4.00  4.31  20.31  0.53
   N  8  8  8  8  8

Table 3: Reproductive Parameters of males - Summary data

   groups  1  2  3  4
 copulatory index (%)     100.0  100.0  100.0  100.0
 fertiliy index (%)     90.0  80.0  90.0  90.0

Table 4: Reproductive Parameters of females - Summary data

   groups  1  2  3  4
 copulatory index (%)     100.0  100.0  100.0  100.0
 fertiliy index (%)     90.0  80.0  90.0  90.0

Applicant's summary and conclusion