Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Mar - 15 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40850/B TE
- Physical state: solid powder
- Colour: red
- Analytical purity: 70.6% all coloured organic constituent, 32.4% main constituent
- Composition of test material, percentage of components: 70.6% all coloured organic constituent, 32.4% main constituent
- Purity test date: 07.09.2010
- Lot/batch No.: BOP 01-10
- Expiration date of the lot/batch: 15.07.2015
- Storage condition of test material: room temperature
- Density: 1.738 g/cm³ at 20°C
- Solubility in Water: 128.1 g/L at 20°C

Test animals

Species:
rat
Strain:
other: Wistar Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 257-304 g; Females: 175-213 g
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 081110)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot. No. 1307), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The amount of test substance for each dose concentration was suspended separately in aqua ad injectionem (sterile water) on each administration day, immediately before the administration. Homogeneity was ensured by using a vortex machine.

VEHICLE
- Justification for use and choice of vehicle: Selection was based on the solubility of the test item.
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 0195A191
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- Females with unsuccessful mating were allowed to mate with other male of the same group.
- Females showing no evidence of copulation up to 14 day mating period were sacrificed 26 days after the last day of the mating period.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each concentration was analysed for nominal concentration. Homogeneity in the vehicle was analysed for the low and high dose concentrations. Samples for nominal concentration verification were taken in Week 1 (first week of pre-mating period), Week 3 (first week of mating), Week 5 (gestation) and Week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the low and high dose preparation in Weeks 1 and 5.
All concentration samples were stored frozen (approx. -20 °C) until the analysis was performed.
Duration of treatment / exposure:
Males: 14 days before mating, 14 days during mating
Females: 14 days before mating, 14 days during mating, 20 days during gestation, 3 days during lactation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
other: yes, concurrent vehicle; the control group was shared with BSL Study no. 110998, another OECD 421 study, which was performed in parallel.
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. A descending sequence of dose levels was selected in order to demonstrate any dose-related response and a NOAEL.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily during weekend/holidays

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before assignment to the experimental groups and on the first day of administration.
Males were weighed weekly during the entire study period.
Females were weighed weekly during the pre-mating period, on Gestation Day 0, 7, 14, 20 and on Post-natal Day 0 (within 24 hours of parturition) and Post-natal Day 4 along with pups.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration and was not measured during the mating period.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
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, runts, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after the completion of mating period (Day 28)
- Maternal animals: All surviving animals were sacrificed on respective PND 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), macroscopic lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspringwere sacrificed at 4 days of age (PND 4).

The animals were subjected to postmortem examinations for gross external abnormalities.
Statistics:
One-way analysis of variance (ANOVA) followed by DUNETT’s multiple comparison test (p<0.05 was considered as statistical significant).
Reproductive indices:
Copulation Index (%) = (No. of rats copulated /No. of pairs) x 100
Fertility Index (%) = (No. of females pregant/No. of females copulated) x 100
Delivery Index(%) = (No. of dams with live newborns/ No. of pregnant dams) x 100
Offspring viability indices:
Viability Index (%) = (No. of live offspring at Day 4/ No. of live offspring at birth) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discoloration of various organs in all dose groups due to deposition of coloured test item (see details below)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
histopathology correlates to discoloration in organs (see details below)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No treatment related clinical signs were observed. There were very few clinical signs recorded in control and treated animals during the study period but these findings were observed transiently post dose administration and had no relevance to treatment.
None of the animals died during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect on body weight was observed in both sexes throughout the complete study period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
No treatment-related effects on testis or epididymidis weight were noted. No other sperm parameters were examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No treatment related effect on precoital interval and duration of gestation was observed.
The copulation index, fertility index and delivery index remained unaffected. No effects on mean No. of corporea lutea, No. of implantation sites, No. of live pups born, % pre- and post-implantation loss were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS):
No statistically significant changes in absolute and relative organ weights were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS):
In males, various macroscopic findings observed were yellow spots on epididymides (C:1/10, LD:1/10, MD:1/10), discoloration of epididymides (HD:5/10 animals), reddish discoloration of kidneys (LD: 4/10, MD: 6/10, HD: 10/10 animals), reddish discoloration of testes (HD: 3/10 animals), small testes and epididymides (HD: 1/10 animals).
In females, discoloration of kidneys (LD: 7/10, MD: 6/10, HD: 10/10 animals), cyst on kidneys (LD: 1/10 animal), ureter dilated (HD: 1/10 animal), dark discoloration of lung (HD: 1/10 animal), hardening of ovary (HD: 1/10 animal).
The discoloration of organs as well as reddish discoloration of the digestive tract noted in almost all treated animals is considered to be attributable to the colour of the test item and as such not a systemic effect due to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS):
There were foamy interstitial macrophages observed in testes of one male of MD group and in 9/10 males of HD group. There were no test item-related histological findings noted in other male and female reproductive organs.
Eosinophilic granules in the tubular epithelium of the renal cortex were seen in all animals of HD group and in a proportion of animals of MD group, but not in animals of LD group although macroscopically red discolored. This finding was considered to be due to test item deposition in renal tubuloepithelial cells. Moreover, in all females of the high dose group, renal corticotubular epithelium was diffusely vacuolated, possibly caused by washout of the test item during the histotechnical procedure.
The lung of one high dose female, being macroscopically dark discolored, showed minimal numbers of eosinophilic alveolar macrophages, considered to represent test item deposition.
All histological findings are due to test item deposition and there were no accompanying findings that would indicate organ damage.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects noted.
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects noted.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING):
No treatment related effect was observed on total No. of pups born, No. of males, No. of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4. Viability index was unaffected.

BODY WEIGHT (OFFSPRING):
No effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4.

GROSS PATHOLOGY (OFFSPRING):
No substance-related gross external findings were observed in any of the treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects noted.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Clinical Observations (P)

Clinical Findings

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Males

Aggressive behavior

0/10

0/10

3/10

0/10

Nasal discharge

1/10

0/10

0/10

0/10

Piloerection

0/10

0/10

0/10

1/10

Salivation

0/10

0/10

1/10

0/10

Alopecia

1/10

0/10

1/10

0/10

Females

Aggressive behavior

1/10

0/10

0/10

0/10

Nasal discharge

0/10

2/10

0/10

0/10

Piloerection

0/10

1/10

0/10

1/10

Alopecia

0/10

1/10

2/10

2/10

 

Table 2: Macroscopic Findings (P)

Findings

Control

100 mg/kg

300 mg/kg

1000 mg/kg

Males

Epididymides: yellow spots

1/10

1/10

1/10

0/10

Epididymides: discoloration (reddish)

0/10

0/10

0/10

5/10

Epididymides: small

0/10

0/10

0/10

1/10

Kidney: discoloration (reddish)

0/10

4/10

6/10

10/10

Testes: discoloration (reddish)

0/10

0/10

0/10

3/10

Testes: small

0/10

0/10

0/10

1/10

Females

Kidney: discoloration (reddish)

0/10

7/10

6/10

10/10

Ureters: dilated

0/10

0/10

0/10

1/10

Lung: discoloration (dark)

0/10

0/10

0/10

1/10

Ovary: hard

0/10

0/10

0/10

1/10

 

Table 3: Reproductive Indices (%)

 

Control

100 mg/kg bw

300 mg/kg bw

1000 mg/kg bw

Copulation Index

100

100

100

100

Fertility Index

60

80

70

60

Delivery Index

100

100

100

100

Viability Index

100

100

100

100

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on reproductive performance.
CLP: not classified
DSD: not classified