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Toxicological information

Toxicity to reproduction

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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
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name of test substance: Pigment Yellow 155
Physical state/ appearance: solid/ yellow
Expiry date: 23 Mar 2021
Storage conditions: room temperature
Specific details on test material used for the study:
- Lot/batch No.: DEB2 146870
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Pigment Yellow 155 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.

VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100mL, respectively

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: indiviually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, littering and lactation behavior of the dams, parturition behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, except during mating period, during gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: Functional observational battery was performed in the first five parental males and females (with litter) per group at the end of the administration period. The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period.


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, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals: Males approximately one week post-mating and females after 4 days of lactation and two weeks thereafter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Oviducts, Peyer’s patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Organn weights: Epididymides, Testes, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.
Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter where analysed by simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy where analysed by pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
Proportions of affected pups per litter with necropsy observations where analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity where analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, post implantation loss
Offspring viability indices:
Viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
yellow discolored feces, no mortality
Body weight and weight changes:
no effects observed
Description (incidence and severity):
except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No animal died prematurely in the present study. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. Except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two males of test group 1 (Nos. 12, 20 mated with females Nos. 112, 120) did not generate F1 pups.
The male fertility index ranged between 80% and 100% (see Tab. 1).
The apparently infertile male animals Nos. 12 and 20 (test group 1; 100 mg/kg bw/d) were not examined histopathologically.
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 3.2, 3.8, 2.9 and 4.2 days in test groups 0-3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. All sperm positive rats delivered pups with the exception of females Nos. 112 and 120 (test group 1), which were mated with male Nos. 12 and 20 did not become pregnant. The female fertility index varied between 80% and 100% (Table 1). Female animals Nos. 112 and 120, which delivered no pups, showed no implantation sites. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.3 days). The gestation index was 100% in all test groups.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.0% in test group 1, 99.1% in test group 3 and 99.2% in control and test group 2. Moreover, the number of stillborn pups was comparable between the groups. The postimplantation loss was 2.3% in test group 0, 0.98% in test group 1, 5.6% in test group 2 and 6.5% in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

ORGAN WEIGHTS (PARENTAL ANIMALS): The weight decrease in absolute epididymis and liver weight in males of test group 2 (300 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw). The decrease in thymus weights of females in test group 3 (1000 mg/kg bw/day) was also regarded to be incidental as no histopathologic correlate could explain the weight decrease.
All other mean weight parameters did not show significant differences when compared to the control groups.

GROSS PATHOLOGY (PARENTAL ANIMALS):Several animals of test group 3 (1000 mg/kg bw) revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related.
Each one male and female of test groups 1 and 2 (100 and 300 mg/kg bw/day) (animals No. 12, 21, 111, 125) revealed either some or all of the following findings: deposition or foci on the lung, deposition on the diaphragm, discoloration and enlargement of the mediastinal lymph nodes, deposition on the thymus, and deposition on the sternum. All these findings were regarded to be treatment related by gavage errors.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS): The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
The discoloration and depositions described for animals No. 12, 21, 111, 125 in several organs of the thoracic cavity were multifocal granulomas with intrahistiocytically located yellow particles, interpreted as test substance. The granulomas were foreign body reactions interpreted as a consequence to a gavage error. The discoloration of the mediastinal lymph nodes was regarded to be the physiologic clearing route of the lung. Particles that were located intraalveolar were transported via macrophages to the regional lymph nodes and caused there the activation of the lymph nodes and the discoloration. Therefore these depositions and discolorations were caused by the test substance due to a gavage error and were not regarded to be an adverse finding.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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): The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% in test group 0, 98.2% in test group 1 and 100% in test groups 2 and 3. Due to the lack of dose response relationship this was assessed as being incidental.

CLINICAL SIGNS (OFFSPRING): The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING): Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
One female runt was seen in test group 0. One male and six female runts were seen in test group 1 (100 mg/kg bw/d); 1 female runt was seen in test group 3 (1000 mg/kg bw/d).

GROSS PATHOLOGY (OFFSPRING): Respectively one male F1 pup of test group 2 (300 mg/kg bw/d) and test group 3 (1000 mg/kg bw/d) showed post mortem autolysis at gross necropsy. Two female pups of test group 1 (100 mg/kg bw/d) were cannibalized. This finding was assessed as being spontaneous in nature and without biological relevance.

OTHER FINDINGS (OFFSPRING): The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature (for details see table 2).

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 10: Fertility index

 

Test group (mg/kg bw/day)

Test groups (mg/kg bw/day

0 (0)

1 (100)

2 (300)

3 (1000)

Male fertility index [%]

100

80

100

100

Female fertility index [%]

100

80

100

100

Table 11: Sex ratio of live F1 pups

PND 0

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Live males [%]

40.3

50.0

50.4

57.4

Live females [%]

59.7

50.0

49.6

42.6

 

 

 

 

 

PND 4

 

 

 

 

Live males [%]

40.3

51.0

50.4

57.4

Live females [%]

59.7

49.0

49.6

42.6

Applicant's summary and conclusion