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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2000-Mar-01 to 2001-Jan-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
extended GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
The standard one-generation fertility study was extended so as to obtain subchronic toxicity data similar to that which would be obtained from a 90-day repeat dose toxicity study.
This extra information was obtained from a satellite subgroup of F0 parental animals (5 male and 5 female rats from each test group). In addition to the standard clinical observations and basic pathology data as required in a one-generation fertility study, blood was collected from the satellite parental animals for haematological and clinical chemistry examinations. After sacrifice additional organ weight determinations and an extended histopathological examination were carried out.
This approach differs from a standard 90-day study in that:
1. As the animals had been dosed during premating, mating, pregnancy and weaning the scheduled terminations for the satellite subgroup were performed after a longer dosing period (approx 20 weeks) than a standard 90-day study.
2. Females in the satellite subgroup having been pregant had undergone physiological changes not experienced by non-pregnant animals in a standard 90-day study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
see below

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: (P) Original Male:28 +/-2 days, Female: 35 +/- 2 days; (P) After sacrafice of animals with lowest and highest body weights Male: 36 +/- 2 days, Female 43 +/- 2 days
- Weight at study initiation: (P) Males: 113.9 (99.3 -129.4)g; Females: 112.3 (97.8 -124.9)g;
- Housing: housed individually in type DK Ill stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area of about 800 cm2 ), with the following exceptions: from day 18 of gestation until day 14 after birth, the pregnant animals and their litters were housed in Makrolon type M Ill cages. TheM Ill cages were again supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/ mouse/hamster meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy)
- Water (e.g. ad libitum): water bottle supplied ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20- 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was weighed in calibrated beakers according to dose group. Double distilled water was filled up to the appropriate volume and mixed with a magnetic stirrer. The test substances were prepared once weekly before dosing.
Details on mating procedure:
- M/F ratio per cage: ratio of 1:1. Exceptions from this general procedure were necessary in a few matings due to premature mortalities of single males (ratio of 1:2 (one male: 2 females)
- Length of cohabitation: maximum of 3 weeks
- Proof of pregnancy: vaginal smear referred to as day 0
- If an F0 generation parental animal had not produced any offspring, it was again mated for not more than 3 weeks with one fertile male or female animal of the control group.
- After successful mating each pregnant female was caged (how): from day 18 of gestation until day 14 after birth, the pregnant animals and their litters were housed in Makrolon type M Ill cages. TheM Ill cages were again supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability of the test substance in doubly distilled water was demonstrated for a period of 7 days at room temperature before the start of the study.
- Homogeneity analyses were not considered necessary since the test substance preparations were true solutions.
- Concentration control analyses were performed at the start of the administration period, at the middle and towards the end of the administration period with samples from all concentrations.
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- 20 mg/kg: selected as the the expected "no observed adverse effect level"
- 150 mg/kg: selected as the intermediate dose level
- 500 mg/kg: selected as the highest dose level
- The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations: morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams, littering behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons in addition to the evaluations in the mornings

BODY WEIGHT: Yes
- Time schedule for examinations: determined on the first day of the administration period and thereafter once a week at the same time of day until the end of the study
- During the mating period the F0 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum.
- Females showing no positive evidence of sperm in vaginal smears during the mating interval were weighed on the same days as the F0 males (once weekly).
- Females with litter were weighed on the day of parturition (day 0 p.p.) and on days 1, 4, 7, 14 and 21 postpartum.
- Females without litter were weighed together with the male animals during the lactation phase.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined: Yes, once a week
- After the 10th week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly on days 0, 7, 14, 20 p.c.
- During the lactation period (animals with litter) food consumption was determined on days 1, 4, 7, 14 p.p.
- Food consumption of the F0-males was not determined any longer after the 10th week through sacrifice.
- There was no determination of food consumption in the females during the mating periods, in the females without positive evidence of sperm
during the programmed gestation phase, or in the females without litters during the lactation phase.
Sperm parameters (parental animals):
Parameters examined in male parental generations: No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring: total number of pups and the number of liveborn and stillborn members of each litter, mortality and morbidity, sex ratio, body weight, clinical symptoms, pups were examined externally and eviscerated, their organs were assessed
macroscopically, all stillborn pups and all pups that died up to weaning were examined externally, eviscerated, and their organs assessed macroscopically
- Number of Litters per Dose/Concentration: Dose: At 0, 20, 150 and 500 mg/kg/day there were 24, 24, 25 and 24 litters respectively delivered


GROSS EXAMINATION OF DEAD PUPS:
- Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
HISTOPATHOLOGY
-Satellite parental animals: all gross lesions, salivary glands (glandula mandibularis and glandula sublingualis), esophagus, stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum colon, rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic, lumbar cord), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose , nasal cavity), aorta, heart, bone marrow (femur), lymph nodes (mesenteric and mandibular lymph nodes), spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus, vagina, epididymides, prostate gland, seminal vesicles (and coagulating glands), female mammary gland, skin, skeletal muscle, sternum (with marrow), femur with knee joint, extraorbital lacrimal glands
-Non-satellite parental animals: vagina, cervix uteri, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulating glands, prostate gland, pituitary gland, liver, kidneys, spleen, all gross lesions

ORGAN WEIGHTS
- Satellite parental animals: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, uterus, ovaries, thymus, spleen, brain, heart
Postmortem examinations (offspring):
SACRIFICE
- All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 after birth or subsequent days) were killed by means of CO2.
- These pups were examined externally and eviscerated, their organs were assessed macroscopically.
Statistics:
DUNNETT-test, FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test
Reproductive indices:
Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index, Live birth index
Offspring viability indices:
Viability index, Lactation index, Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

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)
There were no substance related deaths observed at any dose level. Two animals died (one from group 2 and one from group 4) but these deaths were considered to have been due to gavage dosing error. Retching shortly after administration of the test substance was seen frequently in 15 male and 15 female high dose group animals, and sporadically in 1 or 2 medium dose group animals. Blue discolouration of the organs was observed which was associated with the blue colour of the test substance.

Compared to controls, in high dose animals total bilirubin levels were decreased by 77% and 40% in males and females respectively. There was a dose dependent increase in cholesterol levels in females (reaching 59% in high dose females), whilst in males there was a general (but not fully dose dependent) increase in choloesterol over controls, with high dose males showing an increase of 31%.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During premating and gestation there were no statistically significant differences in bodyweight or bodyweight gain for any of the dose groups. Mean body weights of high dose females during lactation days 0-7 were 8% lower than controls, although in days 14-21 post partum the body weight gains of these animals matched or exceeded control animals such that at day 21 mean body weights were similar to control values. There were no effects on food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male and female mating and fertility indices were not affected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males, the absolute and relative mean adrenal gland weights were statistically significantly increased in the low, medium and high dose groups (absolute by 39%, 23% and 36% respectively), (relative by 31%, 23% and 38% respectively). Absolute kidney weights were increased in high dose males and females by 18% and 17% respectively (which represented a 20% relative increase for both sexes). In low, medium and high dose males the absolute thymus weights was decreased compared to controls by 19%, 15% and 17% (25%, 13% and 15% relative to body weight). In females absolute thymus weights were only reduced in the high dose group (by 14%).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Blue discolouration of the organs was observed and was associated with the blue colour of the test substance. A few gross lesions were noted in the glandular stomach (erosion/ulcer or hyperemia of the mucosa), kidneys (retraction) and skin (areas with alopecia). They all occurred singly or twice, with no indication of a relationship to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
At the top dose, slight degeneration of the kidney cortex tubular was observed in all females, and moderate degeneration was observed in two males. In males only, very few to moderate numbers of basophilic (regenerating) tubuli were recorded in the renal cortex of four of five animals. Based on the extended toxicological investigation conducted on the satellite subgroup animals teh repeat dose toxicity NOAEL is 150 mg/kg/day.

HAEMATOLOGY:
There were no treatment-related haematological changes.

GENERAL TOXICITY:
Clear signs of general toxicity occurred in the high dose F0 parental animals (500 mg/kg body weight/day). General toxicity was substantiated by distinct retching shortly after treatment, statistically significantly lower body weights during the first lactation week, decreases in serum bilirubin and increases in cholesterol levels (possibly as a consequence of a functional impairment of the kidneys), statistically significantly increased mean kidney weights and a degeneration of the tubular cells in the renal cortex. In addition, focal basophilic tubuli were recorded in the renal cortex of male animals, indicative of the repair of altered tubular epithelia. Thus, the kidneys proved to be the target organ after subchronic administration of 500 mg/kg body weight to male and female rats.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(general toxicity)
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on clinical examinations/clinical pathology/organ weights/gross and histophahological findings; the kidneys is considered to be the target organ after subchronic administration
Key result
Dose descriptor:
NOAEL
Remarks:
(fertility)
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no substance related adverse effects to reproductive performance.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
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)
There was no effect on litter size or sex ratio.

CLINICAL SIGNS (OFFSPRING)
The F1 generation pups did not show any clinical signs up to day 21, which could be attributed to the treatment. Only one spontaneous clinical finding (sparse hair) occurred in all pups from one low dose litter (No. 136; 20 mg/kg body weight/day) between days 15 and 19 p.p .

BODY WEIGHT (OFFSPRING)
High dose pup body weight gain on days 1-4 post partum was reduced by up to 23% compared to controls such that mean body weights of these pups were statistically significantly reduced (10%) compared to controls on days 4 and 7 post partum. During the last week of the lactation period (days 14-21 post partum) body weight gain of high dose pups was similar to the control value, although mean body weight remained slightly (3%) below the control value at the end of the study.

SEXUAL MATURATION (OFFSPRING)
There were no significant differences in the offspring development of animals treated with test material compared to controls, as indicated by the group mean age of start and completion of appearance of landmarks of offspring development.

GROSS PATHOLOGY (OFFSPRING)
The differences between the groups concerning pup necropsy findings are finally regarded to be spontaneous in nature and not associated with the treatment of the animals.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
(growth and development of the offspring)
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on transient reduction of mean body weights/body weight gains on the first days after birth (up to about 10%/23% below the control values)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an extended one-generation study according to OECD 415, conducted with a structural analogue of Reactive Blue FC15353, there were no substance related adverse effects to reproductive performance.
Executive summary:

In an extended one-generation reproduction study according to OECD 415, a structural analogue of reactive blue FC15353 was administered to 25 Wistar rats/sex/dose by gavage at dose levels of 0, 20, 150, 500 mg/kg bw/day. 

The following treatment-related findings were observed:

Test group 01 (20 mg/kg body weight/day) F0 parental animals had blue discoloration of feces/kidneys in all/nearly all F0 males and F0 females. There were no substance-related adverse effects to reproductive performance.

Test group 02 (150 mg/kg body weight/day) F0 parental animals had blue discoloration of feces and many organs in all F0 males and F0 females. However, there were no substance related adverse effects to reproductive performance. Test group 02 (150 mg/kg body weight/day) F1 pups had no substance-related adverse effects regarding clinical examinations or gross findings.

Test group 03 (500 mg/kg body weight/day) F0 parental animals had several effects relating to clinical examinations/clinical pathology/organ weights/gross and histophahological findings. These findings included: blue-black/blue discoloration of feces/many organs in all F0 males and F0 females, reduced mean body weights on lactation days 0-7 p.p. (up to about 8%), frequently retching shortly after the test substance administration in the majority of rats in both sexes, decreased total bilirubin in both sexes (satellite parental rats), increased cholesterol in both sexes (satellite parental rats), statistically significantly increased mean absolute (females) and relative (both sexes) kidney weights (satellite parental rats), tubular degeneration in the kidneys of two male and all female satellite parental rats, basophilic (regenerating) tubuli in the renal cortex of four satellite parental males. There were no substance related adverse effects to reproductive performance. Test group 03 (500 mg/kg body weight/day) F1 pups had transient reduction of mean body weights/body weight gains on the first days after birth (up to about 10%/23% below the control values)

The NOAEL for reproductive performance and fertility is 500 mg/kg bw/day for the F0 parental rats. The NOAEL for general toxicity of the test substance is 150 mg/kg bw/day for the F0 parental rats. The NOAEL for developmental toxicity (growth and development of the offspring) is 150 mg/kg bw/day for the F1 offspring.

This study is acceptable and satisfies the guideline requirement for a 1-generation reproductive study according to OECD 415 in rats.