H112323

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Oct. 09, 1995 to Aug. 23, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 415 and Annex V to Council Directive 67/548/EEC, OJEC L133 43-46, 1988 (One-generation reproduction toxicity test) in compliance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Annex V to Council Directive 67/548/EEC, OJEC L133 43-46, 1988 (One-generation reproduction toxicity test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): H112323
- Physical state: Dark blue powder
- Analytical purity: Assumed 100 % w/w
- Lot/batch No.: BX361
- Storage condition of test material: Close container

Test animals

Species:

rat

Strain:

other: Alderley Park (Alpk:APfSD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zeneca Barriered Animal Breeding Unit (BABU), Zeneca Pharmaceuticals, Alderley Park, Macclesfield
- Housing: Housed in litters initially and two males or two females per cage for experimental groups during the pre-mating period. Females were housed individually during pregnancy and lactation and provided with bedding material.
- Diet (e.g. ad libitum): Diet-CT1, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 °C
- Humidity (%): 55±15 %
- Air changes (per hr): At least 15 changes/ h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From: 09-Oct-1995 To: Feb-1996.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared in or 60 kg batches.
- Mixing appropriate amounts with (Type of food): Direct addition of weighed amounts of the test substance to CT1 diet and were mixed thoroughly for 6 min in a Pharma Matrix blender.
- Storage temperature of food: Stored at room temperature
- Amount of test substance for 60 kg diet batch: 60, 300, 1200 g of test substance to achieve the dietary concentration of 1000, 5000, 20000 ppm

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: Sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Immediately separated from the male and individually housed
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity of test substance in the CT1 diet was determined by analysing samples from the low and high dose levels.
METHOD SUMMARY-SPECTROPHOTOMETRY: Samples were extracted with methanol Portions of supernatant were diluted with methanol as appropriate, to give sample solution concentrations within the range of calibration standards and analysed by Ultra Violet/Visible Spectrophotometry (UV/VIS)

Duration of treatment / exposure:

Exposure period: Continuous
Premating exposure period (males): 10 wk
Premating exposure period (females): 10 wk

Frequency of treatment:

Daily in feed

Details on study schedule:

- Mating: F0 parents were mated in different male-female pairings, within dose groups, brother-sister mating was avoided

Doses / concentrations

Remarks:

Doses / Concentrations:
1000, 5000, 20000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

26 rats/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of previous studies in the same strain of rat carried out in the same laboratory and included an anticipated no-observed effect level and a level which achieves the limit dose for evaluation in this type of study 1000 mg/kg/d.

Positive control:

Not used in the study

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before the start of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during premating. Initial weights for the Fo parents were recorded immediately before first feeding
experimental diets. The female rats were weighed on Day 1, 8, 15 and 22 of gestation and Day 1, 5, 8, 11, 15 and 22 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Number and sex of pups, 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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after last dosing.
- Maternal animals: All surviving animals after the last litter of first generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic and abdominal organs and structures.

HISTOPATHOLOGY / ORGAN WEIGTHS
Following organs were weighed and the organ to body weight ratios calculated: kidneys, testes, epididymides.
The 10 males and to females with the lowest individual animal numbers from each group surviving to scheduled termination were selected for submission ofan extended list of tissues

Postmortem examinations (offspring):

Dead or moribund pups were subjected to a gross examination post mortem, abnormalities were recorded and the pups were discarded. All surviving offspring were killed (on Day 22 post partum or soon) and discarded without further examination.

Statistics:

Analysis of variance, analysis of covariance and Fisher's exact test, Student's t-test, two sided

Reproductive indices:

The proportion of successful matings, length of gestation and pre-coital interval.

Offspring viability indices:

% of pups live born, % of pups surviving to Day 22.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

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): Blue colouration was evident on the coat and skin and in the oral cavity of rats of all treatment groups. Blue eyes and blue urine were also observed in mid and high dose treated animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): No evidence of an effect on the bodyweight of rats during premating, gestation and lactation.

FOOD CONSUMPTION AND UTILISATION(PARENTAL ANIMALS): Food consumption was increased compared to controls in males and to a lesser extent, in females receiving 20000 ppm. Food utilisation was less efficient in rats receiving 20000 ppm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No evidence of effects on pre-coital interval, length of gestation, number of animals successfully mating and number of whole litter losses

ORGAN WEIGHTS (PARENTAL ANIMALS): Kidney weights were increased in both sexes receiving 20000 and 5000 ppm. Testes weights were slightly increased in males receiving 20000 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS): Apart from test substance colouration other findings were not treatment related. Dose related blue discolouration of a wide variety of tissues with the majority of animals affected at 20000 ppm and a lesser number at 5000 ppm. Intestinal contents and the tail, limb and other skin sites were also discoloured in some rats at 1000 ppm. Dose related increased incidence of dark kidneys at all dose groups in both sexes and a dose related increased incidence of dark spleens in males at all dose groups.

HISTOPATHOLOGY (PARENTAL ANIMALS): Treatment related increase in the severity of tubular basophilia in the kidneys in males at 20000 and females at 20000 and 5000 ppm.

OTHER FINDINGS (PARENTAL ANIMALS): Microscopic findings in infertile animals: A number of findings were present to explain the infertility in some rats including dystocia, endometritis, vaginitis, or imperforate vagina. However in the majority of rats no pathological change was observed to explain the reproductive failure. There were no treatment related findings observed in these rats with the possible exception of two female rats dosed at 20000ppm with slight tubular basophilia.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive parameters

2

Dose descriptor:

NOAEL

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: For systemic toxicity Based on microscopic changes and increased kidney weights due to re-filtration of the dye in both sexes at 20000 ppm and in females receiving 5000 ppm. There were no effects on the kidney in rats receiving 1000 ppm.

3

Dose descriptor:

NOAEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: For systemic toxicity Based on microscopic changes and increased kidney weights due to re-filtration of the dye in both sexes at 20000 ppm. There were no effects on the kidney in males at 5000 ppm and male and female rats receiving 1000 ppm.

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:

not examined

Histopathological findings:

not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING): Colouration or staining of the test substance in the nostrils and on the skin or coat of the majority of offspring

BODY WEIGHT (OFFSPRING): No evidence of treatment related effects

OTHER FINDINGS (OFFSPRING): Investigations post mortem - offspring: Apart from colouration by the test substance there was no evidence of treatment related findings in those pups which were found dead or killed for humane reasons and were examined macroscopically

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Achieved dose rates

Dose rates (based on nominal dietary levels of H112323) were calculated in terms of mg H1I2323/kg body weight/day. As expected these were at a maximum in the early phase of the pre-pairing period in each generation and declined during the period of rapid growth. The mean dose received was 113, 565 and 2430 mg/kg bw/day in males and 121, 615 and 2569 mg/kg bw/day in females receiving 1000, 5000 or 20000 ppm H112323, respectively. The high dose is double the limit dose level required for a single generation study,

Applicant's summary and conclusion

Conclusions:

Dietary administration of test substance in one generation study to rats resulted in no adverse reproductive effects. No treatment related effect was observed in the reproductive system or in the offspring at dietary levels up to and including 20000 ppm calculating to mean dose levels of 2430 mg/kg bw/day in male and 2569 mg/kg bw/day in female rats

Executive summary:

A reproduction study was conducted in rats by dietary administration of test substance at target dosage levels of 0, 1000, 5000, or 20000 ppm for one generation. The study was conducted according to OECD Guideline 415 and Annex V to Council Directive 67/548/EEC, OJEC L133 43-46, 1988 (One-generation reproduction toxicity test) in compliance with GLP.

Groups of 26 male and 26 female (P₀parents) weanling AIpk: APfSD (Wistar-derived) rats were fed diet containing 0 (control), 1000, 5000 or 20000 ppm test substance. After 10 wk, the animals were mated and allowed to rear the ensuing F1A litters to weaning. Clinical observations, bodyweights, food consumption and utilisation and reproductive performance were performed on all parent animals. Numbers of pups, pup survival, pup and litter weights were assessed in litters/pups. In addition a full range of tissues was taken and examined microscopically from 10 males and 10 females per group at scheduled termination.

Dose rates (based on nominal dietary levels of H112323) were calculated in terms of mg H112323/kg body weight/day. As expected these were at a maximum in the early phase of the pre-pairing period in each generation and declined during the period of rapid growth. The mean dose received was 113, 565 and 2430 mg/kg bw/day in males and 121, 615 and 2569 mg/kg bw/day in females receiving 1000, 5000 or 20000 ppm H112323, respectively. The high dose is double the limit dose level required for a single generation study.

The main clinical observations were related to blue staining of the animals by the test substance. Blue colouration was evident on the coat and skin and in the oral cavity of rats of all treatment groups. Some rats in the 20000 ppm group also had blue eyes and a small number of rats in the 5000 and 20000 ppm groups were also observed to have blue urine. There was no evidence of an effect on bodyweight at any dose level of test substance. Food consumption was increased and food utilisation was less efficient in males receiving 20000 ppm, resulting from the high amount of test compound in the food. There was no evidence of an effect of administration of test substance on any of the reproductive parameters assessed. No adverse effects were observed in the offspring.

Kidney weights were increased in both sexes receiving 20000 and 5000 ppm. However the difference from control was only statistically significant in females at the 5000 ppm dose level. There was a dose related blue discolouration of a wide variety of tissues including the kidneys with the majority of animals affected at 20000 ppm and a lesser number at 5000 ppm. was a treatment related increase in the severity of tubular basophilia in the kidneys. Slight or moderate lesions were present in males at 20000 and females at 20000 and 5000 ppm. This finding is most likely related to a re-absorption of the test substance trough the renal tubuli. Although minimal degrees of this change were not present in the designated control rats at termination an incidence of one rat from the infertile group was observed in males. There were no effects on the kidney in rats receiving 1000 ppm. No treatment related effect was observed in the reproductive system at dietary levels up to and including 20000 ppm.

Under the test conditions, dietary administration of test substance in one generation study to rats resulted in increase in kidney weights with associated microscopic changes at 20000 ppm in both sexes and at females receiving 5000 ppm. No treatment related effects were observed at any dose levels on the reproductive organs or of the offspring. Consequently, the NOAEL for reproductive toxicity was determined to be 20000 ppmcalculating to mean dose levels of 2430 mg/kg bw/day in male and 2569 mg/kg bw/day in female rats.