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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
According to Guideline.TEST ANIMALS- Source: Charles River UK Ltd., England- Age at study initiation: 4-5 weeks - Weight at order: 120 - 150 g- Age after acclimatisation: males 6-7 weeks, females 14-15 weeks: Mating of F1 animals started at an age of 16 weeks - Fasting period before study: No- Housing: Group housed, except for mating period. During pregnancy and lactation period females were housed individually.- Diet (ad libitum): Pelleted SQC rat and mouse No. 3 Breeder- Water (ad libitum): Purified water- Acclimation period: males 12 days, females 6 - 7 weeksENVIRONMENTAL CONDITIONS- Temperature (°C): 18 - 27- Humidity (%): 36 - 62- Air changes (per hr): 16- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
The test article was formulated for dosing as solutions in the vehicle, purified drinking water. For formulation, the weighed quantity of test article was mixed with the appropriate volume of vehicle. Separate formulations were prepared for each dose level. Formulations were prepared once weekly and stored in polycarbonate aspirators at ambient temperature in the animal room during the week of use.
Details on mating procedure:
- M/F ratio per cage: 1:1- Length of cohabitation: Maximum 14 days- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.- Further matings after two unsuccessful attempts: No - After successful mating each pregnant female was caged: Individually- Any other deviations from standard protocol: Due to reduced numbers of litters in each group of the F1a generation the F0 generation was re-paired following weaning of the F1a generation to produce a F1b generation. Females were allowed to rear their offspring to weaning on day 21 post partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two 10 mL samples from the middle of the aspirator of each dose group prepared for weeks 1, 5, 9, 13, 17, 21, 25, 29 and 33 have been quantified via HPLC analysis using an internal standard for quantification. The actual concentrations proved the nominal concentrations.
Duration of treatment / exposure:
P males: 11 weeks before mating, during both mating periods (for F1a and F1b), until necropsy (total 198 days).P females: 2 weeks before mating, during both mating periods (for F1a and F1b), pregnancy and lactation, until necropsy (Day 21 post partum) (total 71 days for F0 to F1a and 114 days for F0 to F1b).F1 males were dosed from birth until approximately 16 weeks of age, during mating, until necropsy (total 110 days).F1 females were dosed from birth until approximately 16 weeks of age, during mating, pregnancy and lactation period (total 119 days).
Frequency of treatment:
Daily
Details on study schedule:
F1 parental animals were mated 16 weeks after selected from the F1 litters.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:30, 100, 300 mg/kg bw/dBasis:other: calculated by test article intake (rough estimate, actual values are rather higher)
Remarks:
Doses / Concentrations:0.03, 0.1, 0.3%Basis:nominal in waterconcentration proven by analytcial examination
No. of animals per sex per dose:
30 (F0 gen), 25 (F1 gen)
Control animals:
yes, concurrent vehicle
Positive control:
Not required.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: YesTwice daily for mortalities and once daily for clinical signs of toxicityBODY WEIGHT: YesMales: at weekly intervals. Females weekly during pre-mating, on days 0, 7, 14 and 20 of pregnancy and on days 0, 4, 7, 14, 21 post partum.FOOD CONSUMPTION: YesAt weekly intervals. Additionally for females from days 1-4, 4-7, 7-11 and 11-14 post partumWATER CONSUMPTION AND COMPOUND INTAKE: YesDaily for males and females during pre-mating, for males at the end of the mating period and for females during pregnancy and lactation. Water consumption was not measured for mating pairs.Other examinations:Blood samples were taken from 10 males and 10 females of each dose group one week prior the F0-F1a and the F1-F2 mating period. Following parameter were assessed: leucocyte differential count, total leucocyte count, A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, creatinine, globulin, total bilirubin, total protein, triglycerides
Estrous cyclicity (parental animals):
Beginning two weeks before the start of the first F0 and the F1 mating period and during all the mating periods until confirmation of maing or end of the relevant periods vaginal smears were taken daily and examined for estrous cycle stage.
Sperm parameters (parental animals):
Parameters examined in male parental generations:testis weight, epididymis weight, sperm count in epididymides, percent motile sperms (incl. calculation of straight line velocity and average pathe velocity), sperm motility, sperm morphology
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 EXAMINEDThe following parameters were examined in F1 and F2 offspring:number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight and body weight gain, physical or behavioural abnormalities, time until developmental milestones are reached (e.g. ear opening, balanopreputial separation of males, vaginal perforation of femals, etc.)FUNCTIONAL OBSERVATIONSOn day 22 post partum the auditory startle habituation response was evaluated using the SR-Screening System on pups selected for special necropsy. At 28 days of age the selected pups were examined for learning potential using a water filled e-maze in two session on consecutive days. A session consisted of six runs. At approximately 28-35 days post partum the selected F1-generation pups were tested for motor activity.GROSS EXAMINATION OF DEAD PUPS:yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. Necropsy was performed on all pups killed prematurely or found dead and for surplus pups after selection for special necropsy and rearing
Postmortem examinations (parental animals):
SACRIFICE- Male animals: All surviving animals after the mating period- Maternal animals: All surviving animals after the last litter of each generation was weaned.GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.ORGAN WEIGHTSFollowing organs were weighed: brain, pituitary gland, liver, adrenals, spleen, thymus, kidneys, testes, epididymides (toatl and cauda), seminal vesicles, prostate gland, coagulating gland, ovaries, oviducts, uterus, cervix, vaginaHISTOPATHOLOGYThe following tissues were prepared for microscopic examination: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, femur, ileum, jejunum, kidneys, liver, mesenteric lymph node, oesophagus, ovaries, pituitary, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, submandibular lymph node, testes, thymus, thyroids, uterus, vagina, gross lessions.Detailed evaluation were further performed on opened stomach to further examine the glandular and non-glandular regions of the stomach for possible signs of irritation.
Postmortem examinations (offspring):
SACRIFICENecropsy was conducted on all pups killed prematurely or found dead and for surplus pups after selection for special necropsy and rearing.GROSS NECROPSY- One male and one female pup from each of the first twenty litters comprising sufficient pups were selected from F1a and F2 litters for special necropsy. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special necropsy comprised assessment of organ weights and fixation in neutral buffered formaldehyde.ORGAN WEIGTHSThe following tissues were weighed during special necropsy: brain, liver, adrenals, spleen, thymus, kidneys, testes, ovaries.HISTOPATHOLOGYThe following tissues were prepared for microscopic examination of the F1 generation: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, femur, ileum, jejunum, kidneys, liver, mesenteric lymph node, oesophagus, ovaries, pituitary, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, submandibular lymph node, testes, thymus, thyroids, uterus, vagina, gross lessions.
Statistics:
Group means and standard deviations were calculated for each observation time. Analysis of variance (ANOVA) was performed on all parameters. Heterogeneity of variance was analysed using Levenes test. Williams test was performed to compare high dose with control at the two-sided 5% level. Kruskal-Wallis ANOVA was performed to assess overall differences between treatment groups followed by Shirley´s test.
Reproductive indices:
Fertility index, gestation index, copulation index, sex ratio
Offspring viability indices:
live birth index, viability indices

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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:No test item related clinical signs of toxicity were observed. 2, 2, 3 and 5 females were found dead or were killed prematurely during the F0 generation phase at 0, 30, 100 and 300 mg/kg bw/d. The observation and timings of these losses do not support any relationship to dosage and mortalities are considered to be not treatment related.BODYWEIGHT:(Transient) reductions in F0 males and females during the first two weeks of the study were considered to be related to palatability issues.BIOCHEMISTRY:Reduced triglyceride levels in F0 females was observed. The values were however within the historical control.SPERM MEASURES:Slight but significantly reduced straight line velocity (VSL) of the sperm was observed at the top dose (19.5 ± 8.8 compared to 25.1 ± 10.9 µm/s) without any significant effects on averaged path velocity (VAP) or total motility. No effect on sperm morphology was observed.BODY WEIGHT AND WATER CONSUMPTION (PARENTAL ANIMALS)No test item related effect on body weight was observed. The water consumption at the top dose was lower compared to the other groups. This is most likely due to palatability problems.REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) No test item related effects observed. ORGAN WEIGHTS:The male F0 generation showed a small but significant reduction in body weight-liver weight ratios, but the corresponding brain related liver weights and the absolute liver weights developed not in a dose dependant way. For the F1 generation where similar results were reported, no dose-response relationship was detected either. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance. REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)No test item related effects on the copulation index was observed. The fertility index was low in each group of the F0 generation. Consequently F0 females were paired after weaning of F1a generation with a different male from the original pairing. The fertility index at 300 mg/kg bw/d was slightly decreased compare to the other groups for F0-F1b females. No effect on fertility was observed in the F1-F2 generation. Thus this finding was considered to be not treatment related. No treatment related effect on duration of gestation and gestation index were observed.GROSS PATHOLOGY (PARENTAL ANIMALS)No test item related effectsHISTOPATHOLOGY (PARENTAL ANIMALS)No test item related effects were observed, except for slight signs of local irritation in the stomach at the top dose.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Remarks on result:
other: Generation: P and F1 (migrated information)

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:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

CLINICAL SIGNS AND MORTALITY:No test item related clinical signs of toxicity or mortality was observed. At the mid dose of the F1a generation a large portion of pups showing poor or no maternal care were observed. 36% of those pups from 6 litters died by day 2 post partum. The same finding was observed at control and low dose pups of the F2 generation. These findings showed no does response and were considered to be not treatment related.BODYWEIGHT:A slight not significant reduction in body weight and body weight gain for males and female pups of the F1a generation at the top dose was observed. As body weight and body weight gain was slightly and not significantly higher in the F1b and the F2 generation this finding was considered to be not treatment related.BIOCHEMISTRY:An increased percentage neutrophil count in F1 males was observed. This finding was not observed in F0 males and was within the historical control. Thus this finding was not treatment related.SEXUAL MATURATION:A significant increase in time taken for sexual development of females at the top dose was observed. For males a not significant increase in time taken for sexual maturation was observed at the top dose. No difference in body weight of these females was observed. No effect on fertility or mating performance was observed.ORGAN WEIGHTS:see 'parental animals'VIABILITY (OFFSPRING): No effects on pup survival (day 4 - 21 post partum), live birth index and mean litter size were observed. Pup survival of the F1a generation (day 0 - 4 post partum) was slightly lower in the treated groups when compared to control. The differences were not significant, showed no does reponse relationship and were not observed for F1b and F2 pups. Therefore this finding was considered to be not treatment related. GROSS PATHOLOGY (OFFSPRING)Pups showing a lack of maternal care and which died or were killed moribundly showed signs of canibalisation and had no milk in the stomach.No treatment related effects were observed.SPECIAL NECROPSYNo treatment related macroscopic findings were observed. HISTOPATHOLOGY (OFFSPRING)No effects were observed.OTHER FINDINGS (OFFSPRING) No adverse treatment related effect on the time to developmental milestones was observed. No treatment related effects on learing ability and functional observational battery were observed.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Remarks on result:
other: Generation: P and F1 (migrated information)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Slight but significantly reduced straight line velocity (VSL) of the sperm was without any significant effects on averaged path velocity (VAP) or total motility. Moreover, in the available subchronic and chronic toxicity studies on various AES the primary sex organs of the males and females did not show evidence for treatment-related adverse effects.The observed reduced triglyceride levels (female) and increased percentage neutrophil counts (males) were slight and within the range of the historical control data.

The male F0 generation showed a small but significant reduction in bodyweight-liver weight ratios, but the corresponding brain related liver weights and the absolute liver weights developed not in a dose dependant way. For the F1 generation where similar results were reported, no dose-response relationship was detected either. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance, additionally underlined by the absence of such effects in the studies for subchronic toxicity mentioned above.

There was evidence of toxicity on pup development at the top dose that was characterised by an increase in the time taken for sexual development of the male (not significant) and female (significant) offspring.This was investigated in more detail in the developmental toxicity study up to 1.5 g/kg bw and no effects were noted there. Considering all these facts the subchronic NOAEL for systemic toxicity and reproduction toxicity can be set to greater than 300 mg/kg bw.

Applicant's summary and conclusion

Conclusions:
In summary, there was no effect of treatment at any dose level on reproduction of the parents or offspring (NOAEL >= 0.3 %; > 300 mg/kg/day).Based on this study an overall NOAEL for systemic effects of 0.3 % (300 mg/kg bw) can be deduced.