Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from March 18, 2009 to October 21, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Humic acids, sodium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 9. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: (P) 8 wks
- Weight at study initiation: Males: cca 332-333 g; Females: cca 210-212 g
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN, ad libitum. Diet was sterilised before using
- Water (e.g. ad libitum): Free access to drinking water (ad libitum).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): Relative humidity 30-70%
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered to the stomach by gavage as the solution in water for injection. The animals were treated 7 days per week at the same time.

VEHICLE
water for injection (Aqua pro injectione)
Manufacturer: Ardeapharma a.s., Ševětín
- Concentration in vehicle: 25, 50 or 100 g/L water
- Amount of vehicle (if gavage): 1 mL per 100 g of body weight.
- Batch No.: 0204190908, 0102220109, 0102050309, 0101030309

Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: sperm in vaginal smear
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined.
The stability of application form was monitored by the analyses of solution of test substance in water. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The solution was prepared by mixing of test substance and water for 5 minutes (cca 400 rpm) in a container for application form preparation. For the determination of stability the samples were taken from the middle of container content after required time intervals (0, 30, 60 and 120 minutes). Time interval 0 min. was the time after 5 minutes mixing. The solution was mixed all the time of monitoring.
The homogeneity of application form was monitored by the analyses of solution of test substance in water prepared by the same way as for the determination of the stability. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The samples were taken after 5 minute mixing from 3 given places - the bottom, the middle and the surface of container content.

The determination of test substance was performed on the basis of measurement of the absorbance of a water solution. Test substance stability and homogeneity were determined by measuring of an absorbance of water solution (application form) in visible range of spectrum.
Duration of treatment / exposure:
The treated groups were administered daily for the following period: males and females – 2 weeks prior to the mating period and then during the mating period.
Pregnant females were administered during pregnancy and till the 3rd day of lactation.
Males were than administered 26 days after mating period and
nonpregnant females 24 days after the last day of the mating period.
Frequency of treatment:
The animals were treated 7 days per week
Details on study schedule:
Mating Procedure
Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:

Basis:
actual ingested
250 mg/kg/day
Remarks:
Doses / Concentrations:

Basis:
actual ingested
500 mg/kg/day
Remarks:
Doses / Concentrations:

Basis:
actual ingested
1000 mg/kg/day
No. of animals per sex per dose:
Dose 250 mg/kg/day: 10 males + 10 females
Dose 500 mg/kg/day: 10 males + 10 females
Dose 1000 mg/kg/day: 10 males + 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for study – 250, 500 and 1000 mg/kg/day were chosen on the basis of the results of the Study No. 27/07/7: Humic acids, sodium salts – Repeated Dose (28 days) Toxicity (Oral), VUOS Report No.0855, 2008.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
Health Condition Control:
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before and during application.

CLINICAL OBSERVATIONS:
Clinical Observations of Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day – at the time of expectation of maximal effect of the test substance.
Animals were observed in natural conditions in their cages.

BODY WEIGHT:
The body weight of animals was recorded on automatic balances with group average computing module on specified days. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of averages in pregnancy and lactation period.

FOOD CONSUMPTION:
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males average values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from average values of each group.
The same way of calculation of average food consumption was used for females in premating period. In pregnancy and lactation period average individual values (grams/animal/day) were calculated for each week of the study. Average food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of average food of pregnant females.
Sperm parameters (parental animals):
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology.

Sperm motility
Sperm samples were taken from the one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades:
1 – fast progressive motility, 2 - slow progressive motility, 3 – no progressive motility, 4 – non-motile sperm.

Sperm morphology
Sperm samples were taken from the one epididymis and sperm morphology were assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck - were recorded.
Litter observations:
PARAMETERS EXAMINED
Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):
Pathological Examination
Parental males were killed at the end of the application period – after 54 days of administration.
Parental females were killed on the 4th day of lactation. Mated females without delivery (nonpregnant or aborted) were killed 27th day confirmed mating.
Then they were macroscopically examined for any structural abnormalities or pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Biometry of Reproduction Organs
The absolute weights of testes, epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

The following tissue and organs were collected from all killed males and females at necropsy and fixed in 4% buffered formaldehyde for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson solution), cervix of uterus, ovaries, uterus and vagina.
Postmortem examinations (offspring):
Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis.
This statistical analysis was used for the results of body weight, biometry of organs and number of pups. Control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.

Reproductive indices:
For each of parental females the following parameters were calculated:
Loss of offspring - individual
Pre-implantation loss: Number of corpora lutea – number of implantations
Post-implantation loss: Number of implantations – number of live births
Post-natal loss: Number of live births – number of alive at postnatal day 4

For each dose group the fertility parameters were calculated.
Fertility parameters group
Percentage mating: (number of females mated / number of females paired) x 100
Fertility index: (number of pregnant females / number of females paired) x 100
Conception index: (number of pregnant females / number of females mated) x 100
Gestation index: (number of females bearing live pups / number of pregnant females) x 100
Percentage of postnatal loss days 0-4 post partum: (number of dead pups on day 4 post partum*/ number of live pups at first litter check) x 100
Note: * without still born pups (dead pups with anaerial lungs)
Offspring viability indices:
Percentage of postnatal loss days 0-4 post partum: (number of dead pups on day 4 post partum*/ number of live pups at first litter check) x 100
Note: * without still born pups (dead pups with anaerial lungs)
Viability index: (number of live pups on day 4 post partum / number of pups born alive+) x 100
Note: + with dead pups with aerial lungs

Results and discussion

Results: P0 (first parental generation)

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

Reproductive function / performance (P0)

Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The application of the test substance did not cause the death of any female or male.
Influence of the test substance on clinical status was not found out in both sexes. Sporadic secretion from nostrils and around eyes and piloerection were observed at all treated and control groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The test substance had not significant effect on growth of parental males.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Slight effect of the test substance on reproductive organs was found out during examination of reproductive system of parental males. Decreased sperm motility with dependence on the dose level was observed at the treated groups. Examination of sperm morphology in males revealed increased percentage of affected sperms at the highest dose level. Decreased sperm motility was probably caused by affected tails. No macroscopic changes were observed during pathological examination.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Examination of reproductive system of parental females revealed insignificant increase of absolute and relative weight of uterus at the middle dose level and insignificant decrease of absolute and relative weight of ovaries at the highest dose level. Histopathological changes (ovary – folikular cyst, uterus – hydrometra, mild atrophy, hyperplasia, dystrophy and apoptosis of superficial cells and accumulation of amorphous eosinophile matter in mesometrium) were found out only sporadically and affected all groups of females. These microscopical affections did not affected fertility of parental females. Hyperplasia of epithelium in vagina was recorded in all groups but more frequently at the highest dose level and could be caused by the test substance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Biometry of organs revealed only statistically insignificant changes.



Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption and compound intake; water consumption and compound intake; gross pathology; organ weights; histopathology; sperm characterization mating index; fertility index; duration of pregnancy;

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

Observation of pups – in day of parturition the average number of pups per litter was well-balanced with the control group at the lowest and middle dose level. At the highest dose level slightly decreased number of pups per litter was recorded. Cannibalism of pups was recorded at the highest dose level in one female in day of parturition. At the end of lactation slight decrease of average number of pups per litter was found at the lowest and middle dose levels (sporadic death of some pups).
The average body weight of pups was unaffected and well-balanced with the control group in day of parturition. Relative well-balanced body weight of pups was also observed at the last day of lactation. Dose related decreased of weight of litters was recorded at treated groups during whole observation period. Sex ratio was relatively well-balanced.
Presence of macroscopic abnormalities – stomach without milk (at the lowest in 3 pups, at the middle dose in 3 pups and at the highest dose in 1 pup), haemorrhage of serosa (at the lowest in 1 pup and at the highest dose in 3 pups), yellow strata of liver (at the lowest dose in 3 pups), predigestion blood in intestine (at the middle dose in 2 pups) were recorded. Other macroscopic findings (haemorrhage of cranium, unaerial lungs and congested peritoneum) were recorded sporadically.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; sex ratio; survival index; viability index;

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproduction data

Reproduction data

Observed parameters

Dose level

0

250

500

1000

 Pairs started (N)

10

10

10

10

 Females showing evidence of copulation (N)

10

10

10

10

 Females achieving pregnancy (N)

8

9

8

10

 Conceiving days 1 – 5 (N)

10

9

10

10

 Conceiving days 6 – 13 (N)

0

1

0

0

 Pregnancy ≤ 21 days (N)

1

2

3

1

 Pregnancy = 22 days (N)

6

5

4

6

 Pregnancy ≥ 23 days (N)

1

2

1

2

 Females with live pups born (N)

8

9

8

9

 Females with live pups at day 4 after parturition (N)

8

9

8

8

 Corpora lutea/pregnant females (average)

19.0

18.0

15.1

17.1

 Implants/pregnant females (average)

15.0

13.8

12.5

13.0

 Live pups/mother at birth (average)

13.6

12.3

12.1

10.8

 Live pups/mother at day 4 after parturition (average)

13.6

11.8

12.0

10.8

 Sex ratio (M/F) at birth (average)

6.6/ 7.0

6.0/ 6.3

4.9/ 7.3

5.6/ 5.1

 Sex ratio (M/F) at day 4 after parturition (average)

6.6/ 7.0

5.6/ 6.2

4.8/ 7.3

5.6/ 5.1

 Litter weight at birth (average)

89.4

77.9

77.9

69.4

 Litter weight at day 4 after parturition (average)

137.0

115.9

115.6

110.5

 Pup weight at birth (average)

6.6

6.4

6.5

6.5

 Pup weight at day 4 after parturition (average)

10.1

9.9

9.7

10.9

ABNORMAL PUPS

 Mothers with 0 (N)

8

4

5

6

 Mothers with 1 (N)

0

3

2

1

 Mothers with ≥ 2 (N)

0

2

1

1

Table 2: Fertility parameters

Fertility parameters

Calculated parameters

Dose level

0

250

500

1000

 Percentage of mating

100

100

100

100

 Fertility index

80

90

80

100

 Conception index

80

90

80

100

 Gestation index

100

100

100

90

 Percentage of postnatal loss

0

4.5

1.0

0

 Viability index

100

95.5

99.0

100

  LOSS OF OFFSPRING

  Pre-implantation (corpora lutea minus implants)

  Pregnant females with 0 – 5 (N)

6

4

8

7

  Pregnant females with 6 - 10 (N)

2

5

0

1

  Pregnant females with 11 - 15 (N)

0

0

0

1

  Pregnant females with ≥ 16 (N)

0

0

0

0

  Pre-natal/post-implantations (implants minus live births)

  Pregnant females with 0 (N)

1

3

5

3*

  Pregnant females with 1 (N)

3

2

2

3*

  Pregnant females with 2 (N)

4

2

1

1*

  Pregnant females with ≥ 3 (N)

0

2

0

1*

  Post-natal (live births minus alive at post-natal day 4)

  Pregnant females with 0 (N)

8

7

7

8*

  Pregnant females with 1 (N)

0

1

1

0*

  Pregnant females with 2 (N)

0

0

0

0*

  Pregnant females with ≥ 3 (N)

0

1

0

0*

Applicant's summary and conclusion

Conclusions:
Administration of the test substance non affected clinical status, growth of parental animals, course of pregnancy and development of pups.
The negative influences of the test substance treatment on reproduction were observed, expressed maily at the highest dose level (limit dose). The following parameters were influenced : sperm quality of parental males (decreased sperm motility, affected tails) and microscopical structure of reproduction organ (vagina - hyperplasia of epithelium) in parental females.
Executive summary:

The test substance was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995.

Methods

   Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in aqua pro injectione using a stomach tube; oral application of rats was made daily. The volume administered was adjusted to 1 mL per of body weight in all doses. Four groups of animals were included in the study - 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). Each group consisted of 10 males and 10 females.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females  – during pregnancy and till the 3rdday of lactation,

males    after mating period – totally for 54 days,

nonpregnant females (mated females without parturition) – for 26 days after the confirmed mating.

   

During the study the clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

Results

Influence of the test substance on clinical status was not found out in both sexes. Secretion from nostrils and around eyes, piloerection and slightly decreased body weight in animals were observed. These findings at treated groups of males and in treated groups of females were only sporadic.  

The test substance had not significant effect on growth and food consumption of parental males and females. Sporadic changes of body weight and body weight increment was observed during study but without statistical significance.

Examination of reproductive system of parental males revealed significant effect on reproductive organs. Decreased sperm motility with dependence on the dose level was observed at the treated groups. Examination of sperm morphology revealed increased percentage of affected sperms at the dose level 1000 mg/kg/day. Statistically insignificant decrease of absolute and relative weight of epididymis at the dose level 250 mg/kg/day and increased absolute and relative weight of prostate glands at the dose level 1000 mg/kg/day was recorded. No macroscopic changes were observed. Histopathological examination of reproductive system of parental males showed increased incidence of lymphocyte infiltration in interstitium and epithelium of epididymides in males of the all dose levels. Vacuolation in pituitary gland was also recorded in all treated groups and the number of affected males was increased with dependence on the dose level. Other microscopic findings were recorded sporadically.

Examination of reproductive system of parental females revealed slightly increased absolute and relative weight of uterus and decreased absolute and relative weight of ovaries without statistical significance. Histopathological examination of reproductive system of parental females revealed increased incidence of microscopic affections in vagina (hyperplasia of epithelium) at the dose level 1000 mg/kg/day. Other microscopic changes (ovary – folikular cyst, uterus – hydrometra, mild atrophy, hyperplasia, dystrophy and apoptosis of superficial cells and accumulation of amorphous eosinophile matter in mesometrium) were recorded sporadically.

Observation of pups – at the dose level 1000 mg/kg/day slightly decreased number of pups per litter was recorded. Cannibalism of pups was recorded also at the dose level 1000 mg/kg/day in one female in day of parturition. At the end of lactation slight decrease of average number of pups per litter was found at the dose levels 250 and 500 mg/kg/day (sporadic death of some pups). The average body weight of pups was unaffected and well-balanced with the control group in day of parturition. Relative well-balanced body weight of pups was also at the last day of lactation. Decreased average weight of litter with dose dependence was recorded at all treated groups during whole observation period. Sex ratio was relatively well-balanced. Presence of macroscopic abnormalities (e.g. stomach without milk, haemorrhage of stomach serosa, yellow strata of liver, predigestion blood in intestine) was found out sporadically at all treated groups.

Reproduction parameters – number of females achieving pregnancy, accompanying conception index, duration of mating, duration of pregnancy of all treated groups was similar to the control group. Gestation index was slightly decreased only at the dose level 1000 mg/kg/day (there were one female which aborted). Slightly decreased viability index and accompanying increased postnatal loss at the dose level 250 mg/kg/day resulted from high post partum mortality of pups. Slightly decreased fertility index was recorded at the dose level 500 mg/kg/day and at the control group.

   

Administration of the test substance Humic acids, sodium salts non affected clinical status, growth of parental animals, course of pregnancy and development of pups.

The negative influences of the test substance treatment on reproduction were observed, expressed maily at the highest dose level (limit dose). The parameters: sperm quality of parental males (decreased sperm motility, affected tails) and microscopical structure of reproduction organ (vagina - hyperplasia of epithelium) in parental females were influenced.      

    

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION in males and females was established as 500 mg/kg body weight/day.

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT of offspring was established as 1000 mg/kg body weight/day.