Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.3.2010 - 25.8.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
During the study the deviations from the guideline limit for relative humidity in animal room were recorded. These relative humidity fluctuations did not affect the welfare of animals and have no impact on the study results.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaction product of Distillates (petroleum), acid-treated heavy naphthenic and calcium oxide
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Smiles notation: not known - UVCB substance
- InChl: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 26.2.2010
- Expiration date of the lot/batch: 26.2.2012
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in PE bottle at room temperature

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., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 10 weeks
- Fasting period before study: none
- 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 (ad libitum): complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN. Diet was sterilised before using.
- Water (ad libitum): drinking water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3°C
- Humidity: 30-70%
- Air changes: approximately 15 air changes per hour
- Photoperiod: 12-hour light/12 hour dark cycle

STUDY TIME SCHEDULE
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 3rd day of lactation,
males – after mating period – totally for 28 days,
nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The application form (test substance suspension in olive oil) was prepared daily just before administration.The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE
- Olive oil (pharmaceutical quality)

Details on mating procedure:
- M/F ratio per cage: Mating 1 : 1 (one male to one female) was used.
- Proof of pregnancy: 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.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
- males and females – 2 weeks prior to the mating period and during the mating period,
- pregnant females – during pregnancy and till the 3rd day of lactation,
- males – after mating period – totally for 28 days,
- nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

Frequency of treatment:
7 days per week at the same time

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
160 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 females and 12 males per group



Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen on the basis of the results of the Dose-range finding experiment with 14-day application period.

Positive control:
No positive control

Examinations

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL:
daily - during the acclimatization and the experimental part

BODY WEIGHT:
- Time schedule for examinations: males - weekly; females - weekly in premating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st and 4th day

FOOD CONSUMPTION:
- Males and females in premating period - (grams/animal/day) calculated from average values of each group (except the mating period)
- Females in pregnancy and lactation period - average individual values (grams/animal/day) - (non-pregnant females not included in calculation)
- Time schedule: males - weekly (except the mating period); females - weekly during premating period, during pregnancy and lactation – on the same days as body weight

MORTALITY:
- Time schedule: daily (for vitality or mortality changes)
Health condition control:
daily - during the acclimatization and the experimental part

LABORATORY EXAMINATIONS:
- vaginal smears: daily in mating period
- pathological examination: males and nonpregnant females - after the end of administration period, parental females and pups - on the 4th day of lactation
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: all males and females after necropsy

Oestrous cyclicity (parental animals):
Not examined

Sperm parameters (parental animals):
Parameters examined in male parental generations: sperm motility and sperm morphology.


Litter observations:
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.

BODY WEIGHT OF LITTER
- Time schedule: 0. or 1st and 4th day

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: at the end of the administration period – after 28 days of administration
- Maternal animals: parental females - on the 4th day of lactation. Mated females without delivery - 26th day after confirmed mating.

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any pathological changes with special attention to the organs of the reproductive systems.

HISTOPATHOLOGY
- The following tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
- For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

ORGAN WEIGHTS (absolute weight)
- Males - testes, epididymis, prostate gland and pituitary gland
- Females - ovaries, uterus (incl. uterine tube and cervix) and pituitary gland

Postmortem examinations (offspring):
SACRIFICE
- On the 4th day of lactation
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs.
- Dead pups were sexed and externally examined; the stomach was examined for the presence of milk.

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. Treated groups were compared with control group.

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)

For each dose group the fertility parameters were calculated.
Fertility parameters - group
Mating index: (number of females with confirmed mating / number of females paired) x 100
Fertility index: (number of pregnant females / number of females with confirmed mating) x 100
Conception index: (number of pregnant females / number of females paired) 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:
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
Description (incidence and severity):
see section Details on result (parental animals)
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see section Details on result (parental animals)
Reproductive performance:
no effects observed
Description (incidence and severity):
see section Details on result (parental animals)

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
The application of the test substance did not cause the death of any female or male.

CLINICAL SIGNS (PARENTAL ANIMALS)
In parental males and females the negative influence of the test substance on clinical status was not found out.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The test substance did not affect growth of parental males and females of all dose levels. The test subsatnce did not affect food consumption of parenteral males anf females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Observation of sperm motility and sperm morphology did not reveal impaired quality of sperm in treated males versus controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The numbers of females achieving pregnancy, the number of females bearing live pups and the number of females with live pups at day 4 after parturition in treated groups were similar to control or higher than control. Also duration of mating and pregnancy were not affected by the test substance treatment. Pre-implantation, post-implantation and post-natal losses of treated groups were well-balanced with the control group. Only gestatiton index was slightly decreased at the highest dose level by the reason of slightly increased number of aborted females.
Decreased number of pups per litter at the highest dose level was accompanied with the significantly decreased number of corpora lutea and decreased number of implantation in females at this dose level. A decrease in litter size could resulted from the decrease in the number of oocytes ovulated.

ORGAN WEIGHTS (PARENTAL ANIMALS)
MALES: Examination of weight of organs proved only one statistically significant difference in treated animals: absolute and relative weight of the prostate gland in the males was decreased at the middle dose level.
FEMALES: No significant difference between treated and control group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
MALES: The test substance had effect on the microscopical structure of the testes without treatment-related damage of spermiogenesis. Histopathological examination of testes of parental males showed increased incidence of irrelevant changes: atrophies of germ epithelium (only sporadic or in less than 10% of the tubules), alteration of tubules in testes and vacuolations of cytoplasm of spermiogonia in males. Above mentioned histopathological changes occurred only in sporadic testicular tubules and did not negatively influence fertility of parental treated males, therefore they were not considered to be adverse.
FEMALES: In the reproductive system affections of ovaries, vaginas were not detected. In the uterus accumulation of ovulatory intraluminal fluid (hydrometra) was found. It is a spontaneous change during the oestrous cycle. Other findings in the uterus were not recorded. No pathological changes were recorded in pituitary glands of all treated and control females.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see section Details on results (offspring)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see section Details on results (offspring)
Histopathological findings:
not examined

Details on results (F1)

OBSERVATION OF PUPS
Number of pups:
Statistical evaluation was performed on the number of live pups. No significant changes were recorded.
Sex ratio of pups:
It was balanced in treated and control groups.

VIABILITY (OFFSPRING)
One pup of each treated and control groups died during lactation period.

CLINICAL SIGNS (OFFSPRING)
No differences in development of pups were observed at the control group and at all treated groups.

BODY WEIGHT (OFFSPRING)
The average weights of the litters and pups at the dose levels 160 and 400 mg/kg/day were relative well-balanced with the control group. At the dose levels of 1000 mg/kg/day, the average weight of the litter was lower compared to control, but the average body weights of pups were statistically significantly higher compared to control group on the first check of litter after parturition. These differences were recorded also on the 4th day of lactation, but without statistical significance.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. No pathologic findings were recorded at control group and at the dose levels 160 and 400 mg/kg/day. Stomach without milk was recorded only in one control pup and two pups at the dose level 400 mg/kg/day. At the dose level 1000 mg/kg/day red foci on kidneys in two pups (from one litter) and oversized teeth in 6 pups (whole one litter) were recorded.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table: Reproduction data

Reproduction data

Observed parameters

Dose level (mg/kg/day)

0

160

400

1000

Pairs started (N)

12

12

12

12

Females showing evidence of copulation (N)

12

12

12

12

Females achieving pregnancy (N)

9

10

9

10

Females with abortion (N)

1

1

1

2

Conceiving days (duration of mating) 1 – 5 (N)

9

11

11

11

Conceiving days (duration of mating) 6 – 13 (N)

3

1

1

1

Pregnancy ≤ 21 days (N)

2

3

4

1

Pregnancy = 22 days (N)

4

5

4

4

Pregnancy ≥ 23 days (N)

2

1

0

3

Females with live pups born (N)

8

9

8

8

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

8

9

8

8

Corpora lutea/pregnant females (average)

12.67

12.10

12.11

9.10

Implantations/pregnant females (average)

12.22

9.40

10.67

7.70

Live pups/mother at birth (average)

12.13

9.44

11.25

8.50

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

12.00

9.33

11.13

8.38

Sex ratio (M/F) at birth (average)

5.00/7.13

5.22/4.22

6.25/5.00

3.75/4.75

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

4.88/7.13

5.11/4.22

6.13/5.00

3.63/4.75

Litter weight at birth (average)

68.41

58.14

67.31

53.89

Litter weight at day 4 after parturition (average)

121.20

92.03

103.28

89.33

Pup weight at birth (average)

5.65

6.17

6.03

6.53

Pup weight at day 4 after parturition (average)

10.11

9.88

9.56

11.46

ABNORMAL PUPS

 

Mothers with 0 (N)

8

9

8

6

Mothers with 1 (N)

0

0

0

1

Mothers with ≥ 2 (N)

0

0

0

1

Note:

(N) – number of animals, M/F – males /females

Table: Fertility parameters

Fertility parameters

Calculated parameters

Dose level (mg/kg/day)

0

160

400

1000

Mating index

100

100

100

100

Fertility index (%)

75.0

83.3

75.0

83.3

Conception index (%)

75.0

83.3

75.0

83.3

Gestation index (%)

88.9

90.0

88.9

80.0

Percentage of postnatal loss

1.03

1.18

1.12

1.49

Viability index

98.97

98.82

98.89

98.53

LOSS OF OFFSPRING

 

Pre-implantation (corpora lutea minus implants)

 

Pregnant females with 0 – 5 (N)

9

8

8

10

Pregnant females with 6 - 10 (N)

0

1

1

0

Pregnant females with 11 - 15 (N)

0

1

0

0

Pregnant females with ≥ 16 (N)

0

0

0

0

Post-implantations (implants minus live births)

 

Pregnant females with 0 (N)

3

6

6

6

Pregnant females with 1 (N)

3

1

0

1

Pregnant females with 2 (N)

2

1

3

2

Pregnant females with ≥ 3 (N)

1

2

0

1

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

 

Pregnant females with 0 (N)

7

8

7

7

Pregnant females with 1 (N)

1

1

1

1

Pregnant females with 2 (N)

0

0

0

0

Pregnant females with ≥ 3 (N)

0

0

0

0

 

Applicant's summary and conclusion

Conclusions:
Body weight, food consumption, clinical status, organs weights and structure of reproductive organs of parental males were not markedly affected by treatment of the test substance.
Body weight, food consumption, clinical status, organs weights and structure of reproductive organs of parental females were also not adversely affected by treatment of the test substance. Reproductive performance – ability of male animals to successfully mate and produce viable offspring was unaffected by the test substance treatment.
The negative influence of the test substance treatment expressed in females of the highest dose level (limit dose): decreased number of corpora lutea, implantations with effect on litter data – decreased number of pups and weight of litters.

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION of males was established at 1000 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION of females was established at 400 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for the DEVELOPMENT of pups was established at 1000 mg/kg body weight/day.

Executive summary:

The test substance - Reaction product of Distillates (petroleum), acid-treated heavy naphtenic and calcium oxide - 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.

Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in olive oil using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per of body weight. Four groups of animals were included in the study - 3 treated groups (doses 160, 400, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 12 males and 12 females. The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen on the basis of the results of the Dose-range finding experiment with 14-day application period.

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 3rd day of lactation,

males – after mating period – totally for 28 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

During the study 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

Parenteral males

Body weight, food consumption, clinical status and macroscopic structure of reproductive organs of treated parental males were unaffected by treatment of the test substance.

A statistically significant difference was detected in the weight of the prostate gland – an absolute and relative weight was statistically significantly increased in males at the dose level of 1000 mg/kg/day. In absence of any changes of microscopic structure of the pituitary gland, the effect could be considered not to be of toxicological importance.

The test substance had effect on the microscopical structure of the testes without treatment-related damage of spermiogenesis. Histopathological examination of testes of parental males showed increased incidence of irrelevant changes: atrophies of germ epithelium (only sporadic or in less than 10% of the tubules), alteration of tubules in testes and vacuolations of cytoplasm of spermiogonia in males. Above mentioned histopathological changes occurred only in sporadic testicular tubules and did not negatively influence fertility of parental treated males, therefore they were not considered to be adverse.

 

Parenteral females

Clinical status, weight and structure of reproductive organs of treated parental females were not significantly affected by treatment of the test substance.

Growth and food consumption of parental females was slightly but insignificantly influenced by the test substance administration.

The number of corpora lutea, implantations, number of pups and accompanied weight of litter were significantly influenced by the administration of the test substance (decrease 1000 mg/kg/day). Sex ratio, average weight of pups and postnatal development of pups were unaffected. Macroscopic abnormalities were described sporadically in pups of the highest dose level.

 

Reproduction parameters

Number of females achieving pregnancy, number of females bearing live pups and number of females with live pups at day 4 after parturition in treated groups were similar to the control or higher than the control groups. Duration of mating and pregnancy were similar in the control and treated females.

Pre-implantation, post-implantation and postnatal losses were relatively well balanced at the treated groups and control group.   

 

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION of males was established at 1000 mg/kg body weight/day.    

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION of females was established at 400 mg/kg body weight/day.     

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