Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May - 08 Sep 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 289-339 g (males) and 170-200 g (females)
- Housing: animals were housed in groups of up to three in open macrolon cages type 2000P (TechniPlast).
- Diet: maintenance diet for rats and mice, No. 1324 TPF, ad libitum. Dams received breeding diet for rats and mice, No. 1314 TPF, ad libitum
- Water: sterilized community tap water, ad libitum
- Acclimation period: 14-17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% sodium carboxymethyl cellulose + 0,1% Polysorbate 80 (Tween 80), diluted in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item and vehicle preparations were conducted weekly; the test item solution was prepared fresh on the day of application.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As the test item’s solubility in water is poor, corn oil was used as an organic solvent for the preceding dose range finding study. Although the solubility of the test item in corn oil was sufficient, its oral application resulted in severe respiratory effects (laboured breathing, wheezing breath sounds) in all rats of the high dose group (1000 mg/kg body mass). The study director and the study monitor concluded that the physical (high surface activity) rather than the chemical properties of the test material when applied orally in this formulation lead to the effects observed in study. Based on an assessment of the physiochemical properties of the test material an adapted formulation from one of the acute toxicological studies was considered safe for application. The test item was applied as an emulsion in a watery solution at the three dosages specified in the study plan.
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until indications for mating were detected
- Proof of pregnancy: vaginal plug / sperm in vaginal referred to as Day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
(P) Males: 2 weeks before mating and continued throughout the mating period until the study was terminated
(P) Females: 2 weeks before mating, the variable time to conception, the duration of pregnancy and at least 4 days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation depended on the female performance: 14 days pre-mating, up to 14 days until mating, an average of 21 days of gestation, and a minimum of 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously performed dose range finding study (OECD 407), the test item was administered in corn oil at three dosages up to 1000 mg/kg body mass over a time period of at least 40 days and produced irritating but no observable toxic effects in the test animals. Although its oral application resulted in severe respiratory effects (laboured breathing, wheezing breath sounds) in all rats of the high dose group (1000 mg/kg body mass), it was concluded that the physical (high surface activity) rather than the chemical properties of the test material lead to the effects observed in study. Therefore, the vehicle formulation was changed as described and 1000 mg/kg was determined as high dose for this study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly and once before beginning of application

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly and once before beginning of application. Females during pregnancy: Day 0, 7, 14, 20, within 24h post parturition and Day 4 post partum.

FOOD CONSUMPTION: Yes
- Time schedule: at least once weekly

WATER CONSUMPTION: Yes
- Time schedule: twice weekly
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weights. In high dose and control animals additionally performed: histology with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, live births, stillborn, post-natal loss, abnormal pups, body weight.

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Animals were euthanased when found to be moribund or when the adequate number oflitters according to guideline OECD 421 was reached.

- Maternal animals: Dams with offspring were sacrificed on Day 4 post-partum or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of: external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: Epididymides, ovaries, testes and organs with macroscopic alterations. Additional findings in non-protocol organs present on slides were only recorded, if they were of pathological significance.
Testes and epididymes of all male adult animals were weighed. The ovaries, testes, epididymes, accessory sex organs and all other organs showing macroscopic lesions of all adult animals were preserved.
Statistics:
The arithmetic mean, standard deviation and median were calculated for all grouped numerical data originating from the monitoring of body mass and gross pathology (organ mass). In addition, the statistical software Graph Pad Prism for Mac, Version 5.01c. was used to calculate detailed column statistics (minimum/maximum data, 75% percentiles, std error, upper and lower confidence interval 95%). The study director pre-selected data sets of the ranked or incidental data for further analysis to those showing at least few alterations. Most statistical hypotheses in this study are characterised best as “many to one”– a vehicle control vs. three treatment groups. Therefore, the adequate analysis method is a One-Way ANOVA (Analysis of variance), followed by a post hoc t-test. With interval-scaled data, the One-Way ANOVA was supplemented by Dunnett’s post-hoc t-test. In case a Bartlett’s test for equal variances indicated that a data set may be heteroscedastic, it was analysed additionally by the Kruskal-Wallis test (rank transformation) and Dunn’s post-hoc t-test. However, the Study Director decided whether the additional test was necessary – data sets of single parameters were assessed as a whole, indications for heteroscedastic data subsets were disregarded. Ordinal-scaled data would have been analysed by the Kruskal-Wallis test, supplemented by Dunn’s t-test. The entire deductive statistics were performed using Graph Pad Prism. The significance level was set to 0.05.
Reproductive indices:
- Pre-implantation loss: corpora lutea - implantations
- Pre-natal loss: implantations - live births

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
all test groups: laboured breathing and wheezing breath sounds; 7 animals died due to test item reaching the respiratory tract, 1 animal died due to an application error
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
male high dose group: reduced mean body weight and reduced relative body weight gain; females high dose group: reduced relative body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
male high dose group: reduced mean body weight and reduced relative body weight gain; females high dose group: reduced relative body weight gain
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
high dose group: mean numbers per dam of corpora lutea slightly and statistically significantly reduced; medium and high dose: weak evidence for a delayed conception

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Laboured breathing and vocalisations, in animals from all groups treated with test item probably due to the high surface activity of the test item, small amounts of test item solution reached the respiratory tract, some leading to fatalities by acute exogenous lipid pneumonia. Eight animals died spontaneously or were killed during the course of the study. Their premature death of most of these animals is considered to be most likely due to an inadvertent deposition of a small amount of the test item at the laryngeal orifice that was inhaled during inspiration. One rat was euthanised due to an application error.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In high dose males, body weight and relative body weight gain were prominently reduced throughout the entire in-life phase with a net mean body mass loss in the first two weeks of application. Females were less affected during pre-mating, and the effect was even less pronounced during the following weeks. The observed tendency of a lowered relative body weight gain in females of the high dose group during gestation and lactation reached the level of statistical significance in data from the day of birth.
A reduction of the mean relative food consumption as well as a strong increase of mean relative water consumption of males and females from the high dose groups was observed during the first two weeks of application (pre-mating). During the following weeks, mean relative food consumption was mildly (sires) or only in parts (dams Day 14, Day 20 of gestation, Day 0 of lactation) affected. However, the mean relative water consumption of males remained prominently raised.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the high dose group, the mean numbers per dam of corpora lutea were slightly and statistically significantly reduced. In the medium and high dose group a weak evidence for a delayed conception was apparent.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The organ mass gave no evidence for toxicological effects of the test item on the sexual organs of Wistar rats.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At gross necropsy, no abnormalities were found that could be related to the administration of the test item.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The histomorphological examination of selected rat organs of the male and female genital system (24 males and 24 females) did not reveal morphological lesions related to the test item.


Effect levels (P0)

open allclose all
Dose descriptor:
LOEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects were considered secondary effects due to parental toxicity
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction of mean relative food consumption and strong increase of mean relative water consumption, reduced mean body weight and body weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
high dose group: significantly raised post natal loss, reduced mean numbers of live pups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduction of body mass with increasing dosage and a statistically highly significant reduction of mean pup body mass and mean litter mass in the high dose group at day four of lactation
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)

VIABILITY (OFFSPRING)
In the high dose group significantly reduced mean numbers of live pups at Day 4 were observed. Data of the numbers of abnormal pups born, or the loss of offspring (pre-implantation and pre-natal) were normal for rats of this strain and age. In conclusion, post-natal loss was significantly raised in the high dose group.

BODY WEIGHT (OFFSPRING)
A tendency of a reduction of body mass for both genders with increasing dosage and a statistically highly significant reduction of mean pup body mass and mean litter mass in the high dose group at day four was apparent. A mild and statistically significant reduction of male mean pup body mass was found at day of birth in the high dose group when compared to the vehicle control.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects are considered to be secondary effects due to parental toxicity
Dose descriptor:
LOAEL
Remarks:
reproduction
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: observed effects are considered to be secondary effects due to parental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Summarised results of the study.

OBSERVATIONS

Dosage (units)

 

 

Vehicle

 

 

Low dose

 

 

Medium dose

 

 

High dose

Pairs started

11*

11#

11#

11#

Totals after first and second mating

Females achieving pregnancy

10

8

9

11

Conceiving Days

1 - 5

10

8

6##

10

Conceiving days 6

0

0

1##

1

Pregnancy = 21 days

0

0

0##

0

Pregnancy = 22 days

8

4

5##

10

Pregnancy ≥ 23 days

2

4

2##

1

Dams with live young born

10

8

9

11

Dams with live young at Day 4 pp

10

8

8

11

Corpora lutea / dam (mean)

16,3

14,1

16,3

12,6

Implantations / dam (mean) 

12,6

9,4

12,6

11,5

Live pups / dam at birth (mean) 

10,7

7,9

9,6

9,6

Live pups / dam at day 4 (mean) 

10,1

6,9

8,9**

5,0

Litter mass Day 0 

64,8

50,3

59,8

48,9

Litter mass Day 4

92,6

67,8

91,4

42,2

No of pups

Live pups born Day 0 (count)

107

63

86

106

Stillborn (count) 

3

3

11

2

Total of pups born Day 0 (count) 

110

66

97

109

Stillborns / pups total (%)

2.8

4.8

12.8

2.8

Sex ratio

Sex ratio d0 (total numbers M / F

54 / 56

33 / 33

49 / 48

54 / 55

Sex ratio d0 (mean, M / F) 

1

1

1

1

Sex ratio d4 (total numbers M / F

56 / 45

31 / 24

38 / 33 **

30 / 25

Sex ratio d4 (mean, M / F) 

1.2

1.3

1.2

1.2

Body mass pups (g)

 

Male pups Day 0 (mean) 

6,1

 

6,3

5,8

5,2

Female pups Day 0 (mean) 

5,8

6,1

5,5

4,9

Male pups Day 4 (mean) 

9,6

10,3

9,6

7,7

Female pups Day 4 (mean) 

9,1

10,0

8,9

7,3

Abnormal pups 

 

 

 

 

Dams with 0 

10

8

9

11

Dams with 1 

0

0

0

0

Dams with ≥ 2 

0

0

0

0

Loss of offspring 

Pre-implantation (corpora lutea minus implant)        

Dams with pre-implantation loss (count) 

10

8

9

11

Pre-implantation loss (mean/group) 

3,7

4,8

3,9

2,4

Pre-natal (implantations minus live births) 

Dams with pre-natal loss (count) 

10

8

9

11

Pre-natal loss (mean/group) 

1,9

1,5

2,9

1,8

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

Dams with post-natal loss (count) 

10

8

9

11

Post-natal loss (mean pups/group) 

0.6

1.0

0.7

4.6

* female E112 invalidated

# one female each died within pre-mating phase;

## not reflecting data of two dams (no sperm plug found during mating phase, but pregnancy achieved)

** not reflecting litter mass data of one dam (E135) at Day 4 (data point lost)

Applicant's summary and conclusion

Conclusions:
No effect on reproductive performance were observed.
Executive summary:

Considering the fact that no abnormalities were found by a gross necropsy and in the following histopathological examination of the reproductive organs, the authors concluded that the negative effects of the high-dose of the test item on Wistar rat reproduction were not caused by its toxic properties on those organs directly, but rather on other secondary local or systemic factors affecting parental animal health.