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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: RccHan:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 266-347g; Females: 181-225g
- Fasting period before study: No
- Housing: Individually in Makrolon type-3 cages
- Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, Switzerland), ad libitum
- Water: Community tap-water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 06 January to 01 March 2011

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel sealed chamber
- Method of holding animals in test chamber: stainless steel wire cage units
- Source and rate of air: 40L/min
- System of generating particulates/aerosols: test material in glass flask, air pumped through flask at 40L/min
- Temperature, humidity, pressure in air chamber: 22.0 to 22.3 degrees C, 37.4 to 52.4%
- Air change rate: 10-15 per hour

TEST ATMOSPHERE
- Brief description of analytical method used: weighing test item reservoir before and after each exposure (nominal). On-line GC (analytical)
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: yes, female housed with male until evidence of mating was observed
- After successful mating each pregnant female was caged: in home cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Weighing test item reservoir before and after each exposure (nominal). On-line GC (analytical)
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 11 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
3 000 ppm (nominal)
Dose / conc.:
2 000 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on 14-Days Dose Range-Finding Inhalation Toxicity Study in the Han Wistar Rat

High dose of 3000 ppm reduced to 2000 ppm from day 12 due to early deaths of 3 females and 1 male at the higher dose.
Positive control:
No

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly. In addition females examined on days 0, 6, 13 and 20 of the gestation period

BODY WEIGHT: Yes
- Time schedule for examinations: Males twice weekly. Females twice weekly during pre-pairing and pairing periods and on days 0, 7, 14 and 20 post-coitum and days 0, 1 and 4 post-partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule - same as body weight

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):
no examined
Sperm parameters (parental animals):
not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, 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 treatment for at least 28 days, when no longer needed for assessment of reproductive effects.
- Maternal animals: All surviving animals on day 5 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
No organs weighed, abnormal tissues only retained.
Statistics:
Means and standard deviations of various data were calculated.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables would be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test [see References (6)] was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Mating performance and fertility
Duration of gestation
Corpora lutea count
Implantation rate and post-implantation loss
Offspring viability indices:
Litter size at first litter check
Postnatal loss days 0-4 post-partum

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Treatment with the test item caused early death of some animals at the high-dose level.

During the treatment with the test item at the dose level of 3000 ppm, two females were found dead either after exposure in the exposure chamber. After reduction of the high-dose level to 2000 ppm on day 12, two additional females were found dead; either in the exposure chamber or in the home cage. For one or two days before death, clinical signs including ruffled fur and decreased activity were observed in some animals.

No further clinical signs or observations were noted in any other animals.

BODY WEIGHT AND WEIGHT GAIN
Treatment with the test item at 3000/2000 ppm caused reduction of body weight gain during the dosing period of up to 10% compared to control in males. This reduction was statistically significant on a number of occasions. For females, similar reductions were noted during the first half of the dosing period, but was comparable to controls during gestation and lactation periods.

No effects on body weighs were observed at the dose levels of 1000 and 100 ppm.

FOOD CONSUMPTION
Treatment with the test item caused a dose-dependent decrease of food consumption in males at all dose levels.

At the dose levels of 3000/2000 and 1000 ppm, decrease of food consumption was statistically significant during the dosing period. Mean food consumption during this period was reduced by about 12% to 17% at the dose level of 1000 ppm and by up to 29% at the dose level of 3000/2000 ppm (percentages refer to the respective values compared to the control group).

At the dose level of 100 ppm, decrease of food consumption was statistically significant from day 5 to 8 and not statistically significant thereafter.

Treatment with the test item caused decrease of food consumption in females at the dose level of 3000/2000 ppm during the first half of the treatment period. This reduction was up to 34% compared to control and was statistically significant on several days. Thereafter food consumptionwas comparable to controls during gestation and lactation periods.

No effects on food consumption of females were observed at the dose levels of 1000 and 100 ppm. Mean food consumption at these dose levels was similar to control values during the entire study period.

MATING PERFORMANCE
No effect on mating performance was noted at any dose level.

DURATION OF GESTATION
No effect on duration of gestation was observed at any dose level.

CORPORA LUTEA COUNT
At the dose level of 3000/2000 ppm, lower number of corpora lutea was noted. Mean number of corpora lutea per dam was 11.3 compared to 13.6 in the control group. The difference was not statistically significant but the value at the high-dose level was below the range of the historical control data; mean historical control values were between 13.8 and 16.8 corpora lutea per dam. For this reason, the effect on corpora lutea was considered to possibly be test item-related.

At the dose levels of 1000 and 100 ppm, meannumbers of corpora lutea per dam were similar to the control value: 13.7 and 14.2, respectively.

IMPLANTATION RATE AND POST IMPLANTATION LOSS
At the dose level of 3000/2000 ppm, a lower implantation rate was noted. Mean number of implantations per dam was 10.5, compared to 12.7 in the control group; this difference was not statistically significant. If expressed as a percentage of the corpora lutea number, implantation rates were similar at the high-dose level and in the control group: 92.9% and 93.4%, respectively. Therefore, the difference in implantation rate was not due to an increased pre-implantation loss but was secondary effect to the lower number of corpora lutea.

At the dose levels of 1000 and 100 ppm, mean numbers of implantations per dam were similar to the control value: 13.2 and 12.4, respectively.
No effect on post-implantation loss was observed at any dose level.

ORGAN WEIGHTS
At the high-dose level in males, terminal body weight at the end of the dosing period was decreased by 9.6% compared to control, this reduction was statistically significant.

In males at the dose level of 3000/2000 ppm, statistically significantly higher by 15% (percentage refer to the value compared to the control group) kidney to body weight ratio was noted. Kidney weight relative to the brain weight was also statistically significantly higher than the respective control values. Absolute kidney weight at this dose level was slightly, not statistically significantly higher than the control value. Although increase of kidney weights was only moderate, this change was considered to be test item-related.

At the dose levels of 3000/2000 and 1000 ppm in males, a dose-dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. No further findings which may have indicated a specific effect of the test item on the central nervous system were noted as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. For this reason the toxicological relevance of this finding remained equivocal. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded

GROSS PATHOLOGY
Treatment of males with the test item at the dose level 3000/2000 and 1000 ppm caused an increased incidence of changes in urinary tract noted during macroscopic examination at scheduled termination as well as decedent animals. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder and thickened mucosa/wall of urinary bladder. At the dose level of 1000 ppm, the following findings were considered to be test item-related: stones in urinary bladder and pelvic dilation.

Treatment of females with the test item at the dose levels of 1000 and 3000/2000 ppm caused an increased incidence of changes in urinary tract found during macroscopic examination of females at scheduled termination as well as decedent females. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder, enlarged kidneys, dilatation of ureter, hemorrhagic contents or hemorrhagic watery fluid, thickened or discoloured mucosa/wall of urinary bladder and urinary bladder distended with urine or dilated. In addition, dark red foci in the vagina and/or thickened mucosa of the vagina were noted. These changes were possibly secondary to the inflammatory changes and lesions in the urethra (identified within histopathological examinations) and therefore were secondary effects to the treatment with the test item.

At the dose level of 1000 ppm, urinary bladder containing stones was found in one female. This finding was considered to be caused by the treatment with the test item.

The following findings were observed only in decedent animals: advanced autolysis, dark red discoloration of the lung, crateriform retractions in the stomach, Peyers patches not visible, enlarged adrenal glands, dark red discoloration of internal organs (including thymus, lungs, ovaries, adrenal glands and mandibular lymph nodes), reddish discoloration of bronchial lymph nodes and watery clear fluid in the abdominal cavity. These findings were considered to possibly arise peri-/post-mortem.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were observed mainly in the urinary organs in both sexes, both in the survivors and decedents, at the dose levels of 1000 and 3000/2000 ppm. The thyroid gland (surviving males) and heart (decedent females) were also considered to be the main target organs.

In the kidney, the following findings were noted in both sexes at the dose levels of 1000 and 3000/2000 ppm or only at the dose level of 3000/2000 ppm: granular deposits in the pelvis and in the tubules, pyelitis (with foreign-body granuloma), urothelial hyperplasia and ulcer/erosion, tubular simple dilation, increased mitoses in the tubular epithelium, pelvic dilation, tubular basophilia, hyaline droplets and mononuclear cell foci. Specific to decedent animals, tubular necrosis/degeneration and slight papillary necrosis, pelvic dilation, tubular basophilia and tubular simple dilation were mainly observed. Granular deposits in the pelvis, pyelitis (with foreign-body granuloma) and urothelial hyperplasia and ulcer/erosion were observed with a low frequency. Fibrosis, inflammatory cell infiltration and mononuclear cell foci were also observed.

In the ureter, the following findings were observed at the dose level of 3000/2000 ppm: granular deposits in the lumen (in males), inflammatory cell infiltration (in males) and luminal dilation (in males and females). Luminal dilation and inflammatory cell infiltration in the surrounding soft tissue were observed in decedent animals.

In the urinary bladder, the following findings were observed at the dose levels of 1000 and 3000/2000 ppm in both sexes: urothelial hyperplasia and increased inflammatory cell infiltration (with foreign-body granuloma) and at the dose level of 3000/2000 in both sexes: granular deposits in the cavity or submucosa, hemorrhage, ulcer and edema (only in males).

In the urethra, marked injury was observed specifically to the decedent females. Granular deposits, necrosis involving the mucosa, submucosa and surrounding tissues, inflammatory cell infiltration, luminal dilation and urothelial hyperplasia were observed in the urethra of all decedent females.
Treatment-related findings in the heart were specific to the decedents. Myocardial necrosis/degeneration, inflammatory cell infiltration, fibrosis, myocardial mineralization and hemorrhage were observed.

Urinary obstruction was indicated in most of the decedent animals by the marked luminal distention of the urethra, distention with urine of the urinary bladder (macroscopically), luminal distention of the ureter, pelvic dilation and tubular simple dilation of the kidney, and was probably caused by the existence of the calculi or the injury including necrosis and inflammation at the lower urinary tract (urethra). Urinary obstruction might have caused uremia and subsequent death. The remarkable heart lesions observed in this study might have caused heart failure and thereby also been the direct cause of death. The heart lesions observed in the decedents can be induced by uremia.

In the thyroid gland, amorphous materials in the colloid were observed in males of all treatment groups with dose-dependency in its severity and incidence. No further indicators of thyroid injury (necrosis, apoptosis, fibrosis, other degenerative changes, etc.), this change was considered to have been caused by metabolic change in the thyroid.

At the dose level of 3000/2000 ppm, treatment-related findings in the liver, hepatocellular hypertrophy and increased mitoses were found in decedents. Hepatocellular hypertrophy was macroscopically correlated with enlargement of the organ. The significance of these changes remains unknown.

Testicular tubular degeneration found in two males at each dose level of 100 and 1000 ppm and in four males at the dose level of 3000/2000 ppm was in most cases unilateral and within the range of historical control data. However, this effect cannot be excluded to be the secondary treatment effect.

Alveolar edema in the lung and congestion in the several organs were observed only in the decedent animals. Although these can be agonal or post mortem change, the possibility of the secondary changes associated with the heart disorder or uremia cannot be excluded.

Cortical hypertrophy of the adrenal gland (macroscopically correlated with enlargement of the organ), atrophy of the spleen (macroscopically correlated with reduction in size of the organ) and atrophy of the thymus and ulcer in the forestomach (macroscopically correlated with mucosal crateriform retractions) were observed only in the decedent animals. No other related findings were observed in the same organs and therefore, these changes were considered to be the secondary effect caused by stress.

In the vagina, mucification with mucus plug was observed in the decedent females. This change may have been caused by the stimulation by the marked inflammatory changes occurred in the urethra or associated with the possible inanition caused by the renal lesions in these animals.


Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the reduction of corpora lutea number and consequent reduction of the number of pups observed at 3000/2000 ppm

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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)

Not included in fertility assessment part of this study

Effect levels (F1)

Remarks on result:
other: Not included in fertility assessment part of this study

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 - Summary of reproductive performance

Group
(ppm)

1
(0)

2
(100)

3
(1000)

4
(3000/2000)

Number of females dead prior to pairing

0

0

0

3

Number of females paired

10

10

10

10

Number of females not mated

0

1

1

1

Number of paired females dead prior to scheduled termination

0

0

0

1

Number of females which reared their pups until day 4 post partum

10

9

9

8

Table 2 - Mean corpora lutea count

 Group (ppm) 1(0)   2(100) 3(1000)  4(3000/2000) 
 Mean  13.6 14.2  13.7  11.3 
 St. Dev.  2.2 1.0  1.1  1.9 
 N  10 10 

Table 3 - test atmosphere concentrations

Group

Achieved Test Atmosphere Concentration (ppm)

Target Test Atmosphere Concentration (ppm)

Test Atmosphere Concentration Relative to Target (%)

2

100 ± 1

(n=37, CV=0.5%)

100

99.9

3

1000 ± 3

(n=40, CV=0.3%)

1000

100.0

4 and 6

From 06 to 17-Jan-2011

2987 ± 55

(n=11, CV=1.8%)

3000*

100.0

From 18-Jan to 14-Feb-2011

2000 ± 9

(n=29, CV=0.4%)

2000*

99.6

* The dose level of 3000 ppm was administered from day 1 to 11 of the pre-pairing period. The dose level of 2000 ppm was administered starting from day 12 of the pre-pairing period onward.

Table 4 - exposure conditions

Group

Temperature [°C]

Relative humidity [%]

Oxygen concentration [%]

1 and 5

22.1 ± 0.1 (n=40)

51.3 ± 1.1 (n=40)

20.3 ± 0.0 (n=40)

2

22.1 ± 0.1 (n=37)

39.5 ± 1.2 (n=37)

20.2 ± 0.0 (n=37)

3

22.4 ± 0.1 (n=40)

38.8 ± 1.4 (n=40)

20.1 ± 0.0 (n=40)

4 and 6

22.2 ± 0.1 (n=40)

42.2 ± 1.9 (n=40)

20.0 ± 0.1 (n=40)

Applicant's summary and conclusion

Conclusions:
Based on the reduction of corpora lutea number and consequent reduction of the number of pups observed at the dose level of 3000/2000 ppm, the NOAEL (no observed adverse effect level) for reproduction was considered to be 1000 ppm.
Executive summary:

In a well-conducted, GLP compliant, OECD 422 study (reliability 1) treatment with the test item at the dose level of 3000 ppm caused early death of two females. After reduction of the high-dose level to 2000 ppm, two further females were found dead. Uremia resulting from the impairment of the urinary tract was indicated as a cause of the early deaths. The early deaths of animals might be due to the initial higher exposure to the test item at the concentration of 3000 ppm. All remaining animals survived the scheduled study period.

During macroscopic and microscopic examinations stones/granular deposits in the urinary tract, injury, inflammation and dilation of urethra, urinary bladder, ureter or kidney were found in females which died before scheduled termination but also in most of the survivors at the dose level of 3000/2000 ppm and some males and females at the dose level of 1000 ppm. In addition, in males and some females at the dose level of 3000/2000 ppm, higher kidney/body weight ratio was noted. It was considered that uremia was probably an important consequence of the urinary tract dysfunction. Uremia was postulated to be a direct cause of the pre-term deaths or, alternatively, to cause heart failure and consequent deaths of males and females at the high-dose level. Impairment of the urinary tract observed in males and females at the dose levels of 3000/2000 and 1000 ppm was considered to be adverse.

In addition to urinary tract, thyroid gland was also considered to be a target organ for the test item-related toxicity. During histopathological examination, amorphous materials in the colloid of the thyroid gland were observed in males of all treatment groups with dose dependency in its severity and incidence. This was considered to have been caused by a metabolic change in the thyroid gland. No further indicators of thyroid injury were observed and therefore the effect on thyroid gland was considered not to be adverse.

Treatment with 2,4,6,8-tetramethylcyclotetrasiloxane caused a reduction of food consumption in males at all dose levels and in females at the dose level of 3000/2000 ppm. Body weight gains and body weights were reduced at the dose level of 3000/2000 ppm in both sexes. Effects on food consumption, body weight gain and body weights were considered not to be adverse.

At the terminal examinations, at the dose levels of 3000/2000 and 1000 ppm in males, a dose dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. Reduction of brain weights is not expected to be a result of systemic toxicity even if body weights are reduced. Within this study, no behavioral or functional dysfunctions which indicated a specific effect of the test item on the central nervous system as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded even if a toxicological relevance of this effect remained equivocal.

At the dose level of 3000/2000 ppm, lower number of corpora lutea was noted. As a consequence, a lower implantation rate and a lower number of living pups at first litter check were noted. Although the differences to the control values were not statistically significant, they were below the range of the historical control values and were therefore considered to be related to the treatment with the test item. These effects occurred at the dose level at which severe maternal toxicity was noted early on in the study and therefore may be secondary to the maternal toxicity. Both possibilities: a reduction of corpora lutea as a specific effect of the test item or as an effect secondary to the maternal toxicity may be taken into consideration. In this view, the reduction of the number of corpora lutea and consequent reduction of number of pups may be an adverse effect.

For this reason, reduction of corpora lutea number and consequent reduction of number of pups was considered to be adverse. No further indication of a test item related effect on reproduction was observed at any dose level; mating performance, fertility, duration of gestation as well as pre- and post-implantation loses in dose groups were similar to the control values.

Based on the reduction of corpora lutea number and consequent reduction of the number of pups observed at the dose level of 3000/2000 ppm, the NOAEL (no observed adverse effect level) for reproduction was considered to be 1000 ppm.