Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10-06-2008 to 12-08-2009
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 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:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc, NC
- Age at study initiation: Minimum nine weeks
- Weight at study initiation: Males: 231.5 to 255.7 g ; Females: 167.0 to 198.8 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages. Pregnant females were housed in shoebox-type cages.
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: Five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.19-23.24
- Humidity (%): 50-67
- Air changes (per hr): 13.3
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 15-06-2008 To: 25-02-2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:DMSD was ground to a fine powder using a mortar and pestle. Dosing solutions were prepared by weighing the appropriate amount of the test substance into a tared container and adding the appropriate amount of corn oil to yield the desired dose level. Solutions were prepared every seven days, based on the stability of the test substance in corn oil.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Most appropriate based on physical and chemical properties of test substance.
- Concentration in vehicle: Not given
- Amount of vehicle (if gavage): Total volume 5ml//kg
- Lot/batch no. (if required): 117K0127
- Purity: No data, used as provided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A GC/FID method was used to verify concentration, stability and homogeneity of the test substance in corn oil. Concentration verification was conducted for the initial dose preparations.
Duration of treatment / exposure:
Toxicity group males and females were treated for 28 and 29 days, respectively. Reproductive phase females were treated to post-partum day 3.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten females in toxicity group; ten females in reproductive toxicity group; ten males to determine reproductive and toxicological endpoints.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a range-finding study
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post-exposure recovery period.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before first dose, and then weekly. Skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (for example excessive grooming and repetitive circling), difficult or prolonged parturition or bizarre behaviour (such as self mutilation, walking backwards) were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined beginning with randomisation into the test groups, on the first day of dosing, at least weekly thereafter, and on the day of sacrifice. During gestation, the reproductive females were weighed on gestation days 0, 7, 14 and 20, within 24 hours of parturition, and on post-partum day four.

FOOD CONSUMPTION:For males, feeder weights were taken on days 1, 8 and 15 during the pre-mating period. For females, feeder weights were taken on days 1, 8, 15, 22 and the day prior to sacrifice.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At terminal sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All males and toxicity group females
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At terminal sacrifice
- Animals fasted: Yes
- How many animals: All males and toxicity group females
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to dosing and during week four of dosing.
- Dose groups that were examined: All males and toxicity group females.
- Battery of functions tested: cage side observations, hand-held observations, open field observations, categorical observations, measurement counts, motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Statistics:
Body weight, food consumption, hematology, and clinical chemistry data, and prothrombin times were analysed using a one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance as determined using a statistical test for normality for homogeneity of variance. If the data do not satisfy the parametric requirements, a Kruskal-Wallis test was used. If the ANOVA or Kruskal-Wallis test was significant (p < 0.05), pair-wise comparisons of the exposed groups to control were made using the Dunnett’s test or a Wilcoxon test, respectively. For variables with multiple measurements across time (motor activity, body weight and food consumption), a repeated measurements ANOVA was performed to determine if a significant time by treatment group interaction exists. Repeated measurements ANOVAs were done using the time (baseline and post-treatment), interval, sex and treatment to look for interactions between the treatment, the time and sex to determine if analysis could be done with the sexes combined. Reproductive parameters with the exception of litter size were analysed using an ANCOVA (Analysis of Covariance) with litter size as the covariate. Litter size was analysed using an ANOVA. Measured continuous Functional Observational Battery (FOB) data were analysed using a combination of ANCOVA with baseline evaluations used as the covariate and repeated measures ANOVA. Categorical FOB data was analysed using the Jonckheere-Terpstra test. Categorical Functional Observational Battery (FOB) data were analysed using the Jonckheere-Terpstra test. This test is used to detect a shift in the categorical observations. Microscopic findings were also analysed using a Cochran-Armitage trend test to indicate an increasing incidence trend with Fischer's Exact tests used to indicate increased incidence over the controls. Clinical signs data were analysed using Mixed Modelling repeated measures.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For the toxicity group males at 500 mg/kg/day, significant abnormal observations (p<0.01) were noted and included soiling in the abdominal region and urogenital soiling. Urogenital soiling was also significant (p<0.05) in males at 250 mg/kg/day. Soiling of the muzzle was a significant abnormal observation (p<0.02) in the toxicity group females at 500 mg/kg/day. Both abdominal soiling and urogenital soiling were significant abnormal observations (p<0.01) in the reproductive group females at 500 mg/kg/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
All but one animal survived to their scheduled necropsy. The animal that died was euthanised following a dosing injury.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant differences across treatment groups in the mean body weights on any day for any of the three groups: males, toxicity group females, and reproductive toxicity group females. With respect to body weight gain, male group 4 animals had a statistically significant decrease in body weight gain (p<0.05) during week 4. The 500 mg/kg/day male group also had a statistically significant decrease (p<0.02) in total gain from day 1 to 29. For toxicology females there was a statistically significant (p<0.02) in body weight gain during week 3. There were no statistically significant differences in body weight gain for the reproductive females in any of the treatment groups: week 1 and 2 pre-mating, gestational weeks 1, 2, 3 and post-partum day 0 to post-partum day 4. Weight gain for the reproductive females in the three treatment groups was not different from control for the interval from day 1 of study to post-partum day 4.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no differences in the average daily food consumption across treatment groups for the reproductive females group or the toxicity female group for any of the measured time periods. In the male group there was a significant difference across treatment groups in week 2, however, there was not a significant difference between control and any of the treatment groups for that week.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant differences noted in the other hematology values or hematology differential data across groups for either sex. No toxicological significance is assigned at this time to any statistically identified differences in hematology parameters since the findings were within or slightly below historical control ranges for this laboratory and the findings did not correlate with a pathological outcome.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were dose-related decreases in male and female alkaline phosphatase and total bilirubin values as well as a dose related decrease in male aspartate aminotransferease values. No toxicologic significance was assigned to any statistically identified differences in clinical pathology since findings were within or slightly below historical control ranges for this laboratory and the findings did not correlate with a pathological outcome.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No statistically significant differences between the control and treatment groups in either sex at the treated time point for all the FOB ranked tests, except for an increase in defecation (males) at 500 mg/kg/day at a significance of p <0.05. There was no dose response associated with this effect, nor did it correlate with any change in the other neurobehavioral tests conducted on the same animals. There were no statistically significant differences between either male or female treatment groups and their respective controls for the FOB continuous test and motor activity. There were no treatment-related changes associated with dimethylsilanediol administration on rat neurobiological function as evaluated with FOB and motor activity parameters.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant differences noted for the following organ weights in toxicity group males; adrenal glands (24% decrease at 500 mg/kg/day at p <0.01), liver (19% increase at 250 and 31% increase at 500 mg/kg/day at p <0.01), testes (12% decrease at 500 mg/kg/day at p <0.01), and thymus (31% decrease at 500 mg/kg/day at p <0.01). There were statistically significant differences for the mean percentage of organ weights relative to body weights for adrenal glands (decrease at p <0.05), liver (increase at 250 and 500 mg/kg/day at p <0.01), and thymus (decrease at p <0.05) for the toxicity group males. There were statistically significant differences noted liver weights in toxicity group females; liver (24% increase at 250 and 61% increase at 500 mg/kg/day at p <0.01). There were statistically significant differences for the mean percentage of organ weights relative to body weights for liver (increase at 250 and 500 mg/kg/day at p <0.01) for the toxicity group females. The increased liver weights in toxicity males and females correlated with the histopathologic finding of centrilobular hyperthrophy. There were no other treatment-related differences in organ weights, absolute and relative (mean %) for toxicity group males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the liver, the finding of discoloration (+/- mottled) was observed more commonly in rats of both sexes administered ≥250 mg/kg/day. Liver enlargement was grossly more notable in females administered 500 mg/kg/day. Lung discoloration was more common in male rats administered ≥250 mg/kg/day. Other findings generally occurred singly and were not considered to be attributable to test article administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: Males
There were two primary liver findings in male rats. In the liver of male rats, the incidence of centrilobular hypertrophy was significantly increased (p <0.01) in 250 and 500 mg/kg/day groups (8/10 animals in each group) over control. The increased incidence of centrilobular hypertrophy identified correlates with the statistically significant increase in relative liver weight observed for these groups. This is a common effect of xenobiotic administration and is considered an adaptive change.

Additionally, there was minimal to moderate brown pigment accumulation in and around bile ducts, with associated bile duct hyperplasia and chronic inflammation in 9/10 high-dose males. Under polarised light some pigment accumulations show birefringence. The finding was split into three components and recorded as 1) brown pigment, 2) periportal chronic inflammation, and 3) bile duct hyperplasia associated with brown pigment accumulation. The severity of the inflammatory and bile duct hyperplasia components generally closely matched the pigment accumulation. The brown pigment was not observed in control animals, at lower dose levels, or in female rats. Periportal chronic inflammation (9/10 animals), hyperplasia in the bile ducts associated with brown pigment accumulation (8/10 animals), and brown pigment accumulation (9/10 animals) showed significant increase in incidence (p<0.01) over control only in the 500 mg/kg/day group. For these microscopic findings in the liver, there were no significant shifts in severity across treatment groups.

Females:
In the liver, the incidence of centrilobular hypertrophy was significantly increased (p <0.01) in the 250 and 500 mg/kg/day female toxicity groups. The increased incidence of centrilobular hypertrophy identified correlates with the statistically significant increase in relative liver weight observed for these groups. This is a common effect of xenobiotic administration and is considered an adaptive change.

For liver periportal vacuolization, only the 500 mg/kg/day toxicity female group was significantly (p <0.02) increased in incidence over the control group. Comparison of the graded animals only showed a significant increase in severity grade across treatment groups for liver vacuolization in females. It is generally but not universally held that this finding in and of itself is not considered adverse unless severe.

Thyroid Gland:
Both the 250 and 500 mg/kg/day male toxicity groups (8/10 and 9/10 animals, respectively) had significantly increased incidence (p <0.01) of thyroid gland follicular hypertrophy than did the control group and the comparison of the graded animals showed a significant increase in severity grade across treatment groups. Hypertrophy of the thyroid follicular epithelium is a common secondary response to increased thyroid hormone catabolism due to up-regulation of hepatic microsomal enzymes in response to xenobiotic administration. The rat is particularly sensitive to this effect due to the species’ lack of a protective carrier protein, thyroid binding globulin.

Prostate Gland
The 500 mg/kg/day group had a statistically significant increase in incidence (p <0.05) in chronic inflammation of the prostate gland over that seen in the control group. Chronic inflammation of the prostate gland is a fairly common spontaneous finding. This finding is not considered to be attributable to test article administration, particularly since the two most severe instances occurred in rats administered 0 and 50 mg/kg/day

Lung
In the lungs of males rats, there was an increasing trend in the observed incidence of pulmonary histiocytosis (aggregates of foamy macrophages). For histocytosis in the lungs there were no pair wise significant differences between any treated group and control. This is a very common spontaneous finding and there was no clear increase in severity associated with dosage. This finding is not considered to be attributable to test article administration.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 3: Absolute and relative organ weights of males and toxicity group females

 

Males

Females

DAILY DOSE
(mg/kg bw/day)

0

50

250

500

0

50

250

500

NUMBER OF ANIMALS

 10

 10

10 

10 

 10

 10

10 

 10

BODY WEIGHT MEAN (g)a

430.83

421.51

420.22

401.2

259.56

256.32

257.99

254.47

BRAIN

 

 

 

 

 

 

 

 

Absolute Weighta

g

1.99

1.97

1.99 

1.97 

1.93

1.92 

1.90 

1.92 

ADRENALS

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.05935*

0.05482

0.05601

0.04514** 

0.071

0.068 

0.072 

0.068 

EPIDIDYMIDES

 

 

 

 

Absolute Weighta

g

1.2160

1.1929

1.1653

1.1224

n.a.b

n.a.b

n.a.b

n.a.b

HEART

 

 

 

 

 

 

 

 

Absolute Weighta

g

1.36 

1.29 

1.34 

1.25 

 0.88

0.85 

0.86 

0.87 

KIDNEYS

 

 

 

 

 

 

 

 

Absolute Weighta

g

2.84

2.86

3.02

2.85

 1.76

1.79 

1.80 

1.74 

LIVER

 

 

 

 

 

 

 

 

Absolute Weighta

g

12.4102*

12.7829

14.8119**

16.2321**

7.4828**

7.4317

9.2845**

12.0684**

PROSTATE GLAND

Absolute Weighta

g

0.8422

0.8371

8244

0.7347

n.a.b

n.a.b

n.a.b

n.a.b

SEMINAL VESICLES

Absolute Weighta

g

1.8543

1.5639

1.7048

1.7136

n.a.b

n.a.b

n.a.b

n.a.b

SPLEEN

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.73 

0.72

0.73 

0.64 

 0.54

0.57 

0.53 

0.52 

TESTES

 

 

 

 

Absolute Weighta

g

3.4618**

3.5100

3.3643

3.0551**

n.a.b

n.a.b

n.a.b

n.a.b

THYMUS

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.4265**

0.4256

0.3509

0.2935**

 0.42

0.44 

0.37 

0.33 

OVARIES

 

 

 

 

Absolute Weighta

g

n.a.b

n.a.b

n.a.b

n.a.b

 0.133

0.134 

0.128 

0.142 

UTERUS

 

 

 

 

Absolute Weighta

 

n.a.b

n.a.b

n.a.b

n.a.b

0.42 

0.44 

0.37 

0.33 

a Group means at the end of terminal necropsy are shown.

b n.a. = not applicable

* p0.05, ** p0.01 (one-way analysis of variance)

Table 4: Incidence of selected pathologies

Parameter

n=5/sex

Dose level (mg/kg bw/day)

Control

61

305

1221

 

Liver: centrilobular hypertrophy

M

0+++

1

8+++

8+++

 

F

0+++

0

8+++

10+++

 

Liver: chronic multifocal inflammation

M

9

8

8

1

 

F

7

9

6

4

 

Liver: Periportal chronic inflammation

M

0+++

0

0

9+++

 

Liver: periportal hepatocellular vacuolation

F

5+++***

4

7

10++

 

Liver: bile duct hyperplasia

M

0+++

0

0

8+++

 

F

-

-

-

-

 

Liver: brown pigment

M

0+++

0

0

9+++

 

F

-

-

-

-

 

Prostate gland: chronic inflammation

M

2+*

6

5

7+

 

F

-

-

-

-

 

Prostate gland: single cell necrosis

M

1

0

0

1

 

F

-

-

-

-

 

Thyroid gland: follicular hypertrophy

M

1+++*

0

8+++

9+++

 

F

0

0

0

1

 

+ indicate a significant trend by the Cochran-Armitage Test across treatment groups (in the control column) or between control and treatment group by Fischer’s Exact Test with p values of +0.05, ++0.02 and +++0.01. * indicate a significant shift in grade across treatment groups (in the control column) with p values of *0.05, **0.02 and ***0.01.

See attachments for result tables.

Applicant's summary and conclusion

Conclusions:
In the combined repeated dose toxicity study with the reproduction / developmental toxicity screening test with dimethylsilanediol, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for general systemic toxicity was concluded to be 250 mg/kg bw/day based on hepatic brown pigment accumulation in and around the bile ducts, with associated bile duct hyperplasia and chronic inflammation at 500 mg/kg bw/day.