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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.09.2007-14.11.2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexamethyldisilazane
- Physical state: Colourless liquid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc, Portage, MI
- Age at study initiation: Minimum of nine weeks
- Weight at study initiation: Males: 291.8 to 354.8 g; Females: 200.5 to 254.9 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages elevated over bedding except during mating when animals were cohoused in the home cage of the male.
- Diet (e.g. ad libitum): Ad libitum, except during exposure period
- Water (e.g. ad libitum): Ad libitum, except during exposure period
- Acclimation period: Seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.04-22.86
- Humidity (%): 33-66
- Air changes (per hr): 11.8-14.3
- Photoperiod (hrs dark / hrs light):


IN-LIFE DATES: From: 27.09.2007 To: 05.03.2008

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000 litre stainless steel and glass Rochester-style inhalation chambers.
- Method of holding animals in test chamber: four levels of 20 individual animal compartments. The animal cage position assignment within each chanber was rotated daily.
- Source and rate of air: Building air (rate: no data)
- Method of conditioning air: Air from a Nash Air Compressor was passed through a series of filters to remove contaminants. This conditioned air was then passed through HEPA and activated charcoal filters before delivery to the chmabers.
- Temperature, humidity, pressure in air chamber: 22 ±3oC, 50 ±20% humidity, no data on pressure
- Air flow rate: No data
- Air change rate: 10-15 chamber volume air changes per hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.
- Samples taken from breathing zone: No data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to seven days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): Individually. Pregnant females were moved into shoebox cages.
- Any other deviations from standard protocol: None evident
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Automated sampling system designed so that test atmosphere continuously pulled from the chamber and delivered to analyser. Analysis was done using a gas chromatograph equipped with a flame ionisation detector to determine the actual chamber concentrations of test substance.
Duration of treatment / exposure:
Exposure period: Males (14 days premating and mating up to 28 days): Females (premating 14 days, mating, gestation, and postpartum days 1-3 up to 42 days
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: Males 28 days; Females up to 42 days
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
Screening study only, so F1 matings were not conducted.
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100 and 400 ppm (0.16, 0.66, and 2.66 mg/L)
Basis:
other: target
No. of animals per sex per dose:
10 males and 20 females (including 10 for toxicity group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Based on previous study.
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality, and daily for clinical signs of toxicity (including abnormalities in nesting)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and then weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: First day of dosing, and at least weekly thereafter, and on day of sacrifice.

FOOD CONSUMPTION:
- 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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice as a terminal procedure.
- Animals fasted: Yes, overnight
- How many animals: All males and toxicity phase females. Details reported in Section 7.5.3

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, details reported in Section 7.5.3
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Testes were weighed and examined microscopically.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No, all animals were killed in Day 4 post-partum for exminations.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: mating, pregnancy, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio of live births/litter size, and sex ratio.  The number of corpora lutea and the number of uterine implantation sites were determined for all females.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 29 days treatment.
- Maternal animals: All surviving animals on day 4 post-partum

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

HISTOPATHOLOGY / ORGAN WEIGHTS: See Section 7.5.3
Postmortem examinations (offspring):
SACRIFICE
- All offspring on day 4 post-partum
- These animals were subjected to gross necropsy examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not conducted
Statistics:
All data analysis was carried out using SAS version  8.2 or 9.1.3. Descriptive toxicology endpoints:  Parametric data was tested using Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant); nonparametric data was tested by Kruskal-Wallis Test followed by Wilcoxon.  Repeated measures ANOVA was applied to data sets of multiple measurements  across time.  Histomorphology:  Cochran-Armitage Trend test for severity with Fisher's Exact Test for  pairwise comparisons.  Kruskal-Wallis was used to test for shifts in  grade with the Wilcoxon test used to test pairwise comparisons.   Functional observational battery:  ANCOVA (baseline as covariate), repeated measures ANOVA, Mixed modeling, and Jonckeere-Terpstra test (categorical endpoints).
Reproductive indices:
Mating and fertilty indices
Offspring viability indices:
None

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:
no effects observed
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:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY: There were no deaths. Clinical signs attributed to test article included uncoordinated gait (not beyond  day 10 or 11) and decreased activity noted immediately after exposure in both sexes from the highest dose group. These clinical signs persisted for some time following the exposure period with animals returning to normal prior to the next day's exposure.

BODY WEIGHT AND WEIGHT GAIN: There was a statistically significant decrease in absolute body weights for females (-15%) with a corresponding decrease in total weight gain compared to controls for both sexes (62 and 25% for females and males, respectively) in the highest dose group.  Absolute body weights for the mid dose group females were identified as statistically decreased (-7%) from controls, however, total and percent weight gain were not different. 

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No adverse effects.

ORGAN WEIGHTS (PARENTAL ANIMALS): Organ weight changes included statistical decreases in absolute epididymides weight in the high dose males and lung weights in the high dose group females, with increases in kidney-to-body weight ratios for  mid and high dose group females and high dose group males.  Increased liver-to-body weight ratios were also noted in the high dose group females.  

GROSS PATHOLOGY (PARENTAL ANIMALS): No adverse findings.

HISTOPATHOLOGY (PARENTAL ANIMALS): Histopathological examination of tissues and organs for control and high exposure animals demonstrated an increased incidence of centrilobular hypertrophy in the liver of high dose group females.  Evaluation of intermediate dose levels confirmed this finding was limited to high dose group females. There were no other test article-related histopathological findings.  

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 ppm
Sex:
male/female
Remarks on result:
other: Generation: General toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
>= 400 ppm
Sex:
male/female
Remarks on result:
other: Generation: Reproductive toxicity (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

There were no adverse findings on any of the reproductive parameters investigated. See Table 1.
One group 2 female was found non-gravid with the remaining females producing litters similar in all respects to control.  A second group 2 female delivered a normal size litter which included one dead, edematous fetus.  This was considered an isolated, spontaneous finding with no additional occurrences observed within this or any other exposure group.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Summary of reproductive parameter findings*

 Parameter  Control  25ppm  100ppm  400ppm
 Length of gestation  21  22 22  22 
 Male pups  8
 Female pups
 Sex ratio  1.2 0.9  0.9  1.0 
 Total pups (mean)  15 15  15  15 
 Initial litter weight (g)  88.1 94.5  98.1  96.2 
 Initial average pup weight (g)  6.2 6.5  6.4  6.5 
 Implantation sites  16 16  16  15 
 Number of corpora lutea  17 17  17  16 
 Number pregnant  10 10  10 

*There were no statistically significant differences found.

Applicant's summary and conclusion

Conclusions:
In a well conducted and documented OECD 422 study (reliability score 1) conducted to GLP, the NOAEL for reproductive toxicity endpoints for hexamethyldisilazane, following whole body inhalation exposure, was ≥400 ppm (2.66 mg/L) in rats.
Executive summary:

In a well conducted and documented OECD 422 study (reliability score 1) conducted to GLP, undiluted hexamethyldisilazane was administered to Sprague-Dawley rats via whole-body vapour inhalation for six hours/day, seven days/week at target concentrations of 0, 25, 100 and 400 ppm. Each exposure group consisted of 10 male and 20 female rats. Females were further divided into a toxicity and reproductive group consisting of 10 animals each (details regarding the repeated dose toxicity elements of the study are presented in Section 7.5.3). Males were used to evaluate toxicity and reproductive parameters. Males were treated for 29 days, and reproductive group females were treated for 14 days prior to mating, throughout mating, gestation and up to and including postpartum day 3. The maximum number of exposures was 42 days. Animals were observed throughout the exposure period for mortality and signs of toxicity. Body weights and food consumption were monitored. Reproductive and developmental parameters evaluated included evidence of mating, pregnancy, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio of live births/litter size and sex ratio. All surving dams and pups were sacrificed on postpartum day 4 and examined for external gross lesions. The number of corpora lutea and the number of uterine implantation sites were determined for all reproductive phase females. There were no adverse effects on any of the reproductive and developmental toxicity parameters investigated, giving a NOAEL of 400 ppm for these endpoints. The NOAEL for repeated dose toxicity for the parents was 100 ppm, based on clinical observation, body weight changes and histological findings (see Section 7.5.3).