Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a two-generation reproductive toxicity study on HMDS, conducted to GLP (WIL Research Laboratories, 2006) the NOAEC for reproductive toxicity is considered to be at least 5000 ppm. 

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09.12.2003 to 20.06.2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: Males (P): 243 - 313 g; Females: 141 - 196 g; Males (F1): 381 - 603 g; Females: 223 - 362 g
- Fasting period before study: None
- Housing: Individually in stainless steel wire-mesh cages, mating in home cage of male, following mating females were removed to plastic maternity cages until lactation day 21, then transferred back to wie-mesh cages.
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3
- Humidity (%): 50± 20
- Air changes (per hr): Minimum 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 03.12.2003 To: 12.12.2005
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: 2.0 m3 stainless steel and glass whole body inhalation chambers
- Method of holding animals in test chamber: None
- Source and rate of air: No data
- Method of conditioning air: No data
- Temperature, humidity, pressure in air chamber: 19-27oC, 34-66%, slight negative pressure, respectively
- Air flow rate: No data
- Air change rate: 12-15 changes/hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes, samples were taken from the approximate middle of each chamber
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days (or until evidence of mating)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): in a plastic maternity cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Duration of treatment / exposure:
At least 70 days prior to mating, throughout mating, gestation through gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia . Premating exposure period (males): 70 days.
Premating exposure period (females): 70 days. Duration of test: appproximately 18 months.
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
- F1 parental animals not mated until approximately 11 weeks (not stated) after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 3 weeks of age.
- Age at mating of the mated animals in the study: 14 -16 weeks.
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
1 600 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the results of a previous study (no details given)
- Rationale for animal assignment (if not random): random
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for appearance, behavior moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly body weights and food consumption were recorded on gestation days (GD) 0, 4, 7, 11, 14 and 20 and on Postnatal days (PND) 1, 4, 7, 14 and 21 for females in the F0 and F1 generations.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to pairing. Ovarian primordial follicle counts were recorded for all F1 females in the control and high exposure groups. 
Sperm parameters (parental animals):
Spermatogenic endpoints (sperm motility [including progressive motility], morphology and numbers) were recorded for all F0 and F1 males. 
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Each litter was examined to determine the number of fetuses, sex, still births runts, survival, litter viability and death. 

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Forty pups/sex/group from the F2 generation were selected for evaluation of surface-righting (PND 5 to day of positive response) and air-righting (PND 13, 17, 21 and 61) reflexes; of these, 20 F2 pups/sex/group were selected for FOB (PND 4, 11, 21, 45 and 60), auditory startle response (PND 20 and 60), locomotor activity (PND 13, 17, 21 and 61) and/or learning and memory assessment (PND 22 or 62). 
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

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

HISTOPATHOLOGY / ORGAN WEIGHTS

Organs examined at necropsy (macroscopic and microscopic): Complete detailed necropsy was conducted. Organ weights: Adrenal glands, brain, epididymis (total and cauda), kidneys, liver, lungs, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands and accessory fluids, spleen, testes, thyroid gland, uterus with oviducts and cervix   Histopathologic evaluation: Adrenal glands, brain, cervix, coagulated glands, epididymis (right), kidneys, liver, lungs, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, thyroid gland, uterus, vagina, vas deferens, all gross (internal) lesions. 
Postmortem examinations (offspring):
Nonselected F1 pups were necropsied on PND 21 or 28, and nonselected F2 pups were necropsied on PND 21.  Selected organs were weighed from F1 and F2 pups (one/sex/litter) that were necropsied on PND 21.  Selected F2 rats not allocated for neuropathology and brain dimension measurements were necropsied following completion of reflex ontogeny evaluations (PND 61) or at study termination (PND 72).  Each surviving  F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F2 pups; selected organs were weighed.  

Organs examined at necropsy (macroscopic and microscopic): Complete detailed necropsy was conducted. Organ weights: Adrenal glands, brain, epididymis (total and cauda), kidneys, liver, lungs, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands and accessory fluids, spleen, testes, thyroid gland, uterus with oviducts and cervix   Histopathologic evaluation: Adrenal glands, brain, cervix, coagulated glands, epididymis (right), kidneys, liver, lungs, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, thyroid gland, uterus, vagina, vas deferens, all gross (internal) lesions. 
Statistics:
Statistical methods: Parametric analysis was screened for homogeneity of variance using Levene's test and normality using Shapiro-Wilk's test. If the data was not homogenous and normal, then the data were analyzed using nonparametric statistics (Kruskal-Wallis ANOVA test followed by the Mann-Whitney U-test). Homogeneous data was analyzed by Chi-Square test with Yates correction factor, One-way ANOVA with Dunnett's test and Kolmogorov-Smirnov test (one-tailed test).  FOB data and histopathological findings were compared to the control group using a two-tailed Fisher's Exact test.  P< 0.05 or P < 0.01.
Reproductive indices:
Reproductive parameters (days between pairing and coitus, length of gestation, evidence of parturation difficulties, mating, fertility, copulation and conception indices, testicular and epididymal sperm counts, sperm production rate sperm motility and morphology, ovarian primordial follicle count), developmental landmarks (e.g., balanopreputial separation and vaginal patency).
Offspring viability indices:
Mean litter size, postnatal survival (postnatal day 0 to 4), postnatal survival for other intervals up to day 21.
Clinical signs:
no effects observed
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No test article-related mortalities or clinical findings were observed in this study. One female in the F1 generation (highest dose group) died of dystocia (17 dead fetused in utero).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Lower weekly body weight gains were noted for F0 males and females in the 1600 and 5000 ppm groups and F1 males in the 5000 ppm group.  Mean body weights in the 1600 ppm group for both the F0 and F1 generations were generally similar to control group values, while those in the 5000 ppm group were reduced throughout the majority of both the F0 and F1 generations. Food consumption was lower for the 5000 ppm group males during the premating period (F0) and throughout the entire generation (F1).  Food consumption for F1 females in the 5000 ppm group was reduced during the first week following weaning (week 17-18) only. 

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): No adverse effects.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No adverse effects.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No adverse effects.

ORGAN WEIGHTS (PARENTAL ANIMALS): Test article-related higher kidney weights were noted for F0 and F1 males in the 1600 and 5000 ppm groups.  Corresponding histopathological effects of HMDS in this study were similar to those previously reported in rats. Higher mean relative liver weights were noted for the F1 males in 1600 and 5000 ppm groups.

GROSS PATHOLOGY (PARENTAL ANIMALS): No adverse treatment-related findings.

HISTOPATHOLOGY (PARENTAL ANIMALS): Test article-related higher kidney weights were noted for F0 and F1 males in the 1600 and 5000 ppm groups.  Corresponding histopathological effects of HMDS in this study were similar to those previously reported (Cassidy et al., 2001) in rats following long-term inhalation exposure at 593 and 5012 ppm.  These findings included hyaline droplets (F0 and F1 males at 5000 ppm) and increased incidence and severity of basophilic tubules in the kidneys (F0 males, F1 males and F1 females at 5000 ppm).  Male rat-specific hyaline droplet (consistent with alpha 2 urinary globulin) nephropathy was associated with the increase in basophilic tubules.  Other test article related microscopic findings, including golden-brown pigment in the periportal areas of the liver for F0 males in the 5000 ppm group and F1 males and females in the 1600 and 5000 ppm groups.  This pigment was accompanied by infiltration of primarily mononuclear inflammatory cells and/or bile duct hyperplasia in the liver in the F0 and F1 5000 ppm groups, and corresponded to higher mean relative liver weights for the F1 males in the 1600 and 5000 ppm groups.  The golden-brown pigment demonstrated bright red birefringence with a central dark Maltese cross under polarized light, characteristic of porphyrin (Churukian, 2002).  In the 5000 ppm group F1 males and females, golden brown pigment was also noted in the medullary macrophages of the mesenteric lymph nodes and alveolar macrophage aggregates were noted for F0 and F1 males and females in the 5000 ppm group. Effects in the lungs were diagnosed as idiopathic rat respiratory syndrome, and were not therefore related to treatment.
Dose descriptor:
NOAEC
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicity: Based on effects in the liver.
Dose descriptor:
NOAEC
Effect level:
>= 5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity: No reproductive toxicity in the parent animals of the P and F1 animals.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING): There were no treatment-related effects in postnatal survival of the F1 litter.


CLINICAL SIGNS (OFFSPRING): None


BODY WEIGHT (OFFSPRING): Offspring body weight gains were decreased for F1 pups in the 5000 ppm group from PND 4-21 and F2 pups in the 1600 and 5000 ppm groups from PND 4 to 14.  In the 5000 ppm group, these reduced body weight gains resulted in substantially lower mean body weights on PND 14 and 21 in both generations (approximately 6% to 7% lower by PND 21); lower body weights continued to be observed for the F2 males and females through PND 56 and PND 35, respectively.  In the F2 pups in the 1600 ppm group, the lower body weight gain during PND 4-14 resulted in significantly lower mean body weights on PND 14 (approximately 7% and 5% for male and female pups, respectively); lower body weights were also observed for the F2 males and females in this group through PND 49 and 35, respectively.


SEXUAL MATURATION (OFFSPRING): Balanopreputial separation and vaginal patency were not affected in the F1 pups.


ORGAN WEIGHTS (OFFSPRING): On PND 21, decreased thymus weights were noted for F2 male pups in the 1600 and 5000 ppm groups and F2 female pups in the 5000 ppm group only. There were no such findings in the F1 pups.


GROSS PATHOLOGY (OFFSPRING): There were no remarkable findings in the F1 and F2 pups that were found dead or euthanised in extremis. There were no abnormal findings in the F1 and F2 pups.


HISTOPATHOLOGY (OFFSPRING): No treatment-related effects.


OTHER FINDINGS (OFFSPRING): F1 adult females and their offspring exhibited no exposure-related effects during the FOB assessments. No test article related effects on air-righting reflex or learning and memory were noted for F2 offspring. The only test substance-related effect on attainment of developmental landmarks noted for F2 pups was a slight delay in attainment of the surface righting response for females in the 5000 ppm group (65% attained the response on PND 5 versus 90% in the control group).
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicity: Based on effects in the liver.
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
1 600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on decreased body weight gains in the F2 generation animals at 5000 ppm.
Reproductive effects observed:
not specified

There were no statistically significant offspring weights at any dose level. Statistically significant (p<0.05 or p<0.01) lower mean pup body weight gains were noted for males in the 400, 1600 and 5000 ppm groups and for females in the 400 and 5000 ppm groups from PND 4 to PND 7. Male and female pup weight gain in the 5000 ppm group continued to be slightly lower (not statistically significant) than the control group during PND 7 to 14 and 14 to 21. As a result of the lower weight gain, mean pup body weights in the 5000 ppm group were lower (7.2% to 7.3%) than control group values on PND 21; the difference from the control group for the females was statistically significant (p<0.05). Because the lower weight gain in the 400 and 1600 ppm groups did not persist, the lower mean body weight gains in these groups during PND 4 to 7 were not considered test article-related. No other differences in pup body weight or body weight gain relative to the control group were noted in the 400, 1600 or 5000 ppm groups. Mean male and female pup body weights and body weight changes in the 100 ppm group were unaffected by test article exposure throughout the postnatal period. The NOAEC for F1 developmental toxicity was 1600 ppm due to decreased offspring weights at 5000 ppm. There were no gross anomalies in the pups. The NOAEC for developmental neurobehavioral endpoints was 1600 ppm due to the lack of habituation exhibited in the locomotor activity assessments and delayed attainment of the surface righting response in the 5000 ppm group F2 females, and decreases in average and peak acoustic startle response on PND 20 in the 5000 ppm group F2 males and females. The NOAEC for neuropathologic endpoints was 5000 ppm.

Table 1 - SUMMARY OF OFFSPRING WEIGHTS (F1- pre-weaning) [G] (LITTER AS EXPERIMENTAL UNIT)      

GROUP:                

0 PPM           

100 PPM           

400 PPM          

1600 PPM          

5000 PPM 

PND 1

MALES               

MEAN                 

7.0               

6.9         

7.1 

7.0 

6.9

S.D.  

0.59

0.54

0.90

0.49

0.70

N 

27              

29

26

27                 

25

FEMALES               

MEAN                   

6.6

6.5

6.7

6.6

6.6

S.D.                  

0.60

0.58

0.78

0.47

0.67

N

27

29

26

27

25

PND 4 (BEFORE SELECTION)

MALES

MEAN                   

10.0

9.9

10.5

9.8

10.2

S.D.                  

1.08

1.00

1.44

0.99

1.12

N 

27

29

26

27

25

FEMALES

MEAN                   

9.6

9.4

9.8

9.3

9.7

S.D.                  

1.06

1.0.4

1.32

0.98

1.23

N 

27

29

26

27

25

PND 7

MALES

MEAN                   

14.3

14.0

14.3

13.7

13.6

S.D.                  

1.63

1.79

1.82

1.59

1.25

N 

27

28

26

27

25

FEMALES

MEAN                   

13.7

13.2

13.4

13.0

13.1

S.D.                  

1.57

1.80

1.78

1.56

1.31

N 

27

28

26

27

25

PND 14

MALES

MEAN                   

25.8

26.0

26.1

24.9

24.4

S.D.                  

2.33

2.18

2.46

2.88

2.09

N 

27

27

25

27

25

FEMALES

MEAN                   

24.9               

25.0               

24.8               

24.0               

23.6

S.D.                  

2.28               

1.93               

2.43               

2.56               

2.27

N 

27                                                                      

27

25

27

25    

PND 21

MALES

MEAN                   

41.3               

42.0               

40.9               

39.5               

38.3               

S.D.                  

4.42               

4.19               

5.09               

4.22               

4.13

N 

27                                                                                

27

25

27

25

FEMALES               

MEAN                   

40.1               

40.2               

38.8               

37.9               

37.2*               

S.D.                  

4.30               

3.91               

4.62               

3.99               

4.06

N 

27                                                                     

27

25  

27 

25

PND = POSTNATAL DAY 

MODIFIED STATISTICS USED. 

* = Significantly different from the control group at 0.05

 

  Table 2 - SUMMARY OF OFFSPRING WEIGHT CHANGES [G] (F1- pre-weaning) (LITTER AS EXPERIMENTAL UNIT)

GROUP:                

0 PPM           

100 PPM           

400 PPM          

1600 PPM          

5000 PPM 

PND 1-4 (BEFORE SELECTION)

MALES               

MEAN                 

3.0                

3.0                

3.3                

2.8                

3.2

S.D.  

0.62               

0.67               

0.75               

0.64               

0.74

N 

27                                                                             

29 

26

27

25 

FEMALES               

MEAN                   

2.9                

2.8                

3.1                

2.7                

3.1               

S.D.                  

0.62               

0.68               

0.75               

0.64               

0.79                

N

27                                                                      

29

26

27

25

PND 4 TO 7

MALES

MEAN                   

4.4                

4.1                

3.8+               

3.9+               

3.5++

S.D.                  

0.68               

1.09               

0.97               

0.95               

0.71

N 

27                                                                            

28

26 

27  

25

FEMALES

MEAN                   

4.2                

3.8                

3.6+               

3.7                

3.4++

S.D.                  

0.67               

1.01               

0.89               

0.93               

0.67                

N 

27                                                                            

28

26

27

25

PND 7 TO 14

MALES

MEAN                   

11.4               

11.9               

11.9               

11.2               

10.8

S.D.                  

1.42               

1.44               

1.28               

1.83               

1.11

N 

27                                                                   

27

25

27   

25

FEMALES

MEAN                   

11.2               

11.6               

11. 4               

11.0               

10.5

S.D.                  

1.41               

1.20               

1.22               

1.64               

1.23                

N 

27                                                 

27

25   

27                 

25    

PND 14 TO 21

MALES

MEAN                   

15.5               

16.0               

14.8               

14.6               

13.9

S.D.                  

2.61               

2.74               

3.17               

2.06               

2.48

N 

27                 

27                                                  

25

27  

25

FEMALES

MEAN                   

15.2               

15.2               

14.1               

14.0               

13.6               

S.D.                  

2.41               

2.66               

2.85               

2.28               

2.28                

N 

27                                                                           

27

25

27

25

PND = POSTNATAL DAY MODIFIED STATISTICS USED. 

* INDICATES PARAMETRIC ANALYSIS AND + INDICATES NON-PARAMETRIC ANALYSIS. 

Conclusions:
In a two-generation reproductive toxicity study on HMDS, conducted to GLP (reliability score 1) the NOAEC for parental toxicity relevant to humans was 400 ppm based on microscopic liver findings in the F0 males of the 5000 ppm group and F1 males and females in the 5000 and 1600 ppm groups. F0 and F1 reproductive performance was not affected at any concentrations. In the F2 generation, offspring body weight gains were persistently decreased in the 5000 ppm groups from PND 4-14, and this lead to decreased body weights at late as PND 49 in male offspring. The few “findings” at the 1600 ppm and lower exposure levels lacked consistency (along the time line), were of insufficient magnitude, and insufficiently dose-responsive. F1 adult females and their offspring exhibited no exposure-related effects during the FOB assessment. Based on the results of this study the NOAEC for HMDS for parental reproductive toxicity was considered to be at least 5000 ppm. The NOAEC for neonatal toxicity was considered to be 1600 ppm due to decreased offspring weights at 5000 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
33 200 mg/m³
Study duration:
subchronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key reproductive toxicity study (WIL Research Laboratories, 2006) Sprague-Dawley rats (30 animals/sex/dose) were exposed to HMDS at a concentration of 100, 400, 1600 or 5000 ppm, 6 hours/day, 7 days/week. Exposure started at least 70 days prior to mating, throughout mating, during gestation through to gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. F1 parents were selected from litters at 3 weeks of age and mated at between 14 and 16 weeks of age. Observations and examinations were conducted to OECD test guideline 416. The NOAEC for parental toxicity relevant to humans was 400 ppm based on microscopic liver findings (golden-brown pigment in the periportal areas) in the F0 males of the 5000 ppm group and F1 males and females in the 5000 and 1600 ppm groups. F0 and F1 reproductive performance was not affected at any concentration. Based on the results of this study the NOAEC for HMDS for parental reproductive toxicity is considered to be at least 5000 ppm (33200 mg/m³).

An additional one-generation reproductive toxicity study supports the finding that HMDS does not have an adverse effect on reproductive parameters, as there were no adverse effects in Sprague-Dawley rats up to a concentration of 5000 ppm.

Using the Rat Uterotrophic Assay (McKim et al 2001) as an indicator of estrogenic potential, HMDS administration at a dose level of 1200 mg/kg bw/day did not result in an estrogenic response in the Sprague Dawley rat. When HMDS was co-administered with EE there was a small but statistically significant reduction in uterine weight gain. The biological ramification of this could not be assessed in the study. The lack of an effect at a high dose level in this sensitive assay is significant.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study, conducted according to the appropriate OECD test guideline and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose tested of 3000 ppm (equivalent to 19923.93 mg/m³) (Charles River, 2018).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jan 2018 to ADD WHEN AVAILABLE
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
whole-body inhalation route of exposure used. Justification for route of exposure was given.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 10–11 weeks old
- Weight at study initiation: between 208 and 250 g on Gestation Day 0
- Fasting period before study: none
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during inhalation exposure periods.The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during inhalation exposure periods. Periodic analysis of the water was performed.
- Acclimation period: 5 days CONFIRM WHEN AVAILABLE

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Remarks:
The route of administration was whole body inhalation exposure because the intended use of the test substance indicates that this would be a likely route of human exposure.
Vehicle:
air
Remarks:
humidified, filtered
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted using 500-L glass and stainless steel whole body exposure chambers.
- Method of holding animals in test chamber: whole-body exposure
- Source and rate of air: Air supplied to the whole-body chambers was provided from an in-house compressed nitrogen source or a HEPA- and charcoal-filtered, temperature and humidity-controlled supply air source.
- Method of conditioning air: not applicable
- System of generating particulates/aerosols: not applicable. Vapors of hexamethyldisiloxane were generated using a stainless steel bead column-type vaporization system. The column for each test substance chamber was filled with various sized glass beads and heated using heat tape controlled using a temperature controller and J-type thermocouple. A pump equipped with a piston head was used to deliver liquid test substance from the reservoir to the top of the bead column. Nitrogen was metered to the bottom of the bead column at a 90° angle using a regulator and rotameter-type flowmeter. Vaporization occurred as the test substance flowed over the surface of the heated beads while the nitrogen flowed up through the column.
- Temperature, humidity, pressure in air chamber: Temperature and humidity wre 19°C to 25°C and 30% to 70%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups.
- Air flow rate: not applicable
- Air change rate: not applicable
- Method of particle size determination: not applicable
- Treatment of exhaust air: All chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: Nominal exposure concentrations were calculated for all test substance exposure chambers. The nominal concentration was calculated based on the total amount of test substance consumed during the exposure (as weighed prior to and at the termination of the generation) and the total volume of air passed through the chamber during exposure. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation. Exposure atmospheres were sampled and analyzed at approximately 45-minute intervals using a gas chromatograph (GC) with flame ionization detection (FID). Samples were collected from the approximate animal-breathing zone of each exposure chamber via heated stainless steel tubing. Sampling and analyses were performed as follows. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. The ln-quadratic equation based on the GC calibration curve was used to calculate the measured concentration in ppm.
- Samples taken from breathing zone: yes (see above)

VEHICLE (if applicable)
- Justification for use and choice of vehicle: humidified, filtered air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure atmospheres were sampled and analyzed at approximately 45-minute intervals using a gas chromatograph (GC) with flame ionization detection (FID). Samples were collected from the approximate animal-breathing zone of each exposure chamber via heated stainless steel tubing. Sampling and analyses were performed as follows. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. The ln-quadratic equation based on the GC calibration curve was used to calculate the measured concentration in ppm.
Details on mating procedure:
TO BE ADDED WHEN FINAL REPORT AVAILABLE
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily
Duration of test:
from Gestation Day 6 to 20, inclusively
Dose / conc.:
100 ppm
Remarks:
target concentration
Dose / conc.:
1 000 ppm
Remarks:
target concentration
Dose / conc.:
3 000 ppm
Remarks:
target concentration
Dose / conc.:
131 ppm (nominal)
Dose / conc.:
1 225 ppm (nominal)
Dose / conc.:
3 533 ppm (nominal)
Dose / conc.:
99 ppm (analytical)
Dose / conc.:
1 007 ppm (analytical)
Dose / conc.:
2 964 ppm (analytical)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a previously conducted two-generation reproduction study in which rats were exposed via whole-body vapor inhalation to target concentrations of 0, 100, 400, 1600, or 5000 ppm. The NOAEL for neonatal toxicity was considered to be 1600 ppm due to decreased offspring weights at 5000 ppm. Unlike the acute studies, there are no defined limit concentrations in 28-day subacute inhalation toxicity studies. The maximum concentration tested should consider: 1) the maximum attainable concentration, 2) the “worst case” human exposure level, 3) the need to maintain an adequate oxygen supply, and/or 4) animal welfare considerations. In the absence of data-based limits, the acute limits of the United Nations Globally Harmonized System of Classification and Labelling of Chemicals may be used (i.e., up to a maximum concentration of 5 mg/L for aerosols, 20 mg/L for vapors and 20,000 ppm for gases). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity. Therefore, a high exposure level of 3000 ppm was selected for this study.
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning on the day of receipt and lasting through Gestation Day 21

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Gestation Days 0 (by supplier), 5, and 6–21 (daily).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured on Gestation Days 5–21 (daily).

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

OTHER: ORGAN WEIGHTS
- Kidneys, liver and lungs were weighed at necropsy for all scheduled euthanasia animals.

TISSUE COLLECTION AND PRESERVATION
- Representative samples of the kidneys, liver, lungs and all gross lesions were collected from all animals and preserved in 10% neutral-buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [one-half of the fetuses in each litter ]
- Skeletal examinations: Yes: [one-half of the fetuses in each litter ]
- Head examinations: Yes: [all per litter ]
Statistics:
Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid animals were excluded from statistical analyses.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance treated group to the control group by sex.
Maternal body weights and body weight changes (absolute and net), and food consumption, gravid uterine weights, organ weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance treated groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and postimplantation loss, and fetal sex distribution) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
Not calculated
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance related clinical observations were noted at the daily examinations or 1–2 hours postexposure at any dosage level. Findings noted in the test substance exposed groups, including red material around the nose and hair loss on forelimb(s) and hindlimb(s), occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 100, 1000, and 3000 ppm groups survived to the scheduled necropsy on Gestation Day 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100, 1000, and 3000 ppm groups were unaffected by test substance exposure.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 1000, and 3000 ppm groups was unaffected by test substance exposure.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on organ weights in any exposure group. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 21, no test substance-related internal findings were observed at exposure concentrations of 100, 1000, and 3000 ppm. Macroscopic findings observed in the test substance exposed groups occurred infrequently and/or in a manner that was not dose related.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
>= 3 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: (equivalent to 19923.93 mg/m³ based on MW of 162.38 )
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
An external malformation, curly tail, was noted in one fetus in the 3000 ppm group. This malformation was noted in a single fetus, and therefore was not considered test substance related. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were observed in fetuses in this study.
The mean litter proportion of 14th rudimentary rib(s) in the 3000 ppm group (34.9% per litter) was statistically significantly higher than the concurrent control group (5.0% per litter) and the maximum mean value in the Charles River Ashland historical control data (14.57% per litter). This difference was considered test substance-related; however supernumerary ribs, such as 14th rudimentary ribs, have been found to resolve during maturation in rats.In the absence of other fetal effects, it is believed that a 14th rudimentary rib would not be considered a toxicologic concern or that these extra ribs would affect the quality of life of these animals, and therefore was considered to be nonadverse.
No other test substance-related skeletal developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral developmental malformations were observed in fetuses in this study. No test substance-related visceral developmental variations were noted.
The only finding noted, renal papilla(e) not developed and/or distended ureter(s) was noted similarly in the control group and the difference in the mean litter proportion was not were not statistically significant compared to the concurrent control group, and the value was within the range of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed was noted for 2, 1, and 3 fetuses in the control, 1000, and 3000 ppm groups, respectively. Dark red contents in the thoracic cavity, surrounding the lungs was noted for one fetus in the control group. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test article-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Other effects:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
>= 3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: (equivalent to 19923.93 mg/m³ based on MW of 162.38 )
Abnormalities:
no effects observed
Developmental effects observed:
no

ADD TABLES WHEN AVAILABLE

Conclusions:
In the prenatal developmental toxicity study, conducted according to the appropriate OECD test guideline and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose tested of 3000 ppm (equivalent to 19923.93 mg/m³ based on MW of 162.38 ).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
19 923.93 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key prenatal developmental toxicity study, conducted according to the appropriate OECD test guideline and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose tested of 3000 ppm (equivalent to 19923.93 mg/m³) (Charles River, 2018).

Animals were exposed via 6-hour whole-body inhalation once daily during Gestation Days 6–20.

The following parameters and end points were evaluated in this study:  clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, intrauterine growth and survival, gross necropsy findings, organ weights, and fetal examinations.

All animals survived to the scheduled necropsy on Gestation Day 21.  There were no test substance related clinical observations at the daily examinations or 1–2 hours post exposure at any exposure level.

Mean maternal body weight gains, body weights, food consumption, net body weights, net body weight gains, and gravid uterine weights at 100, 1000, and 3000 ppm were unaffected by test substance exposure.

There were no test substance-related macroscopic findings at the scheduled necropsy at any exposure level.

Intrauterine growth and survival were unaffected by the test substance at all exposure levels.  A test substance-related higher mean litter proportion of the skeletal variation 14th rudimentary rib(s) was noted in the 3000 ppm group compared to the control group.  Supernumerary ribs, such as 14th rudimentary ribs, have been found to resolve during maturation in rats. In the absence of other fetal effects, it is believed that a 14th rudimentary rib would not be considered a toxicologic concern or that these extra ribs would affect the quality of life of these animals, and therefore were considered nonadverse.  No other test substance-related effects on fetal morphology were noted at any exposure level.

In a reproductive toxicity study (WIL Research Laboratories, 2006) Sprague-Dawley rats (30 animals/sex/dose) were exposed to HMDS at a concentration of 100, 400, 1600 or 5000 ppm, 6 hours/day, 7 days/week. Exposure started at least 70 days prior to mating, throughout mating, during gestation through to gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. F1 parents were selected from litters at 3 weeks of age and mated at between 14 and 16 weeks of age. Observations and examinations were conducted to OECD test guideline 416.

There were no statistically significant offspring weights at any dose level. Statistically significant (p<0.05 or p<0.01) lower mean pup body weight gains were noted for males in the 400, 1600 and 5000 ppm groups and for females in the 400 and 5000 ppm groups from PND 4 to PND 7. Male and female pup weight gain in the 5000 ppm group continued to be slightly lower (not statistically significant) than the control group during PND 7 to 14 and 14 to 21. As a result of the lower weight gain, mean pup body weights in the 5000 ppm group were lower (7.2% to 7.3%) than control group values on PND 21; the difference from the control group for the females was statistically significant (p<0.05). Because the lower weight gain in the 400 and 1600 ppm groups did not persist, the lower mean body weight gains in these groups during PND 4 to 7 were not considered test article-related. No other differences in pup body weight or body weight gain relative to the control group were noted in the 400, 1600 or 5000 ppm groups. Mean male and female pup body weights and body weight changes in the 100 ppm group were unaffected by test article exposure throughout the postnatal period. The NOAEC for F1 and F1 developmental toxicity was 1600 ppm due to decreased offspring weights at 5000 ppm. There were no gross anomalies in the pups. The NOAEC for developmental neurobehavioral endpoints was 1600 ppm due to the lack of habituation exhibited in the locomotor activity assessments and delayed attainment of the surface righting response in the 5000 ppm group F2 females, and decreases in average and peak acoustic startle response on PND 20 in the 5000 ppm group F2 males and females. The NOAEC for neuropathologic endpoints was 5000 ppm.


Justification for classification or non-classification

The available studies do not suggest that hexamethyldisiloxane should be classified for adverse effects on fertility or offspring development according to Regulation (EC) No 1272/2008.