Hexamethyldisiloxane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a two-generation reproductive toxicity study on hexamethyldisiloxane (HMDS; CAS 107 -46 -0), 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

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 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.  Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals.  In the 5000 ppm group F1 males and females, 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.

Remarks on result:

other: (equivalent to 2657 mg/m³ based on MW of 162.38)

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.

Remarks on result:

other: (equivalent to 33207 mg/m³ based on MW of 162.38)

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.

Remarks on result:

other: (equivalent to 2657 mg/m³ based on MW of 162.38)

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.

Remarks on result:

other: (equivalent to 10626 mg/m³ based on MW of 162.38)

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 (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 in rats conducted according to the OECD Test Guideline 414 and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose of hexamethyldisiloxane (HMDS; CAS 107 -46 -0) tested of 3000 ppm (equivalent to 19924 mg/m³) (Charles River Laboratories, 2018).

In a prenatal developmental toxicity study in rabbits conducted according to the OECD Test Guideline 414 and in compliance with GLP, the NOAEC for maternal toxicity and developmental toxicity was concluded to be at least 3300 ppm (equivalent to 21916 mg/m³ based on a molecular weight of 162.38 g/mol) based on no observed adverse effects when time-mated New Zealand White rabbits were exposed to hexamethyldisiloxane (HMDS) via whole-body exposure for 6 hours/day during Gestation Days 7–28 (Charles River Laboratories, 2021).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jan 2018 to 14 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system 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

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 19924 mg/m³ based on MW of 162.38 )

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

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 19924 mg/m³ based on MW of 162.38 )

Abnormalities:

no effects observed

Developmental effects observed:

no

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 ).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 October 2020 to 22 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Saint Constant, QC, Canada
- Age at study initiation: 5.5 to 7 months old
- Weight at study initiation: 3108-5383g
- Fasting period before study: no
- Housing: Randomly housed individually in stainless steel cages with perforated flooring elevated above ground corncob bedding. All rabbits received approximately 10 g/day of Certified Timothy hay from arrival through euthanasia. Hay was provided separate from the daily feed allotment and was not weighed at the time of food consumption data collection.
- Diet (e.g. ad libitum): Pellets from PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322; gradually incremented during the period prior to exposure and 180 ± 5 g/day during the study (except during the daily 6-hour exposure period).
- Water (e.g. ad libitum): Municipal tap water, by reverse osmosis and ultraviolet irradiation, ad libitum via an automatic watering system (except during the daily 6-hour exposure period).
- Acclimation period: the study report states that the animals were acclimated, but the exact number of days is not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16°C to 22°C
- Humidity (%): 30% to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES:
From: 07 or 08 October 2020 To: 29 or 20 October 2020 (Phase 1)
From: 02 or 03 November 2020 To: 24 or 25 November 2020 (Phase 2)

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: Four, approximately 1500L stainless steel and glass whole-body exposure chambers. One chamber was dedicated to each group for the duration of the study and for the inhalation exposures of Phase 1 and Phase 2.
- Method of holding animals in test chamber: Animals selected for exposure were transferred to exposure caging in the colony room and transported to Exposure Room 2. Animals were exposed for the requisite duration and then returned to their home cages in the animal colony room. Animals were individually housed in standard exposure batteries of appropriate size for the whole-body exposure chamber in use. Batteries were rotated on a daily basis within the exposure chamber.
- Source and rate of air: Air supplied to the chambers was provided from the Inhalation Department breathing quality, in-house compressed air source and and/or a HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source and in-house nitrogen source.
- Method of conditioning air: For exposure of the test substance-treated groups, a vapor exposure was generated from a bead column. For exposure of the filtered air control group, supply air and compressed nitrogen were mixed to achieve a similar nitrogen and total airflow rate to that in the high test substance-exposed groups. Presence for aerosol was evaluated during exposures in all chambers using a Casella CEL-712 Microdust Pro (Casella CEL; Bedford, UK).
- System of generating particulates/aerosols: Not relevant.
- Temperature, humidity, pressure in air chamber: Temperature (Mean 21.0-21.6 °C in Phase 1 and Mean 20.8-21.5 °C in Phase 2) and humidity (Mean 49.8-50.6% in Phase 1 and Mean 49.0-51.0% in Phase 2) were monitored continuously using a display and a Vaisala probe; Negative pressure within the chamber was monitored continuously using a Dwyer Magnehelic Indicating Transmitter pressure gauge.
- Air flow rate: Mean 303-307 in Phase 1; Mean 301-306 in Phase 2; Air flow (ventilation rate) was monitored using a sharp edge orifice meter and Dwyer Magnehelic® Indicating Transmitter pressure gauge.
- Air change rate: 10 air changes per hour (250 LPM)
- Method of particle size determination: Not relevant.
- Treatment of exhaust air: All chamber exhausts were directed to 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: gas chromatograph (GC). 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.
- Samples taken from breathing zone: yes, a minimum of 6 times during each animal exposure

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure atmosphere are sampled and analysed using gas chromatography, a miminum of 6 times during each animal exposure, then characterised by nominal concentrations. Samples were collected from the approximate animal-breathing zone of the exposure system via 1/8-inch stainless steel tubing. 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.
Nominal exposure concentrations were calculated based on the total amount of test substance consumed during each 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 chamber ventilation rate by the duration of generation.
The following equation was used: Concentration (ppm) = WT x MV x 10⁶/ GMW x CVR x DUR
Where:
WT = Weight of test substance consumed, grams
MV = Molar volume at 730 mmHg and 21ºC, 25.11 L/mol
106 = ppm conversion factor
GMW = Gram molecular weight of test substance, 162.38 g/mol
CVR = Daily mean chamber ventilation rate, LPM
DUR = Duration of generation, minutes

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant; time-mated female rabbits [Gestation Day 1 or 2 (Phase 1) and Gestation Day 3 or 4 (Phase 2)]

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

Daily

Duration of test:

Gestation Days 7-28; for a total of 22 exposures

Dose / conc.:

0 ppm (nominal)

Remarks:

Control Group - Group 1 (Filtered air)

Dose / conc.:

300 ppm (nominal)

Remarks:

Group 2

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Group 3

Dose / conc.:

3 300 ppm (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

24 animals per group (12 animals in each group were assigned to Phases 1 and 2).

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The exposure levels for the current study were selected by the Sponsor Representative based on the results of a previous dose range-finding prenatal developmental toxicity study where 5 time-mated animals per groups were exposed to the test substance via whole-body exposure once daily during Gestation Days 7–28 at concentrations of 0, 1000, 2000, or 3300 ppm. No effects on survival and no noteworthy clinical observations or macroscopic findings were noted at any exposure level. Mean body weights, body weight gains, and food consumption for all groups were generally comparable to the control group; any noteworthy changes did not occur in an exposure-related manner and/or were a continuation of effects noted prior to the initiation of exposure. Higher (13.3% and 17.2%; not statistically significant) mean absolute liver and kidney weights were noted in the 3300 ppm group compared to the control group. No effects on intrauterine survival were noted at any exposure levels.
In the absence of data-based limits for rabbits, the acute limits of the United Nations Globally Harmonized System of Classification and Labelling of Chemicals should be used (i.e., up to a maximum concentration of 20 mg/L for vapors). Based on nominal laboratory temperature and pressure in Ashland, OH, 20 mg hexamethyldisiloxane/L corresponds to approximately 3093 ppm. Therefore, the target concentration for the highest test substance-treated group was 3300 ppm. The target concentrations for the low- and mid-exposure levels (300 and 1000 ppm, respectively) were selected to reveal any exposure-related trends.
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, on all animals (within their cage)
- Time schedule: At least twice daily (morning and afternoon), beginning upon arrival through termination.

DETAILED CLINICAL OBSERVATIONS: Yes, on all animals (removed from the cage)
- Time schedule: Once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia.

BODY WEIGHT: Yes, on all animals
- Time schedule for examinations: Gestation Days 0 (by supplier), 5, and 7–29 (daily)

FOOD CONSUMPTION: Yes, on all animals
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: Gestation Days 5–29 (daily)

WATER CONSUMPTION: Not measured

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Cranial, thoracic, abnominal and pelvic cavities were opened and the contents examined. The uterus of each dam was excised, and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights (including the cervix) were obtained and recorded. The uterus of each dam was opened, and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss. The liver and kidneys were collected, weighed, and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Gross lesions were also collected and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison.

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

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: No data

Statistics:

Means, standard deviations, percentages, numbers, and/or incidences were reported, as appropriate by dataset. Calculated values on the Provantis tables may not be reproducible from the individual values presented because all calculations are conducted using non-rounded values.
Data obtained from nongravid animals were excluded from statistical analysis. Inferential statistical analysis (Parametric/Non-Parametric or Non-Parametric depending on the variables) were used.

Indices:

Pre-implantation loss, post-implantation loss, sex ratio (% males), litter % of foetuses with abnormalities, were included where applicable.

Historical control data:

Charles River has historical data on the background incidence of foetal malformations and developmental variations in this species from the same strain and source.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted at the daily examinations or within 2 hours postexposure at any concentration. Clinical observations noted in the test substance-exposed groups were noted infrequently, similarly in the control group, and/or in a manner that was not concentration-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals in the control, 300, 1000, and 3300 ppm groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 and 3300 ppm groups, lower mean body weight gains or mean body weight losses were generally noted throughout the exposure period (Gestation Days 7–10, 10–13, 13–20, and/or 20–29), and consequently when the overall exposure period (Gestation Days 7–29) was evaluated compared to the control group; the differences were not statistically significant. A statistically significant lower mean body weight gain was noted at 1000 ppm on Gestation Day 14–15. The effects on mean body weight change in the 1000 and 3300 ppm groups were considered test substance-related, but not adverse, because the differences were not noted in a clear concentration-related manner and the absence of a noteworthy impact on mean absolute body weights and the overall health and well-being of the animals.
Mean gravid uterine weights, adjusted body weights, and adjusted body weight gains in the 1000 and 3300 ppm groups were unaffected by test substance exposure. Mean maternal body weights, body weight gains, gravid uterine weight, adjusted body weights and adjusted body weight gain in the 300 ppm group were unaffected by test substance exposure. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 300, 1000, and 3300 ppm groups, lower mean maternal food consumption, evaluated as g/animal/day, was generally noted throughout the exposure period compared to the control group; differences were statistically significant during Gestation Days 10–13 and 13–20 at all concentrations, as well as when the entire exposure period (Gestation Days 7–29) was evaluated at 1000 ppm. The lower mean food consumption corresponded with the lower mean body weight gains/body weight losses at 1000 and 3300 ppm and was considered test substance-related, but nonadverse, at all concentrations based on the lack of effects on mean absolute body weights and/or body weight gains and because differences were not noted in a clear concentration-related manner.

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

Endocrine 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):

Mean absolute liver and kidney weights in the 300, 1000, and 3300 ppm groups were unaffected by test substance exposure. Differences from the control group were slight, not statistically significant, and/or not noted in a concentration-related manner.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were observed at concentrations of 300, 1000, and 3300 ppm. Macroscopic findings observed in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not concentration-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance exposure at concentrations of 300, 1000, and 3300 ppm.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean litter proportions of pre-implantation loss was 17.42%, 12, 26%, 12.13% and 19.04% in Group 1, 2, 3, and 4 respectively
Mean litter proportions of post-implantation loss was 12.76%, 8.67%, 5.80%, 8.44% in Group 1, 2, 3 and 4, respectively

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

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

With the exception of 2 animals each in the control, 1000, and 3300 ppm groups, all animals were gravid.

Other effects:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 3 300 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse general and developmental effects in maternal animals

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

equivalent to 21916 mg/m3 based on MW of 162.38 g/mol

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 and 3300 ppm groups, slightly lower (5.1% to 10.8%) mean foetal weights (male, female, and combined sexes) were noted compared to the control group. However, the differences were not statistically significant, not noted in a clear exposure-related manner, and
the values were generally within the ranges of the Charles River Ashland historical control data, and, therefore, were not considered test substance-related.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of foetuses (litters) available for morphological evaluation were 196(21), 243(24), 233(22), and 217(22) in the control, 300, 1000, and 3300 ppm groups, respectively. Malformations were observed in 3(3), 5(5), 7(7), and 3(3) foetuses (litters) in these same
respective dose groups and were considered spontaneous in origin. No test substance-related external malformations were noted in foetuses in this study.
Malformations were noted for 2(2) and 1(1) foetuses (litters) in the 300 and 1000 ppm groups, respectively. In the 1000 ppm group, Foetus No. 3511-08 was noted with hyperflexion of the right forepaw. In the 300 ppm group, Foetus No. 2506-11 was noted with an omphalocele and Foetus No. 2511-12 was noted with forepaw ectrodactyly and forepaw syndactyly. These malformations were not considered test substance-related because they were noted for single foetuses in a non-concentration-related manner. No other external malformations were noted for foetuses in this study. No external developmental variations were observed in foetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related skeletal malformations were noted for foetuses in this study. Skeletal malformations were noted for 2(2), 3(3), and 1(1) foetuses (litters) in the 300, 1000, and 3300 ppm groups, respectively. At 3300 ppm, Foetus No. 4520-06 was noted with a lumbar hemivertebra. At 1000 ppm, Foetus Nos. 3509-10 and 3520-09 were noted with supernumerary lumbar vertebra and Foetus No. 3512-04 was noted with branched, fused, and supernumerary ribs and supernumerary thoracic arch. At 300 ppm, Foetus No. 2511-12 was noted with absent and/or fused forepaw phalanges and absent metacarpal (corresponding with the external malformations of ectrodactyly and syndactyly noted for this fetus), and Foetus No. 2518-10 was noted with branched and supernumerary ribs and supernumerary thoracic arch. These malformations were not considered test substance-related because they were noted infrequently, were not noted in a clear exposure-related manner, and/or the differences in the mean litter proportions were not statistically significant compared to the concurrent control group.
No test substance-related skeletal developmental variations were noted. In the 3300 ppm groups, a statistically significantly higher mean litter proportion of unossified talus was noted compared to the concurrent control group; however, this developmental variation was not considered test substance-related because it was noted for 3 low-weight foetuses in 3 litters (Nos. 4506-06 [12.6 g], 4508-08 [14.6 g], and 4612-08 [14.6 g]). Other skeletal developmental variations were noted infrequently, similarly in the control group, were not noted in a concentration-related manner, 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:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related visceral malformations were noted for foetuses in this study. Visceral malformations were noted for 3(3), 2(2), 3(3), and 2(2) foetuses (litters) in the control, 300, 1000, and 3300 ppm groups, respectively. In the 3300 ppm group, Foetus No. 4509-06 was noted with persistent truncus arteriosus and a membranous ventricular septal defect of the heart, and Foetus No. 4607-06 was noted with a muscular ventricular septal defect of the heart and a malpositioned right kidney. In the 1000 ppm group, Foetus No. 3501-05 was noted with persistent truncus arteriosus and a membranous ventricular septal defect of the heart, Foetus No. 3506-07 was noted with a retroesophageal right subclavian artery, and Foetus No. 3519-02 was noted with persistent truncus arteriosus and a muscular ventricular septal defect of the heart. In the 300 ppm group, Foetus No. 2515-02 was noted with a large heart and small lung, and Foetus No. 2523-03 was noted with a retroesophageal right subclavian artery. These malformations were not considered test substance-related because they were noted infrequently, similarly in the control group, were not noted in a concentration-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. In the control group, 2 foetuses (Nos. 1510-05 and 1524-02) were noted with persistent truncus arteriosus and a membranous ventricular septal defect of the heart, and Foetus No. 1523-01 was noted with a muscular ventricular septal defect of the heart. No other visceral malformations were noted for foetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in a concentration-related manner, 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.

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

>= 3 300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects in foetuses

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

equivalent to 21916 mg/m3 based on MW of 162.38 g/mol

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a prenatal developmental toxicity study in rabbits conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEC for maternal toxicity and developmental toxicity was concluded to be at least 3300 ppm (equivalent to 21916.32 mg/m3 based on a molecular weight of 162.38 g/mol) based on no observed adverse effects when time-mated New Zealand White rabbits were exposed to hexamethyldisiloxane (L2) via whole-body exposure for 6 hours/day during Gestation Days 7–28.

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

19 924 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 in rats conducted according to the OECD Test Guideline 414 and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose tested of 3000 ppm (equivalent to 19924 mg/m³) HMDS. 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 these findings were considered nonadverse.  No other test substance-related effects on fetal morphology were noted at any exposure level (Charles River Laboratories, 2018).

In the key prenatal developmental toxicity study in rabbits conducted according to the OECD Test Guideline 414 and in compliance with GLP, time-mated female New Zealand White rabbits were exposed daily via whole-body exposure for 6 hours/day during Gestation Days 7–28, for a total of 22 exposures to HMDS. This study was conducted in 2 phases due to limitations on the total number of animals that could be placed in the stainless steel whole-body exposure chambers at 1 time (maximum of 12). The following parameters and endpoints were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, organ weights, macroscopic examinations, intrauterine growth and survival, and foetal morphology. Target exposure concentrations were 300, 1000, and 3300 ppm.

The target dose levels were selected based on the results of the dose range finder wherein no remarkable effects were noted on intrauterine growth, survival, macroscopic findings, mean maternal body weights, body weight gains, gravid uterine weights and food consumption at any exposure concentration in the five maternal rabbits exposed to the test substance via whole-body exposure for 6 hours/day during Gestation Days 7–28, and where no external malformations or developmental variations in foetuses were noted (Charles River Laboratories, 2020).

The exposure levels for the dose range finder study were selected based on the results of a previous repeat-exposure toxicity study where 3 non-pregnant rabbits per group were exposed to the test substance via whole-body exposure once daily (6 hours per day) for 14 consecutive days at exposure concentrations of 0 and 3300 ppm (Groups 1 and 2, respectively) or for 7 consecutive days at exposure concentrations of 1000 and 2000 ppm (Groups 3 and 4, respectively) following review of the in-life study data after 5 days of exposure. No effects on survival and  noteworthy clinical observations were noted at any exposure level throughout the respective exposure periods.

In the main prenatal developmental toxicity study, the overall mean analyzed exposure concentrations were 301, 1007, and 3302 ppm HMDS, respectively, during Phase 1 and 299, 1002, and 3285 ppm HMDS, respectively, during Phase 2. All animals survived to the scheduled necropsy on Gestation Day 29. There were no test substance-related clinical observations at the daily observations or within 2 hours following exposure at any concentration. In the 1000 and 3300 ppm groups, lower mean body weight gains, with correspondingly lower mean food consumption, were noted when the entire exposure period (Gestation Days 7–29) was evaluated compared to the concurrent control group. Lower mean food consumption compared to the control group (without any correlating effects on mean body weight gains) was also noted in the 300 ppm group generally throughout the exposure period. The effects on body weights and/or food consumption in the 300, 1000, and 3300 ppm groups were considered test substance-related, but not adverse, because differences were not noted in a clear concentration-related manner and based on the small or no effect on mean absolute body weights. Mean adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 300, 1000, and 3300 ppm groups were unaffected by test substance exposure. At the scheduled necropsy, no test substance-related macroscopic findings were noted at any exposure concentration. Mean absolute liver and kidney weights in the 300, 1000, and 3300 ppm groups were unaffected by test substance exposure. Intrauterine growth and survival and fetal morphology were unaffected by test substance exposure at all concentrations.

In conclusion, based on the lack of adverse findings at any concentration tested, an exposure concentration of 3300 ppm (the highest exposure concentration tested; equivalent to 21916 mg/m³ based on a molecular weight of 162.38 g/mol) was considered to be the no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and developmental toxicity when time-mated New Zealand White rabbits were exposed to hexamethyldisiloxane (HMDS) via whole-body exposure for 6 hours/day during Gestation Days 7–28 (Charles River Laboratories, 2021).

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.