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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key inhalation two-generation reproductive toxicity study in Sprague-Dawley rats (WIL Research Laboratories, 2001a), the NOAEC for reproductive toxicity was 300 ppm, based on reduced fertility indices and reduced mean live litter sizes, and the NOAEC for general systemic toxicity was 300 ppm, based on reduced body weight gain in adult animals.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20.12.1996 to 09.03.2009 (experimental termination, as study completion date not given)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
This study evaluated the potential toxicity of whole-body vapor inhalation of octamethylcyclotetrasiloxane (D4) on reproductive capabilities in exposed F0 andF1 parental animals and the potential effects on neonatal survival, growth, and development of the F1 and F2 offspring.

Amendment to the Final Report: 2009-10000-60443.
The following changes were made:
1) A summarization of the results of the additional microscopic examinations specified in protocol amendment XIII were added to the abstract and conclusions of the report.
2) The experimental termination date was updated due to additional microscopic examinations. In addition, the experimental completion date was not presented in the original final report.
3) The results of the additional microscopic examinations specified in protocol amendment XIII were added. In addition, the morphology of the pigment was further discussed as part of the additional examinations, and it was not determined if the microscopic findings in the liver were adverse or non-adverse.
4) The additional reference was cited in the microscopic section regarding the results of the histopathologic evaluation of the liver slides.
5) Following re-examination, it was determined that no pigment was present in the liver for female no. 61179-08.
6) An additional protocol amendment was prepared following the issuance of the final report for the purpose of performing additional histopathologic evaluation of the liver slides for all animals noted with brown pigment.
GLP compliance:
yes
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, North Carolina
- Age at study initiation: (P) 16 wks; (F1, first pairing) 14-15 wks: (F1, second pairing) 26-27 wks.
- Weight at study initiation: (P) Males: 372-605 g; Females: 215-346 g; (F1, first pairing) Males: 293-619 g; Females: 199-343 g; (F1, second pairing) Males: 379-701 g; Females: 220-460 g.
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages (after mating in plastic maternity cages with nesting material)
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ±2.2
- Humidity (%): 40-70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14.01.1997 To: 25.04.2000
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each group of animals was exposed in a 2m3 stainless steel and glass whole-body inhalation chamber.
- Source and rate of air: No data
- Method of conditioning air: No data
- Temperature, humidity, pressure in air chamber: 20-26 oC, 30-60% humidity
- Air change rate: 12-15
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: no data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: No data
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material.

F0 animals were mated once to produce the F1 generation; F1 parental animals were mated twice to produce two F2 litters. In addition, the F1 males were mated with unexposed females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations within each chamber were measured approximately every 35 minutes, yielding approximately 10 data points, during each daily exposure period by a validated gas chromatographic method.
Duration of treatment / exposure:
Exposure period: F0 and F1 males and females were exposed at least 70 days prior to mating, throughout mating, gestation (to gestation day 20), lactation, with the exception of lactation days 0-4, until euthanization.
Starting on PND 22, F1 weanlings were exposed to D4 as described for the  F0 generation.
The F2 pups were not directly exposed to D4.
Duration of test: ca. 39 months.
Frequency of treatment:
6 hr/day, 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 4 days of age.
- Age at mating of the mated animals in the study: F1 first mating: 14-15 wks; F1 second mating: 26-27 wks; F1 treated males with untreated females: Males: 28-29 wks and females 12 wks.
- 30/pups/sex were selected on PND 28 to comprise the F1 generation.
Dose / conc.:
70 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
Dose / conc.:
700 ppm (nominal)
Dose / conc.:
71 ppm (analytical)
Remarks:
F0 generation
Dose / conc.:
298 ppm (analytical)
Remarks:
F0 generation
Dose / conc.:
502 ppm (analytical)
Remarks:
F0 generation
Dose / conc.:
700 ppm (analytical)
Remarks:
F0 generation
Dose / conc.:
71 ppm (analytical)
Remarks:
F1 generation
Dose / conc.:
301 ppm (analytical)
Remarks:
F1 generation
Dose / conc.:
502 ppm (analytical)
Remarks:
F1 generation
Dose / conc.:
702 ppm (analytical)
Remarks:
F1 generation
No. of animals per sex per dose:
30/sex/group
Control animals:
other: yes, exposed to filtered air
Details on study design:
- Dose selection rationale: Based on previous reproductive toxicity studies.

Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for appearance, behaviour, moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: F0 and F1 males: weekly beginning with initiation of exposure and continuing until sacrifice. F0 and F1 females: weekly beginning at initiation of exposure and continuing until evidence of copulation. Then on gestation days 0, 7, 10, 14 and 20, and on lactation days 1, 4, 7, 14 and 21. After weaning (lactation day 21), weekly body weights were recorded for F0 females until scheduled necropsy. For the F1 females body weights were recorded beginning after weaning of the F2a litters on lactation day 21 and continued until evidence of copulation was observed after the second pairing. Once evidence of mating was observed, F1 body weights were recorded on gestation days 0, 7, 10, 14 and 20, and on lactation days 1 and 4.

FOOD CONSUMPTION: Yes, on a weekly basis, but during the mating period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

FUNCTIONAL OBSERVATION BATTERY: Yes, of all F1 female rats on gestation day 10 and lactation day 20.

reproductive parameters (days between pairing and coitus, fertility, fecundity, length of gestation, pup growth and survival, testicular and epididymal sperm count, 
sperm production rate, sperm motility and morphology, ovarian primordial follicle count), developmental landmarks (e.g., balanopreputial separation and vaginal patency), developmental neuropathology, selected organ weights, gross pathology, histopathology.

NEUROPATHY: yes
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of the oestrus cycle for each F0 and selected F1 females, beginning 21 days prior to pairing and continuing until evidence of copulation was observed. Following weaning of the F2a litters and a rest period of at least 16 days, daily vaginal smears were performed for each F1 female for 15 days prior to the second pairing with the F1 males.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations: epididymal weights, testicular and epididymal sperm count, sperm production rate, sperm motility and morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: pup appearance and behaviour, pup growth and survival, developmental landmarks (anogenital distance on PND 1 for all F1 and F2a pups, balanopreputial separation in F1 and F2a males, vaginal patency in F1 and F2a females), sex ratio, developmental neuropathology and neurobehavioral evaluations.


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
All surviving animals. F0 adults: following the selection of the F1 generation and completion of detailed clinical obs; F1 males: following completion of breeding with unexposed females; F1 females: on lactation day 4, on post-mating day 25, or 25 days following completion of the second breeding period.

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

HISTOPATHOLOGY / ORGAN WEIGHTS (control and high concentration groups, as well as animals from F0 and F1 parental animals that died spontaneously or in extremis: Organs examined at necropsy (macroscopic and microscopic): Complete gross necropsy was conducted. Organ weights:  Adrenals, prostate, brain, seminal vesicles with epididymides (total and cauda), coagulating glands, heart, kidneys, spleen, liver, testes, lungs, thymus, ovaries, thyroid, pituitary, and uterus.
Histopathologic evaluation: complete histopathologic evaluation of tissues and organs including  adrenal glands, brain, cervix, coagulating gland, epididymis (right caput, corpus, and cauda), kidneys, liver, lungs, ovaries, penis, pituitary gland, prepuce, preputial gland, prostate, seminal vesicles, testis (right), thyroid, uterus, vagina, and vas deferens, all gross (internal) lesions.
Postmortem examinations (offspring):
SACRIFICE
Prior to the weaning of the F1 pups, 30 pups/sex/group were randomly selected for the F1 parental generation and for evaluation of developmental landmarks. All surviving non-selected F1 weanlings were sacrificed on PND 21 or 28 and necropsied with an emphasis on developmental morphology evaluations.
Similarly, prior to weaning of the F2a pups, 30 pups/sex/group were randomly selected for neurobehavioural testing, developmental landmarks, and/or neuropathology and brain weight/dimension measurements. In addition, 10 F2 pups/sex/group were selected for brain weight measurements and/or neuropathology on PND 11. All remaining F2a weanlings were sacrificed and necropsied on PND 21, with an emphasis placed on developmental morphology evaluations. F2a pups not allocated for neuropathology and brain examinations received a complete necropsy on PND 70.
F2b and F2c litters were sacrificed and discarded on PND 4.


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


HISTOPATHOLOGY / ORGAN WEIGHTS: The brains, spleens and thymus glands from all unselected F1 and F2a weanlings necropsied on PND 21 or 28 were weighed. Additional microscopic examinations (Birefringence of pigment under polarized light) were performed for F1.
Statistics:
Statistical methods:
Chi-Square test with Yates' correction factor for parental mating and fertility indices
One-way ANOVA with Dunnett's test for the following endpoints:  Pre-coital intervals, Parental weekly,
gestational and lactational body weight and food consumption data, Gestation lengths, Sperm numbers, 
Sperm production rates, Organ weights*, Ovarian primordial follicle counts, Numbers of pups born**, 
Live litter sizes**, Anogenital distances**, Pup body weights**, Continuous FOB data, Startle response data, 
Balanopreputial separation, Vaginal patency
Fisher's Exact Test - Scalar/descriptive FOB data Two-way repeated measures ANOVA - Motor activity data
Kruskal-Wallis test with Mann-Whitney U test - Sperm motility, Sperm morphology, Pup sexes at birth
(% males per litter)**, Postnatal survival**
Kolmogorov-Smimov test (one-tailed test) - Histopathological findings * for PND 21 organ weights, 
the litter was the experimental unit of evaluation **litter used as experimental unit
Reproductive indices:
Mating and fertility indices: male and female mating index; male and female fertility index;
Offspring viability indices:
Live litter size, postnatal survival between birth and PND 0 or PND 4, postnatal survival for all other intervals,
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
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:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): F0: In the control, 500 and 700 ppm groups, 1/30, 1/30 and 1/30 males and 0/30, 0/30 and 4/30 females, respectively, were found dead between weeks 1 and 16. An additional 1/30 males in the 700 ppm group was authanised in extremis on day 17. Two females of the 700 ppm group died of dystocia on days 96 and 94. The other two females in the 700 ppm group died on gestation day 22 and study day 117. Liver necrosis played a part in one death, and the other was due to kidney failure. Ejaculatory plugs were noted in an exposure-related manner in most of the males throughout the exposure. The significance of this finding is unknown. No other significant clinical signs were noted at any D4 concentration. 
F1: 1/30 and 1/30 females died in 500 and 700 ppm groups between weeks 19 and 43. There were no clinical signs in animals that survived to scheduled necropsy.

BODY WEIGHT: during the first week of exposure, statistically significant reductions in mean body weight gain were observed in males and females in the 700 ppm group and in females in the 500 ppm group in the F0 animals only. Mean body weight gain was reduced (statistically significant) during gestation in the 700 ppm group in both the F0 and F1 parental animals.

FOOD CONSUMPTION: Weekly food consumption was reduced during week 0-1 in the 700 ppm group males, consistent with the reduced body weight gain in this group. Food efficiency was reduced in the 700 ppm group during gestation days 14-20 and the overall gestation period. This finding was consistent with the reduced body weight gain observed in this group at the end of the gestation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Microscopic evaluation of the ovaries, uterus, vagina, mammary gland and pituitary gland from the 0, 70, 300, 500, and 700 ppm F1 females suggested a subtle non-exposure responsive effect, characterized by perturbation of the estrous cycle and accelerated reproductive senescence in the F1 (but not F0) females at 70, 300, and 500 ppm, with a more obvious effect at 700 ppm.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No adverse effects were observed on male functional reproductive parameters, on male spermatogenic endpoints, on microscopic evaluation of male reproductive tissue, or when the D4-exposed F1 males were mated with the unexposed, nulliparous females, demonstrating that the reproductive toxicity observed was due to D4 exposure of the female.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Extended parturition and/or dystocia were observed in two and three F0 females in the 500 and 700 ppm groups, respectively, and in one F1 dam each in the 300, 500, and 700 ppm groups. Two of the three F0 700 ppm group dams and the one F1 500 ppm group female died as a result of the dystocia. Fertility indices (OECD and EPA methods of calculation) were statistically significantly reduced in the 700 ppm group for the first F1 mating period. In the second F1 mating period, male and female fertility indices were statistically significantly reduced in the 500 and 700 groups (EPA method). The male and female fertility indices for the second F1 mating were also reduced in a non-exposure responsive manner in the 70 and 300 ppm groups using both methods of calculation, although the differences from the control group were not statistically significant.

ORGAN WEIGHTS (PARENTAL ANIMALS): Statistically significant increases in mean kidney weights (absolute and relative to final body weight and relative to brain weight) were observed in males in the 500 and 700 ppm groups. A statistically significant increase (relative to body weight) was also observed in the 300 ppm males. Increased mean liver weights (statistically significant; absolute and relative to final body weight and relative to brain weight) were observed in females in the 300, 500 and 700 ppm groups and in males of the 700 ppm group.

GROSS PATHOLOGY (PARENTAL ANIMALS): No adverse findings at scheduled sacrifice.

HISTOPATHOLOGY (PARENTAL ANIMALS): For scheduled deaths: The incidence of tubular mineral mineralisation in the kidneys of the 500 and 700 ppm group males (6/29 and 12/28 males) was increase relative to controls (0/29 males). The severity of the effect was minimal in the 500 ppm and moderate in the 700 ppm group. This mineralisation was considered to be exposure-related, but was not adverse. The incidence of centrilobular hepatocyte hypertrophy was increased in females in the 500 and 700 ppm groups (8/30 and 6/26) relative to controls (1/30). This finding was considered to be exposure-related, but not adverse.
Increased alveolar histiocytosis was observed in males of the 500 and 700 ppm groups and females in all exposure groups. This effect was attributed to treatment. However, since the effect is an incidental finding in older rats, the increase was considered to represent D4-related exacerbations of spontaneous lesions.
Examinations of the ovaries, uterus, vagina, mammary gland and pituitary gland and the establishment of the phase of the oestrus cycle for each female in the control and 700 ppm group revealed no exposure-related effects.

FUNCTIONAL OBSERVATION BATTERY: No adverse treatment-related findings.
Key result
Dose descriptor:
NOAEC
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reductions in body weight gains.
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
VIABILITY (OFFSPRING): Statistically significant reductions in mean live litter sizes and mean number of pups born were observed in the 500 and 700 ppm groups for the F0 animals, and statistically significant reductions were noted for the first mating period in the F1 animals for the mean live litter size in the 500 and 700 ppm groups and for mean number of pups born in the 700 ppm group. Slight decreases (not statistically significant) were also observed in both of these parameters in the 300 ppm group for the F0 generation. In the F1 generation, reductions (not statistically significant) in mean numbers of pups born were observed in the 500 ppm group for the first mating and the 700 ppm group for the second mating. In addition, mean live litter sizes were slightly reduced (not statistically significant) in the 70 and 300 ppm groups for the first mating and in all groups for the second mating. The reductions following the second mating period occurred in a non-exposure responsive manner in the 70, 300 and 500 ppm groups.
When the F1 males were paired with unexposed females, no effects on reproductive performance were observed. In the F1 generation, mating indices were reduced in the 700 ppm group for the first and second matings (statistically significant for the females in both matings and for males in the second mating).

CLINICAL SIGNS (OFFSPRING): No adverse effects.

BODY WEIGHT (OFFSPRING): Mean F1 pup weights were increased in the 700 ppm group compared with the controls on PND 1 and 4. These increases were considered to be related to the reduced mean live litter size in this group. Mean pup body weights were not significantly affected by exposure on PND 7, 14, 21 and 28.

SEXUAL MATURATION (OFFSPRING): No adverse effects were observed at any exposure level on anogenital distance, vaginal patency, and preputial separation.

ORGAN WEIGHTS (OFFSPRING): Mean absolute and relative organ weights (brain, spleen and thymus) of the unselected F1 weanlings euthanised on PND 21 were comparable to the control group values. At the PND 28 necropsy of unselected weanlings, mean spleen weights were reduced in males in the 300, 500 and 700 pp groups and in females of the 500 and 700 ppm groups. These decreases were generally statistically significant and could have been related to exposure to D4.

GROSS PATHOLOGY (OFFSPRING): No internal findings that could be attributed to parental exposure to D4 were noted at the necropsies of pups that were found dead prior to weaning or PND 22 and 28. At scheduled necropsy of unselected F1 weanlings on PND 21 and 28, there were no exposure-related findings.

HISTOPATHOLOGY (OFFSPRING): Brown pigment in the bile duct areas of the liver was observed in an exposure-related manner in the F1 generation males and females in the 300, 500 and 700 ppm groups. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals, and this birefringence often showed in only parts of the pigment deposits.

OTHER FINDINGS (OFFSPRING): No developmental neurotoxicity was observed in the F2a generation.
Key result
Dose descriptor:
NOAEC
Remarks:
Reproductive effects
Generation:
F1
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced fertility indices and reduced mean live litter sizes.
Reproductive effects observed:
not specified
Conclusions:
In a guideline, GLP, inhalation two-generation reproductive toxicity study in rats (reliability score 1), statistically significant reductions in mean live litter sizes and mean number of pups born were observed in the 500 and 700 ppm D4 groups for the F0 animals, and statistically significant reductions were noted for the first mating period in the F1 animals for the mean live litter size in the 500 and 700 ppm groups and for mean number of pups born in the 700 ppm group. When the F1 males were paired with unexposed females, no effects on reproductive performance were observed. In the F1 generation, mating indices were reduced in the 700 ppm group for the first and second matings (statistically significant for the females in both matings and for males in the second mating). Fertility indices were statistically significantly reduced in the 700 ppm group for the first F1 mating period. In the second F1 mating period, male and female fertility indices were statistically significantly reduced in the 500 and 700 ppm groups. The male and female fertility indices for the second F1 mating were also reduced in a non-exposure responsive manner in the 70 and 300 ppm groups, although the differences from the control group were not statistically significant. Microscopic evaluation of the ovaries, uterus, vagina, mammary gland and pituitary gland from the 0, 70, 300, 500, and 700 ppm F1 females suggested a subtle non-exposure responsive effect characterized by perturbation of the estrous cycle and accelerated reproductive senescence in F1 (but not F0) females at 70, 300, and 500 ppm, with a more obvious effect at 700 ppm.

In addition, some hepatic brown pigment accumulation in the bile duct was observed in the F1 generation males and females in the 300, 500 and 700 ppm groups. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals, and this birefringence often showed in only parts of the pigment deposits.

The NOAEC for reproductive toxicity and general systemic toxicity was therefore 300 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 640 mg/m³
Study duration:
subchronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key inhalation two-generation reproductive toxicity study in Sprague-Dawley rats (WIL Research Laboratories, 2001a), statistically significant reductions in mean live litter sizes and mean number of pups born were observed in the 500 and 700 ppm D4 groups for the F0 animals, and statistically significant reductions were noted for the first mating period in the F1 animals for the mean live litter size in the 500 and 700 ppm groups and for mean number of pups born in the 700 ppm group. When the F1 males were paired with unexposed females, no effects on reproductive performance were observed. In the F1 generation, mating indices were reduced in the 700 ppm group for the first and second matings (statistically significant for the females in both matings and for males in the second mating). Fertility indices were statistically significantly reduced in the 700 ppm group for the first F1 mating period. In the second F1 mating period, male and female fertility indices were statistically significantly reduced in the 500 and 700 ppm groups. The male and female fertility indices for the second F1 mating were also reduced in a non-exposure responsive manner in the 70 and 300 ppm groups, although the differences from the control group were not statistically significant. Microscopic evaluation of the ovaries, uterus, vagina, mammary gland and pituitary gland from the 0, 70, 300, 500, and 700 ppm F1 females suggested a subtle non-exposure responsive effect characterized by perturbation of the estrous cycle and accelerated reproductive senescence in F1 (but not F0) females at 70, 300, and 500 ppm, with a more obvious effect at 700 ppm. The NOAEC for reproductive toxicity was therefore 300 ppm based on reduced fertility indices and reduced mean live litter sizes, and the NOAEC for general toxicity was 300 ppm based on reduced body weight gain in adult animals.

In addition, some hepatic brown pigment accumulation in the bile duct was observed in the F1 generation males and females in the 300, 500 and 700 ppm groups. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals, and this birefringence often showed in only parts of the pigment deposits.

Findings in six supporting inhalation reproductive toxicity studies in Sprague-Dawley rats are in agreement with the findings in the key study (WIL Research Laboratories, 1996 a & b, 1997 a, b & c and 1998).


Effects on developmental toxicity

Description of key information

In the key inhalation developmental toxicity study in New Zealand white rabbits (International Research and Development Corporation, 1993), D4 did not affect fetal developmental and the NOAEC for this endpoint was therefore greater than the highest concentration tested (500 ppm; 6066 mg/m3). The NOAEC for maternal toxicity was 300 ppm based on reduced food consumption in the highest dose group.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
some details missing, but this does not affect the quality of the study
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products, Inc. Kalamazoo, Michigan.
- Age at study initiation: Approx. 27.5 weeks
- Weight at study initiation: 2871-3569 g at insemination
- Fasting period before study: No data
- Housing: Suspended stainless steel cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 37 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F):60-69
- Humidity (%): 49-79
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 08.09.1992 To: 09.10.1992
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All animals were exposed simultaneously in four stainless steel and glass exposure chambers with a volume of approximately 16 cubic meters each. One chamber was used for each group for the duration of the study, and the animals remained in the chamber 24 hours per day.
- Source and rate of air: HVAC system separate from the general laboratory system.
- Method of conditioning air: filtered
- Temperature, humidity, pressure in air chamber: Controlled, but no further details
- Air flow rate: No data
- Air change rate: No data
- Treatment of exhaust air: No data

TEST ATMOSPHERE
- Brief description of analytical method used: Gas-phase infrared spectrometry using a MIRAN 1A
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each chamber was sampled by drawing the exposure atmosphere into the MIRAN through Teflon sample lines. A solenoid valve-based sampling system and the recording of exposure concentrations from the MIRAN were controlled by a Hewlett-Packard 3388A laboratory computer system such that each chamber was analysed once per hour.
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: No data
- Day of insemination referred to as day 0 pregnancy
Duration of treatment / exposure:
day 6 - 18 of gestation
Frequency of treatment:
daily for 6 h
Duration of test:
29 days
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
No. of animals per sex per dose:
20 females
Control animals:
other: yes, concurrent treatment with filtered air
Details on study design:
- Dose selection rationale: Based on range-finding study results
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily throughout the study for mortality and signs of overt toxicity.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0, 6, 9, 12, 15, 19, 24 and 29.


FOOD CONSUMPTION: Yes
- Individually for the following intervals: 0-6, 6-9, 9-12, 12-15, 15-19, 19-24, 24-29, 6-19 and 0-29.


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The uterus and ovaries were exposed and pregnancy status was determined. The abdominal and thoracic cavities and organs of the females were examined for gross adverse changes. Gross lesions were preserved for possible histopathological examination. Uteri from females that appeared nongravid were opened and examined for detection implantations. Maternal liver weights were also measured and recorded.

OTHER: Females showing signs of abortion were euthanised on the day such evidence was observed and the aborted tissue examined and preserved for possible histopathological examination.
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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
All statistical analyses compared the exposure groups with the control group; mean values were noted where p≤0.05 and p≤0.01 occurred. All means were accompanied by standard deviations. All statistical tests were performed by a VAX computer, SAS statistical software, as well as in-house software, were utilised for analyses.
Indices:
No indiced calculated
Historical control data:
No data described
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
No substance related mortality; no adverse antemortem or necropsy findings; no substance related effects on body weight gain at any exposure group.
Statistically significant reductions in maternal food consumption were noted in the highest exposure group during the first and second exposure intervals (gestation days 6-9 and 9-12) when compared with the controls. Food consumption was also slightly reduced, relative to the control group, in that group during the third interval (gestation days 12-15) and during the overall exposure interval (gestation days 6-19). These reductions were considered to be treatment-related.
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean postimplantation loss (resorptions) was slightly increased in the highest exposure group when compared with the controls, but were well within the historical control range. This finding was not attributed to treatment. There were no treatment-related differences in the number of viable fetuses per dam or mean fetal body weight. There were no treatment-related malformations or developmental variations.
Dose descriptor:
NOAEL
Effect level:
>= 500 ppm
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In the key inhalation developmental toxicity study in rabbits, conducted using a protocol comparable to OECD 414 and GLP (reliability score 1), D4 did not affect fetal developmental and the NOAEC for this endpoint was therefore greater than the highest concentration tested (500 ppm). The NOAEC for maternal toxicity was 300 ppm based on reduced food consumption in the highest dose group.
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
6 066 mg/m³
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Five reliability 1 studies conducted in the same year are available for developmental toxicity (International Research and Developmental Corporation, 1993a, b, c, d & e). Two of these studies are range-finding studies, so were excluded from being key studies (International Research and Developmental Corporation, 1993d & e). One of the studies (International Research and Developmental Corporation, 1993b) tested a restricted number of animals, so was also excluded from being the key study.

Either one of the remaining two studies could have been selected as the key study (International Research and Developmental Corporation, 1993a & c).

In the key inhalation prenatal developmental toxicity study (International Research and Developmental Corporation, 1993a) New Zealand white rabbits (20 animals/dose) were exposed to D4 whole body at concentrations of 100, 300, 500 ppm on gestation days 6 to 18. There was no substance related mortality; no adverse antemortem or necropsy findings, and no substance related effects on body weight gain were observed in any exposure group.

Statistically significant reductions in maternal food consumption were noted in the highest exposure group during the first and second exposure intervals (gestation days 6-9 and 9-12) when compared with the controls. Food consumption was also slightly reduced, relative to the control group, in that group during the third interval (gestation days 12-15) and during the overall exposure interval (gestation days 6-19). These reductions were considered to be treatment-related. Mean post implantation loss (resorptions) was slightly increased in the highest exposure group when compared with the controls, but were well within the historical control range. This finding was not attributed to treatment. There were no treatment-related differences in the number of viable fetuses per dam or mean fetal body weight. There were no treatment-related malformations or developmental variations. Therefore, the NOAEC for developmental toxicity was 500 ppm. The NOAEC for maternal toxicity was 300 ppm based on reduced food consumption in the highest dose group.

In the supporting inhalation prenatal developmental toxicity study (International Research and Developmental Corporation, 1993c) CD rats (30 animals/dose) were exposed to D4 whole body at concentrations of 100, 300, 700 ppm on gestation days 6 to 15. No deaths occurred and there were no exposure-related signs of toxicity. There was statistically significantly decreased body weight gain in the highest exposure group over gestation days 6 -16, 9 -12 and over the entire gestation period (gestation days 0 -20). All animals in the high exposure group lost weight on gestation days 6 -9. A statistically significantly reduced food consumption occurred in the high exposure group during exposure group and over the entire gestation period. Pregnancy rates were not different to controls in all exposure groups. Only one animal of the low dose group had whole litter resorption, a finding that was not considered to be treatment-related. All c-section parameters, including embryolethality, litter sizes, sex distribution of fetuses and fetal body weight were comparable between control values and treated groups. The mean maternal liver weights in the treated groups were comparable with those of the control group. There were no treatment-related malformations or developmental variations. The NOAEC for maternal toxicity was 300 ppm, based on reduced food consumption and body weight gain. The NOAEC for developmental toxicity was 700 ppm.

The other supporting studies also gave negative results for effects on development, including teratogenicity.


Toxicity to reproduction: other studies

Additional information

The effects on fertility associated with inhalation exposure to high vapour concentrations of D4 are attributed to an effect on the female and specific to exposure surrounding the ovulatory phase (WIL Research Laboratories, 1999). Research to further define these associations has demonstrated that D4 exposure increases the incidence of females expressing a delayed/suppressed pre-ovulatory LH surge and ovulation (Control: 21%; 700 ppm D4: 58%; 900 ppm D4: 69%) (Dow Corning Corporation, 2002a; WIL 2001b). The pre-ovulatory LH surge and ovulation are critically connected in the rat as is well established in the literature. An insufficient or blocked pre-ovulatory LH surge fails to induce ovulation in the rat and results in the spectrum of fertility effects as seen with D4.

Differences in the regulation of the pre-ovulatory LH surge and the criticality of timed events with regard to rat versus human support the conclusion that the observed high-dose effect of D4 on female rat fertility should not be considered relevant to humans. Therefore, the current understanding of estrus cyclicity and neural/hormonal regulation of ovulation in humans suggests that the effects of D4 on fertility as seen in the SD rat are not relevant.

A more detailed discussion of the justification for the above conclusion is attached to this endpoint summary.

Justification for classification or non-classification

D4 has a harmonised classification as Reproductive Category 2 in Annex VI of Regulation 1272/2008. This was based on the available data at the time for its demonstrated effects on female fertility in rats. However, subsequent research has shown the fertility effects are associated with exposure of the female rat during the critical ovulatory phase and that exposure during the ovulatory phase induces a delay/suppression of the pre-ovulatory LH surge and subsequent ovulation. Differences in the regulation of the pre-ovulatory LH surge and the criticality of timed events with regard to rat versus human support that the observed high-dose effect of D4 on female rat fertility should not be considered relevant to humans. The available data suggest that D4 does not require classification for effects on development or male fertility.

Additional information