Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The potential effects of D5 on the dopamine receptor, estrogenic or androgenic activity were investigated. D5 was demonstrated to act as D2-receptor agonist but was without effect in all other cases.

In a two-generation reproductive toxicity study (WIL Research Laboratories Inc, 1999) conducted to appropriate EPA test guidelines and to GLP, no parental toxicity in the F0 and F1 generations was observed at exposure concentrations of 30, 70, and 160 ppm D5. F0 and F1 reproductive performance was not affected at any concentration. No test-substance-related total litter losses occurred, and no neonatal toxicity was evident in the F0 and F1 generations at concentrations of 30, 70, and 160 ppm. Based on the results of this study, the NOAEC for parental toxicity, reproductive toxicity, and neonatal toxicity is considered to be at least 160 ppm (actual 2496mg/m3).

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.10.1996 to 22.02.1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
EPA OPP 83-6 (Developmental Neurotoxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan.
- Age at study initiation: 45 days (F1 rats prior to pairing were 13-15 weeks old)
- Weight at study initiation: (P) Males: 138-232 g; Females: 115-193 g; (F1 prior to pairing) Males: 304-576 g; Females: 196-321 g
- Fasting period before study: No
- Housing: Wire-mesh cages or plastic maternity cages
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2.2
- Humidity (%): 40-70
- Air changes (per hr): Approximately 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 25.10.1996 To: 21.08.1998
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: Each group of animals was exposed in a 2 m3 stainless steel and glass whole body inhalation chamber.
- Method of holding animals in test chamber: cages
- Temperature, humidity, pressure in air chamber: 18-26oC, 30-60%
- Air change rate: 12 to 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): plastic maternity cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposures within each chamber were measured 9 to 11 times during each daily exposure period by a validated gas chromatographic method.
Duration of treatment / exposure:
Exposure period: F0 and F1 males and females: At least 70 days prior to mating, throughout mating, gestation (to gestation day 20), lactation,
with the exception of lactation days 0-4, until scheduled euthanasia. F1 pups were exposed from weaning through sexual maturity, breeding, gestation.
Duration of test: ca. 18 months
Frequency of treatment:
6 hr/day, 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Dose / conc.:
478 mg/m³ air (analytical)
Remarks:
31 ppm (analytical conc.)
Dose / conc.:
1 094 mg/m³ air (analytical)
Remarks:
71 ppm (analytical conc.)
Dose / conc.:
2 496 mg/m³ air (analytical)
Remarks:
162 ppm (analytical conc.)
No. of animals per sex per dose:
30/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random
- F2 pups were randomly selected on PND 4 for neuropathological and/or neurobehavioural evaluations.
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: weekly for all animals and on gestation day (GD) 0, 7, 10, 14, and 20 and PND 1, 4, 7, 14, and 21 for females in the F0 and F1 generations.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. FC was also recorded on GD 0, 7, 10, 14, and 20 and PND 1, 4, 7, 14, and 21 for females in the F0 and F1 generations.

WATER CONSUMPTION: No

FUNCTIONAL OBSERVATION BATTERY (FOB): On all females F1 rats on gestation day 10 and lactation day 20.
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of oestrus cycle for each F0 and selected F1 females, beginning 21 days prior to pairing and continuing until evidence of copulation was observed. For females with no evidence of copulation, smearing was continued until termination of the mating period.
Sperm parameters (parental animals):
Parameters examined in all P, F1 and F2 male parental generations: testicular and epididymal sperm count, sperm production rates, sperm motility, sperm 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: When parturition was judged complete, litters were sexed and examined for gross malformations and the numbers of stillborn and live pups were recorded. Individual gestation durations were calculated using the date delivery initiated. Each litter was examined twice daily for survival, and all deaths were recorded. Litters were also examined for any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14 and 21, at weekly intervals thereafter until scheduled euthanasia. On PND 1, the anogenital distance was measured for all F1 pups in the control and high dose groups, and for all F2 pups. Pups were weighed on PND 1, 4, 7, 14 and 21. Individual body weights were also recorded for selected F2 rats at weekly intervals. Pup sexes were individually determined on PND 0, 4 and 21.

FOB: Thirty pups/sex/group from the F2 generation were selected for developmental landmarks, neurobehavioural testing, neuropathy, brain weights and/or brain dimension measurements.

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 F0 and F1 parental animals after the last litter of each generation was weaned.

GROSS NECROPSY: On all parental animals dying spontaneously, euthanised in extremis, or surviving to the scheduled sacrifice.
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: Histopathology (on all F0 and F1 adult animals from the control and high-exposure groups and from all F0 and F1 parental animals dying spontaneously or euthanised in extremis): 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. 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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 or 28 days of age. Selected F2 rats not allocated for neuropathy and brain dimension measurements were necropsied on PND 70 and selected organs were weighed.
- These animals were subjected to a macroscopic examination, with emphasis placed on developmental morphology evaluations.

HISTOPATHOLOGY / ORGAN WEIGHTS: Including all pups that died after PND 4 and prior to weaning.

F2 NEUROPATHOLOGICAL EXAMINATION: Brain weights and brain region dissections: At the scheduled evaluations (PND 11 and 70), 10 and 16 F2 pups/sex/group, respectively, selected for neuropathology and/or brain weight measurements were euthanised. Brains and carcasses of F2 rats selected only for brain weight measurement were discarded following weighing of brain regions.
Neuropathology: At the PND 11 evaluation, 6 selected F2 pups/sex/group were allocated for neuropathological analysis. Brain tissue was subjected to histopathological examination. At study termination (PND 70), 6 selected F2 rats/sex/group were euthanised. The central and peripheral nerve tissues were dissected and preserved. Brain weights and dimensions were recorded. Any observable gross changes, abnormal coloration, or lesions of the brain and spinal cord were recorded. Nerve tissues were examined histopathologically.
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

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:
Male (female) mating index (%): No. of males (females) with evidence of mating/No. of paired animals x100.
Male (female) fertility index: No. of males siring a litter (females confirmed pregnant)/No. of males (females) used for mating x100.

Because the protocol specified that each female was to be paired with only one male, the mating and fertility indices are identical for both sexes.
Offspring viability indices:
Live litter size: total viable pups Day 0/No. of litters with viable pups Day 0 x100.
postnatal survival between birth and PND 0 or PND 4 (pre-selection)(%/litter): Σ(viable pups per litter on PND 0 or 4/No. of pups born per litter)/No. of litters per group x 100.

Postnatal survival for all other intervals (%/litter): Σ(viable pups per litter at end of interval N/viable pups per litter at start of interval N)/ No. of litters per group. Where N = PND 0-1, 1-4, 4-7, 7-11, 11-14 or 4-21.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Maternal data with dose level (with NOAEL value): No adverse effects were noted in any endpoint in the F0 or F1 parental
animals at any dose level tested.

• Mortality and day of death: No exposure-related increases in mortality were reported.
• Number pregnant per dose level: mating indices for the 0, 30, 70, and 160 ppm dose groups were 100%, 83.3%, 93.3%, and 93.1%
• Number aborting: no animals aborted
• Duration of Pregnancy: No exposure-related differences in time to coitus or in length of pregnancy were reported.
• Body weight: No exposure-related changes in body weight were reported.
• Food/water consumption: No exposure-related changes in food consumption were reported.
• Description, severity, time of onset and duration of clinical signs: No exposure-related changes in clinical signs were reported.
• Gross pathology incidence and severity: No exposure-related increase in gross pathological findings was
reported. Increased incidences of minimal alveolar histiocytosis were noted in the F0 females and the F1 males and females in the 30, 70, and 160 ppm groups. While the increased incidences in these groups were attributed to test article exposure, the finding was not considered to be an adverse effect as it was graded minimal in all groups (including the control group). Moreover, no associated histopathological lung lesions or differences in
mean lung weights were observed at any exposure level. This finding was considered to be a compensatory response to material delivered to the lungs via the inhalation route.
• Organ weight changes, particularly effects on total uterine weight: No exposure-related changes in organ
weights were reported.
• Histopathology incidence and severity: No exposure-related increase in histopathological findings was reported.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity, reproductive performance or neonatal toxicity in the P generation was seen at exposure concentrations of 30, 70, and 160 ppm.
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Fetal data with dose level (with NOAEL value): No adverse effects were noted in any endpoint in the F1 or F2 generation
animals at any dose level tested.

• Pup data, provide at a minimum qualitative descriptions of responses where dose related effects were seen:
Ø Litter size and weights: Litter sizes were comparable across groups.
Ø Number viable (number alive and number dead): Pup survival was unaffected by exposure to D5. One F0 female in the 160 ppm group had total litter loss on lactation day 0. However, this female delivered only one pup. Because no exposure-related decreases in postnatal survival of the F1 and F2 litters were noted at any concentration, the single occurrence of total litter loss in the 160 ppm group was not attributed to test substance exposure. Mean pup body weights and the general physical condition of the F1 and F2 pups were similar in control, 30, 70, and 160 ppm groups both before and after weaning.
Ø Sex ratio: Sex ratios were comparable across groups in the F1 and F2 generations.
Ø Grossly visible abnormalities: No treatment-related gross external malformations were reported.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity, reproductive performance or neonatal toxicity in the F1 generation was seen at exposure concentrations of 30, 70, and 160 ppm.
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No F2 developmental neurotoxicity was evident at any concentration.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
In a two-generation reproductive toxicity study (reliability score 1) conducted to appropriate EPA test guidelines and to GLP, no parental toxicity in the F0 and F1 generations was observed at exposure concentrations of 30, 70, and 160 ppm. F0 and F1 reproductive performance was not affected at any concentration. No test-substance-related total litter losses occurred, and no neonatal toxicity was evident in the F0 and F1 generations at concentrations of 30, 70, and 160 ppm. No F2 developmental neurotoxicity was evident at any concentration. Based on the results of this study, the NOAEL for parental toxicity, reproductive toxicity, neonatal toxicity, and developmental neurotoxicity is considered to be at least 160 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
2 420 mg/m³
Study duration:
subchronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study for reproductive toxicity was conducted to EPA OPPTS 870.3800 (Reproduction and Fertility Effects) and to EPA OPP 83-6 (Developmental Neurotoxicity Study), which is similar to OECD test guideline 416 (WIL Research Laboratories Inc, 1999). Sprague-Dawley rats (30 animals/sex/dose) were exposed, whole body, to D5 vapour 6 hours/day, 7 days/week at concentrations of 30, 70 and 160ppm (actual analytical concentrations of 478, 1094 and 2496mg/m3). The F0 and F1 males and females were exposed to D5 at least 70 days prior to mating, throughout mating, gestation (to Day 20), lactation (except days 0 -4), then until scheduled euthanasia. F1 pups were exposed from weaning, through sexual maturity, breeding and gestation. Observations and examinations were conducted according to the guideline. In addition, a neuropathological examination of the brain and spinal cords of animals from the F2 generation were conducted on postnatal days 11 and 70. There were no signs toxicity in adult animals of the F0 and F1 generations, nor was there any adverse effect on the reproductive performance. For offspring, litter sizes and weights were comparable across the doses, as were the number of viable pups and the sex ratios. In addition, there were no treatment-related gross external malformations observed. The neuropathological examination did not discover any adverse effects of D5 on the brain region weights, and there were no observable gross changes, abnormal colouration, or lesions of the brains, spinal cords and nerve tissues. Based on the results of this study, the NOAEC for parental toxicity, reproductive toxicity, and neonatal toxicity is considered to be at least 160 ppm (actual 2496mg/m3).

There is one additional reproductive toxicity study (WIL Research Laboratories Inc, 1996) that supports the findings of the key study in that no adverse effects on reproductive parameters were observed up to an exposure concentration of 132ppm (2034 mg/m³).


Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

The potential for D5 to act at the dopamine receptor was investigated in a reliability 2 study (Dow corning Corporation, 2005) which demonstrated that it acts as a dopamine D2-receptor agonist. The estrongenic activity of D5 was investigated in ovariectomised Fischer F344 or Sprague-Dawley rats in reliability 2 studies (Dow Corning Corporation, 2004a; Dow Corning Corporation, 2004b). Both studies demonstrated that D5 did not have any estrogenic, or anti-estrogenic activity.

An in vitro reliability 2 study (Dow Corning Corporation, 2005) evaluated the potential of D5 to act as a progesterone receptor ligand, and found no evidence that it did. Two further reliability 4 studies demonstrated that D5 did not produce an androgenic or anti-androgenic response (Dow Corning, 2003) and that D5 was not a direct competitor at the active estrogen receptor alpha site (Dow Corning, 2002).

Justification for classification or non-classification

Based on the available data D5 does not require classification for toxicity to reproduction according to Regulation (EC) No. 1272/2008.