Registration Dossier

Administrative data

Description of key information

Inhalation exposure to D4 induced an increased incidence of endometrial adenomas and cystic endometrial epithelial hyperplasia in the uteri of female Fischer 344 rats exposed to 700 ppm for 24 months (Battelle, 2004). There were no carcinogenic findings in male rats. The study authors indicated the NOAEL for carcinogenic effects was 150 and ≥700 ppm in females and males, respectively. However, based on fundamental differences between rats and humans with respect to the development of reproductive function, brain regulation of LH secretion, and the mechanism of reproductive aging and the hormonal environment of reproductive senescence the NOAEC for carcinogenic effects relevant to humans is ≥700 ppm (equivalent to ≥8492 mg/m3based on a molecular weight of 296.62).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
41.01.1999 to 16.08.2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: approximately 154-157 g; Females: approximately 111-112 g
- Fasting period before study: No
- Housing: Individually in stainless steel wire cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 9-10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74 ±4oF
- Humidity (%): 50±20%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 21.01.1999 To: 01.02.2001
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: During the exposure period, rats were housed in whole-body exposure chambers 24 hours/day. The rats were exposed to D4 in four chambers/sex. After the 12 months of exposure, the recovery group was removed from th eexposure chambers and moved to the recovery room for their remaining time on the study.
- Source of air: room
- Method of conditioning air: Single HEPA filter
- Temperature, humidity, pressure in air chamber: 74 ±4oF, 50±20%,
- Air change rate: 15 ±3 air changes/hour
- Treatment of exhaust air: Filtered (fibreglass roughing, duct prefilter, dual HEPA)


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber and room concentrations of D4 were determined using on-line GC/FID. All exposure and control chambers were monitored once every 30 minutes throughout the 6-hour exposure.
Duration of treatment / exposure:
up to 24 months
Frequency of treatment:
6 hours/day, 5 days/week
Post exposure period:
12 months recovery in one group
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
30 ppm (nominal)
Dose / conc.:
150 ppm (nominal)
Dose / conc.:
700 ppm (nominal)
No. of animals per sex per dose:
96
Control animals:
other: yes, filtered air
Details on study design:
There were 4 subgroups of rats per sex and exposure concentration:
Subgroup A was scheduled for necropsy after 6 months of exposure (Tissue Level Study; 6M/6F per dose).
Subgroup B was scheduled for necropsy after 12 months of exposure (Chronic Toxicity Study; 10M/10F per dose).
Subgroup C was scheduled for necropsy at 24 months on study, after 12 months of exposure and a 12-month recovery period (Chronic Recovery Study; 20M/20F per dose).
Subgroup D was scheduled for necropsy after 24 months of exposure (Oncogenicity Study; 60M/60F).
Positive control:
No positive control used in this study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to exposure start, at the time of randomisation, weekly during the 14 weeks, every four weeks thereafter, and at scheduled necropsies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to exposure and two weeks prior to sacrifice
- Dose groups that were examined: groups B and D (10 rats/sex/conc), control and 700 ppm group

HAEMATOLOGY, CLINICAL PATHOLOGY AND URINALYSIS: Yes (Table 1)
- Time schedule for collection of blood: Dependent on subgroup, see below.
- Anaesthetic used for blood collection: Yes (70% CO2)
- Animals fasted: Yes
- Metabolism cages used for collection of urine: Yes
Subgroup B: prothrombin time (PTT) and activated partial thromboplastin time (APTT) at scheduled sacrifice.
Subgroup C: Full clinical pathology at 3, 6, 9 (clinical chemistry only) and 12 months (10 rats/sex/group).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
 A complete necropsy was performed on all rats including those dying or sacrificed due to moribund condition, with an exception of rats in Subgroup A.

Subgroup A: Plasma, fat, and liver samples were collected for determination of D4 content. Pituitary and liver weights were recorded. A number of tissues were collected and stored: Cervix, kidneys, larynx, liver, lungs, mammary gland, nasal cavity, ovaries, penis, pharynx, pituitary gland, prepuce, preputial gland, prostate gland, seminal vesicles, testes (with epididymides), thyroid gland, trachea, uterus, vagina and zymbals gland.

Subgroups B, C and D: At scheduled sacrifices, weights were collected on protocol-specified organs (adrenal glands, brain, liver, lungs, kidneys, ovaries, spleen, testes, epididymides, heart, uterus and pituitary gland - up to and including 12-month interim sacrifice only). Complete histopathology was performed on specified tissues for all early removal rats (except subgroup A) and for all rats in the control and 700 ppm exposure groups. If a treatment-related lesion was suspected at 700 ppm, the potential target organ was examined in all exposure groups. Histopathologic evaluations were performed on the lungs, liver, kidneys, nasal cavities, tissue masses, and gross lesions from all rats and uterus from female rats in the 10, 30, and 150 ppm groups.
Other examinations:
None reported
Statistics:
The data were first assessed for homogeneity using Bartlett's test. If the data were homogenous, Dunnett's test was used to determine statistical significance. If the data were non-homogeneous, a Modified t-test was used to determine statistical significance. The statistical significance was evaluated at the 0.05 and 0.01 levels of significance.
Homogeneity of variance of each parameter or analyte was determined by exposure group and gender using Hartley's Fmax (equal n) or Cochran's C test (unequal n). If homogeneity was rejected, that data set was analysed using the Behren's-Fisher t-test. If the data set was homogeneous it was analysed using Dunnett's two-tailed t-test. The statistical tests were evaluated at the 0.05 and 0.01 levels of significance.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS, MORTALITY, BODY WEIGHT AND BODY WEIGHT GAINS: Effects of D4 exposure on in-life parameters (survival, clinical signs, and body weights) were, overall, minimal. One exception was that the 700 ppm male rats in Subgroup D had significantly lower 2-year survival and terminal body weight than the concurrent controls. The decreases in survival and body weight of 700 ppm male rats were apparent mainly during the last few months on study. The in-life parameters of other D4-exposed Subgroup D male and female rats were comparable to the controls. The survival of Subgroup C when assessed after 12 months of recovery showed no significant difference between the exposed and the control groups of either sex. There was no early death in either Subgroup A or B prior to their scheduled sacrifices. There were no clinical signs that were clearly associated with D4 exposure.

OPHTHALMOSCOPIC EXAMINATION: Ocular examination conducted two weeks prior to the scheduled sacrifices for Subgroups B and D did not reveal eye lesions clearly associated with D4 exposure.

CLINICAL PATHOLOGY: Overall erythrocyte and urinalysis parameters of either sex were not affected by D4 exposure. Leukocytosis was consistently observed in both sexes of rats exposed to 700 ppm at all time points, resulting from increased lymphocytes. There was an exposure-related decrease in aspartate aminotransfefase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) activities in D4-exposed rats of both sexes at 3, 6, 9, and 12 months of exposure.  These decreases were frequently present in a dose-related manner, in particular at the 6- and 9-month time points. No clear toxicological significance of this decrease in serum enzymes was identified relative to histopathology findings.

TISSUE CONCENTRATIONS: After 6 months of exposure (Subgroup A), D4 concentrations in plasma, liver, and fat tissues increased with increasing
D4 exposure concentration. Female rats had consistently higher D4 concentrations in these tissues than male rats at all exposure concentrations, except at 700 ppm. Differences in the D4 concentration in different types of fat were observed mainly at lower exposure concentrations.

ORGAN WEIGHTS: Weight increases in the liver, kidney, and uterus were of particular interest. At 6 months on study (Subgroup A), the absolute liver weight tended to increase with increasing D4 exposure concentration and the difference was statistically significant at 700 and at >30 ppm for
females and males, respectively, relative to the concurrent controls. At 12 months (Subgroup B), the absolute liver weights significantly increased at 150 and 700 ppm compared to the controls for both sexes and the relative liver weights (normalized either to body or brain weight) generally increased with increasing exposure concentrations.  This liver weight increase might be associated with centrilobular hypertrophy of hepatocytes diagnosed in 700-ppm males in Subgroup B. The absolute and/or relative kidney weights increased in some exposed males and females at 12 months but the differences were statistically significant mainly at 700 ppm when compared with the controls. For Subgroup D (24 months of exposure), the absolute and/or the relative liver and kidney weights significantly increased in both sexes exposed to 700 ppm above the controls.  Increases in kidney weights may reflect increases in severity of chronic nephropathy from these groups of rats. In males, increased liver weight was associated with increased centrilobular hypertrophy of hepatocytes at 700 ppm. However this liver lesion did not accompany the weight increases observed in Subgroup D females. Twenty four months of D4 exposure resulted in a substantial increase in the absolute and relative uterus weights at 700 ppm compared to the controls. The uterine weight increase may be related to endometrial epithelial hyperplasia observed microscopically. Although at 24 months the body weight-normalized testes weight and the body weight-normalized heart weight in females increased above the controls at 700 ppm, there were no associated concurrent histopathologic lesions in these groups of either sex. In Subgroup C increased weight of liver, kidney, and/or uterus was observed in a few exposed animals. However the weight differences from the controls were overall less than those differences in Subgroup D, likely because of 12 months of recovery.

HISTOPATHOLOGY: Non-neoplastic findings are reported in Section 7.5, and are therefore not included here. Only neoplastic findings are reported in this summary. Endometrial adenomas were present in sections of uterus from 4/60 (7%) female rats exposed to 700 ppm for 24 months, while incidence of this lesion in the concurrent control group was 0/59. A statistical analysis of these data indicated a significant (p < 0.05) positive trend. Incidence in historical control F344 females is 2/1109 or 0.2% (NIEHS website: 2003). Tumour incidence for Vehicle Control NTP 2000 Diet (Rats), http://ntp-server.niehs.nih.gov/Main Pages/ntp_hcrs.html, Version 02/27/03). There was a clear increase in the incidence and mean severity of endometrial epithelial hyperplasia in females exposed to 700 ppm for up to 24 months compared to concurrent controls. Statistical analysis of the incidence of endometrial epithelial hyperplasia in all groups indicated a significant increase (p < 0.01) in the incidence in female rats exposed to 700 ppm and a significant positive trend, providing further evidence for a proliferative effect of 700-ppm exposure on the endometrial epithelium. These data are interpreted as evidence for induction of endometrial epithelial hyperplasia and adenomas by exposure to 700 ppm for 24 months. One endometrial adenoma was diagnosed in a Subgroup C rat necropsied following 12 months of exposure to 30 ppm and 12 months of recovery, and one endometrial adenocarcinoma was diagnosed in a Subgroup C rat exposed to 150 ppm. The incidence of these tumours was not statistically significant compared to concurrent controls. Incidence of endometrial epithelial hyperplasia was increased in all exposed groups compared to controls in Subgroup C, but this increase was not dose-related and was not statistically significant except for the group exposed to 10 ppm. Therefore tumours and hyperplasia of endometrial epithelium in Subgroup C are not considered to be related to D4 exposure. There was a low incidence of hyperplasia of squamous epithelium lining the cervical lumen in Subgroup D exposed to D4 for up to 24 months and in the recovery Subgroup C exposed to 700 ppm. Although some of the increases in incidence of this lesion were statistically significant, this lesion is not considered to be related to D4 exposure. The cervical mucosal epithelium may have wide variations in normal structure reflecting estrous cycle changes related to aging. Uterine stromal lesions were observed frequently in controls and exposed groups in Subgroup D, with no statistical differences in incidence and no clear indication of an effect of exposure. The incidence of endometrial stromal polyps was increased in exposed Subgroup C compared to controls; this increase was statistically significant (p < 0.05) compared to controls in recovery groups exposed to 10, 150, or 700 ppm. However, this increase was not dose-related and not seen in Subgroup D; therefore the noted increase in incidence of stromal polyps was not considered to be related to D4 exposure.
Dose descriptor:
NOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on increased uterine weight, increased incidence of endometrial cell hyperplasia, and an increased incidence of endometrial adenomas at 700 ppm.
Dose descriptor:
NOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on chronic nephropathy at 700 ppm
Conclusions:
In summary, inhalation exposure to D4 for up to 24 months (study reliability score 1) induced the following notable effects in male and female rats:
* Reduced 2-year survival and terminal body weight of male rats exposed to 700 ppm
*Lymphocytic leukocytosis in both sexes of rats exposed to 700 ppm
*A dose-related decrease in selected serum enzymes (AST, ALT, CK, and/or LDH) in both sexes of rats
*A dose-related increase in D4 concentration in plasma, liver, and fat tissues at 6 months; sex difference was noted at lower exposure concentrations
*Increases in absolute and/or relative weight of liver, kidney, and uterus of D4-exposed rats, especially at 700 ppm
*Increased incidence of endometrial adenomas and endometrial epithelial hyperplasia in the uteri of rats exposed to 700 ppm for 24 months
* Minimal to mild goblet cell hyperplasia in the nasal mucosa of rats exposed to 700 ppm for 12 or 24 months
*Minimal hyperplasia of squamous epithelium in the nasal vestibule in rats exposed to 700 ppm for 12 months
* Increased incidence of suppurative rhinitis in males exposed to 700 ppm for 12 months
*Increased incidence and severity of intracytoplasmic eosinophilic globules in the nasal epithelium of both sexes of rats exposed to 700 ppm and females exposed to 150 ppm for 24 months
*Increased severity of chronic nephropathy in both sexes of rats exposed to 700 ppm for 24 months
*Increased incidence of centrilobular hypertrophy of hepatocytes in male rats exposed to 700 ppm
for 12 or 24 months.
The NOAEL for carcinogenic effects was 150 and ≥700 ppm in females and males, respectively.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
8 492 mg/m³
Study duration:
chronic
Species:
rat

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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


Justification for classification or non-classification

Though the mode of action responsible for induction of uterine tumours in the female F344 rat has not been confirmed, the subtlety of the effects following exposure to D4 may prevent further assessment and definition of a precise mode of action. However, the available data provide important insight into the potential human relevance of the uterine tumours in rats. Relevant findings include:   

  • D4 has not been shown to be mutagenic or genotoxic in the in vitro and in vivo experimental models designed to evaluate this potential.
  • No tumours were associated with chronic D4 exposure to male F344 rats and no organs other than the uterus developed treatment related tumours in female F344 rats following chronic D4 exposure.
  • Uterine cystic endometrial epithelial hyperplasia and adenoma in the female F344 rat arose during the 2nd year of exposure, a period of marked changes in physiology and onset of a reproductive senescence that is unique to the F344 rat and distinctly different from human and often associated with an increased endogenous estradiol from ovarian cysts.
  • Uterine cystic endometrial epithelial hyperplasia and adenoma in the female F344 rat that arose during the 2nd year of exposure was only seen at the high dose exposure indicating a threshold response.
  • The uterine changes reported were benign and non-metastatic, with no evidence of progression to malignancy, even at the terminal sacrifice following two years of exposure. Unlike glandular epithelial focal hyperplasia, cyctic epithelial hyperplasia is generally considered not a precursor lesion to malignancies.
  • The apparent affinity of D4 for estrogen receptor and progesterone receptor is low to non-existent as determined in various in vitro and in vivo experimental efforts. It is also uncertain if the demonstrated weak estrogenic responses observed with D4 are involved in the uterine effects that developed in the aging F344 rat in response to chronic D4 exposure (either in the chronic bioassay or in the chronic aged animal study) as there were no other indications of a weak estrogenic response in either males (chronic bioassay) or females (chronic bioassay and chronic aged animal study) from these studies.
  • Although demonstration of D4 as a direct dopamine agonist was not achieved, there were slight alterations in the dopamine activation pathway and modulation of prolactin levels following exposure to D4 that may be suggestive of partial agonist/antagonist activity but the subtlety of these changes prevent further assessment.
  • It is well established that D4 exposure inhibits ovulation and can prolong exposure of the uterine endometrium to endogenous estrogen in the rat. In addition, in the chronic aged animal study, D4 exposure produced a higher percentage of days for which the vaginal lavages exhibited a more estrogenic character. The higher percentage of days in proestrus/estrus in the D4 group appeared to be the result of prolonged estrogenic phases during the first half of the study (consistent with the LH surge study) followed by increased cycling (i.e., greater numbers of times in proestrus/estrus) during the second half of the study. If alteration of the LH surge with subsequent prolonged exposure of the uterine endometrium to endogenous estrogen is responsible for cystic endometrial hyperplasia and adenomas, it is considered unlikely this would occur in the human due to the marked differences in reproductive function, brain regulation of LH secretion, and the mechanism of reproductive aging and the hormonal environment of reproductive senescence in the rat verses humans. (Plant, 2012).   

D4 should not be classified as a human carcinogen on the basis of:

  • A lack of carcinogenic outcome in males exposed to D4 for 24 months at up to and including 700 ppm.
  • No evidence for direct mutagenic/genotoxic potential.
  • The many fundamental differences between rats and humans with respect to the development of reproductive function, brain regulation of LH secretion, and the mechanism of reproductive aging and the hormonal environment of reproductive senescence. These differences make the F344 female rat a poor surrogate for assessment of female human reproductive organ hazard assessment associated with exposure to D4. 

In summary, the available information suggests that the induction of benign proliferative endometrial lesions in the rat after chronic D4 inhalation has no relevance to human. Due to the absence of any appreciable direct hormonal activity of D4, the induction of cystic endometrial hyperplasia and the significant trend for an increased incidence of uterine endometrial adenoma observed across D4 dose levels in the two-year inhalation study are likely due to interferences of D4 with rat estrous cycle control that are only seen at doses that exceed the metabolic capacity of animals and are not relevant to humans.