Registration Dossier

Administrative data

Description of key information

In the key repeated dose inhalation study, exposure to D4 for up to 24 months (Batelle, 2004) resulted in a NOAEC for general toxicity of 150 ppm, based on chronic nephropathy. The NOAEC for local respiratory effects was also 150 ppm based on findings in the nasal cavity.

In a three-week dermal exposure study (Bayer AG, 1988) in rabbits conducted using a protocol similar to OECD 410 and GLP, there were no adverse effects and therefore the dermal NOAEL was ≥ 1 ml/kg bw/day (equivalent to 960 mg/kg bw/day).

There are no reliable data that define a NOAEL for the oral route.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.01.1999 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
Guideline:
other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- 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: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: NA
Details on inhalation 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 the exposure 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
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: 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: Neoplastic findings are reported in Section 7.7. Non-neoplastic findings were as follows: The primary target organs affected by D4 exposure included: respiratory tract, kidney, and liver.

Inhalation exposure to D4 for 12 or 24 months increased the incidence of goblet cell hyperplasia in the nasal mucosa of rats of both sexes exposed to 700 ppm. However, the increased incidence in D4-exposed Subgroup C was small, indicating that goblet cell hyperplasia was related to D4 exposure and the exposed tissues returned to near control levels when exposure was stopped for 12 months. Inhalation exposure to 700 ppm for 12 months induced minimal hyperplasia of squamous epithelium in the nasal vestibule of exposed rats of both sexes. This lesion was present in much lower incidence in D4-exposed groups necropsied after 24 months of exposure, suggesting that this minimal lesion returns to normal with continued exposure. A viable alternative explanation is that the subtle increase in thickness of squamous epithelium in the nasal vestibule induced by exposure to 700 ppm is not discernible in rats necropsied after 24 months of exposure because of increases in and variability of thickness of the nasal squamous epithelium related to aging. The increase in minimal or mild suppurative rhinitis observed in Subgroup B exposed to 700 ppm for 12 months is statistically significant in males. Statistical analysis of the incidence of suppurative rhinitis in both sexes of D4-exposed groups indicated a positive trend related to D4 exposure. Incidence of suppurative rhinitis at 24 months (Subgroup D) was much lower, with statistical significance only in females and no evidence of a dose-response relationship. Incidence was also lower in recovery Subgroup C, with no statistical differences. Therefore suppurative rhinitis is considered to be related to exposure to 700 ppm for 12 months, but not related to exposure for 24 months or in recovery Subgroup C. Despite statistical indications of differences in incidence, eosinophilic globules in the nasal epithelium are not considered to be related to D4 exposure for Subgroup C. The diagnosis of eosinophilic globules in Subgroup B males was clearly treatment-related, but was questionable in Subgroup B females. The incidence was also clearly treatment-related in Subgroup D males and females. The presence of a dose-related increase and statistical results indicating a significant increase in eosinophilic globules in both sexes of rats exposed to 700 ppm and females exposed to 150 ppm for 24 months provide good evidence that the eosinophilic globules in these groups are related to D4 exposure for 24 months (Subgroup D). There was a slightly increased and statistically significant incidence of minimal to mild chronic inflammation in the subpleural alveoli of female rats exposed to 700 ppm for up to 24 months. This increase was not present in similarly exposed male rats. The low incidence and the fact that this lesion is frequently observed in unexposed aged Fischer rats leads to the conclusion that it is not related to D4 exposure. Several primary or metastatic tumours were present in the thoracic cavity of control and D4-exposed rats, with no evidence of an effect of exposure. The primary mesothelioma observed in the thoracic cavity of a D4-exposed rat is interpreted as an atriocaval mesothelioma unrelated to D4 exposure.

Exposure to D4 for up to 24 months appeared to increase the severity of chronic nephropathy in Subgroup D of both sexes exposed to 700 ppm, compared to the concurrent controls. Statistical analysis supported a significant increase in both sexes at 700 ppm. Although statistical analysis indicated significant increases compared to controls in recovery Subgroup C males exposed to 700, 150, or 30 ppm and females exposed to 150 ppm, these differences are not considered toxicologically significant because of the relatively small difference in the mean severity of nephropathy compared to the controls, and lack of evidence of a dose-response relationship. Thus the only treatment-related renal effect appeared to be the increases in severity of chronic nephropathy seen in both sexes in response to 24 months of exposure to 700 ppm.

Centrilobular hypertrophy of hepatocytes observed in males exposed to 700 ppm for 12 or 24 months is considered to be related to D4 exposure. Statistical analysis of the data confirms the significance of these differences. Hypertrophy of hepatocytes due to induction of cytochrome P450 enzymes has been well characterized as an adaptive response to D4 exposure.

The increased incidence of hematopoietic proliferation in spleens from females exposed to 700 ppm for 24 months was confirmed by statistical analysis. This lesion is considered to be secondary to haemorrhage from proliferative lesions in this group rather than a direct effect of exposure to D4. The increase in hematopoietic proliferation in males exposed to 30 ppm, although statistically different, is not considered to be related to D4 exposure.

There were microscopic lesions in other tissues in which the incidence and/or severity was increased or decreased in exposed groups versus controls of the same sex, and statistical analysis indicated significant differences in incidence/severity. These differences were reflective of random variation background lesions in aging rats and not considered to be of toxicologic significance.
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.
Remarks on result:
other: Based on a molecular weight of 296.62, 150 ppm is equivalent to 1820 mg/m3.
Dose descriptor:
NOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEC for local respiratory effects based on findings in nasal cavity at 700 ppm
Remarks on result:
other: Based on a molecular weight of 296.62, 150 ppm is equivalent to 1820 mg/m3.
Critical effects observed:
not specified
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 two-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 NOAECs for carcinogenic effects were 150 and ≥700 ppm in females and males, respectively. The NOAEC for general toxicity was 150 ppm, based on chronic nephropathy. The NOAEC for local respiratory effects was also 150 ppm based on findings in the nasal cavity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 820 mg/m³
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.01.1999 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
Guideline:
other: EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
- 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: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: NA
Details on inhalation 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 the exposure 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
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: 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: Neoplastic findings are reported in Section 7.7. Non-neoplastic findings were as follows: The primary target organs affected by D4 exposure included: respiratory tract, kidney, and liver.

Inhalation exposure to D4 for 12 or 24 months increased the incidence of goblet cell hyperplasia in the nasal mucosa of rats of both sexes exposed to 700 ppm. However, the increased incidence in D4-exposed Subgroup C was small, indicating that goblet cell hyperplasia was related to D4 exposure and the exposed tissues returned to near control levels when exposure was stopped for 12 months. Inhalation exposure to 700 ppm for 12 months induced minimal hyperplasia of squamous epithelium in the nasal vestibule of exposed rats of both sexes. This lesion was present in much lower incidence in D4-exposed groups necropsied after 24 months of exposure, suggesting that this minimal lesion returns to normal with continued exposure. A viable alternative explanation is that the subtle increase in thickness of squamous epithelium in the nasal vestibule induced by exposure to 700 ppm is not discernible in rats necropsied after 24 months of exposure because of increases in and variability of thickness of the nasal squamous epithelium related to aging. The increase in minimal or mild suppurative rhinitis observed in Subgroup B exposed to 700 ppm for 12 months is statistically significant in males. Statistical analysis of the incidence of suppurative rhinitis in both sexes of D4-exposed groups indicated a positive trend related to D4 exposure. Incidence of suppurative rhinitis at 24 months (Subgroup D) was much lower, with statistical significance only in females and no evidence of a dose-response relationship. Incidence was also lower in recovery Subgroup C, with no statistical differences. Therefore suppurative rhinitis is considered to be related to exposure to 700 ppm for 12 months, but not related to exposure for 24 months or in recovery Subgroup C. Despite statistical indications of differences in incidence, eosinophilic globules in the nasal epithelium are not considered to be related to D4 exposure for Subgroup C. The diagnosis of eosinophilic globules in Subgroup B males was clearly treatment-related, but was questionable in Subgroup B females. The incidence was also clearly treatment-related in Subgroup D males and females. The presence of a dose-related increase and statistical results indicating a significant increase in eosinophilic globules in both sexes of rats exposed to 700 ppm and females exposed to 150 ppm for 24 months provide good evidence that the eosinophilic globules in these groups are related to D4 exposure for 24 months (Subgroup D). There was a slightly increased and statistically significant incidence of minimal to mild chronic inflammation in the subpleural alveoli of female rats exposed to 700 ppm for up to 24 months. This increase was not present in similarly exposed male rats. The low incidence and the fact that this lesion is frequently observed in unexposed aged Fischer rats leads to the conclusion that it is not related to D4 exposure. Several primary or metastatic tumours were present in the thoracic cavity of control and D4-exposed rats, with no evidence of an effect of exposure. The primary mesothelioma observed in the thoracic cavity of a D4-exposed rat is interpreted as an atriocaval mesothelioma unrelated to D4 exposure.

Exposure to D4 for up to 24 months appeared to increase the severity of chronic nephropathy in Subgroup D of both sexes exposed to 700 ppm, compared to the concurrent controls. Statistical analysis supported a significant increase in both sexes at 700 ppm. Although statistical analysis indicated significant increases compared to controls in recovery Subgroup C males exposed to 700, 150, or 30 ppm and females exposed to 150 ppm, these differences are not considered toxicologically significant because of the relatively small difference in the mean severity of nephropathy compared to the controls, and lack of evidence of a dose-response relationship. Thus the only treatment-related renal effect appeared to be the increases in severity of chronic nephropathy seen in both sexes in response to 24 months of exposure to 700 ppm.

Centrilobular hypertrophy of hepatocytes observed in males exposed to 700 ppm for 12 or 24 months is considered to be related to D4 exposure. Statistical analysis of the data confirms the significance of these differences. Hypertrophy of hepatocytes due to induction of cytochrome P450 enzymes has been well characterized as an adaptive response to D4 exposure.

The increased incidence of hematopoietic proliferation in spleens from females exposed to 700 ppm for 24 months was confirmed by statistical analysis. This lesion is considered to be secondary to haemorrhage from proliferative lesions in this group rather than a direct effect of exposure to D4. The increase in hematopoietic proliferation in males exposed to 30 ppm, although statistically different, is not considered to be related to D4 exposure.

There were microscopic lesions in other tissues in which the incidence and/or severity was increased or decreased in exposed groups versus controls of the same sex, and statistical analysis indicated significant differences in incidence/severity. These differences were reflective of random variation background lesions in aging rats and not considered to be of toxicologic significance.
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.
Remarks on result:
other: Based on a molecular weight of 296.62, 150 ppm is equivalent to 1820 mg/m3.
Dose descriptor:
NOAEC
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEC for local respiratory effects based on findings in nasal cavity at 700 ppm
Remarks on result:
other: Based on a molecular weight of 296.62, 150 ppm is equivalent to 1820 mg/m3.
Critical effects observed:
not specified
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 two-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 NOAECs for carcinogenic effects were 150 and ≥700 ppm in females and males, respectively. The NOAEC for general toxicity was 150 ppm, based on chronic nephropathy. The NOAEC for local respiratory effects was also 150 ppm based on findings in the nasal cavity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 820 mg/m³
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October to November 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that the report was in German, and a proper translation could not be performed, therefore some details are not presented.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Züchter Hacking Churchill, Huntingdon, England.
- Age at study initiation: No data
- Weight at study initiation: Males: 3.2 kg; Females: 3.1 kg.
- Fasting period before study: No data
- Housing: Conventional cages
- Diet (e.g. ad libitum):Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2
- Humidity (%): approximately 50%
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 15th October 1984 To: November 1984
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Flanks (shaven intact skin)
- % coverage: No data
- Type of wrap if used: No wrap, open exposure
- Time intervals for shavings or clippings: as required


REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with water
- Time after start of exposure: six hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1, 0.3 and 1.0 ml/kg bw/day.
- Constant volume or concentration used: various volumes of neat test substance applied.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
5 days/week
Dose / conc.:
96 mg/kg bw (total dose)
Remarks:
0.1 ml/kg bw
Dose / conc.:
190 mg/kg bw (total dose)
Remarks:
0.3 ml/kg bw
Dose / conc.:
960 mg/kg bw (total dose)
Remarks:
1 ml/kg bw
No. of animals per sex per dose:
Five
Control animals:
yes
Details on study design:
- Dose selection rationale: No data (might have been missed in translation)
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: To investigate the reversibility of effects.
- Post-exposure recovery period in satellite groups: Two weeks for highest dose group.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on week days, and once daily at weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily, and scored according to the Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: Needs translation

FOOD CONSUMPTION:
- Food consumption was measured once per week, by calculating the amount of food not eaten by the animals. Food consumption per seven days was calculated for each week.


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: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, and at the end of the observation period.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals (except only 4 and 3 males in 0.1 and 0.3 ml/kg groups, respectively)
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, and at the end of the observation period.
- Animals fasted: No
- How many animals: All animals (except only 4 and 3 males in 0.1 and 0.3 ml/kg groups, respectively)
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period and observation period (16 hour collection period).
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table 1 were examined.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
None reported.
Statistics:
NEEDS TRANSLATION
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
There were no treatment-related effects.
Dose descriptor:
NOAEL
Effect level:
>= 1 other: ml/kg bw
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: NOAEL was ≥ 1 ml/kg bw (equivalent to 960 mg/kg bw/day).
Critical effects observed:
no
Conclusions:
In a three-week dermal exposure study in rabbits conducted using a protocol similar to OECD 410 and GLP (reliability score 2), there were no adverse effects and therefore the dermal NOAEL was ≥ 1 ml/kg bw/day (equivalent to 960 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
960 mg/kg bw/day
Study duration:
subacute
Species:
rabbit

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October to November 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that the report was in German, and a proper translation could not be performed, therefore some details are not presented.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Züchter Hacking Churchill, Huntingdon, England.
- Age at study initiation: No data
- Weight at study initiation: Males: 3.2 kg; Females: 3.1 kg.
- Fasting period before study: No data
- Housing: Conventional cages
- Diet (e.g. ad libitum):Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2
- Humidity (%): approximately 50%
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 15th October 1984 To: November 1984
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Flanks (shaven intact skin)
- % coverage: No data
- Type of wrap if used: No wrap, open exposure
- Time intervals for shavings or clippings: as required


REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with water
- Time after start of exposure: six hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1, 0.3 and 1.0 ml/kg bw/day.
- Constant volume or concentration used: various volumes of neat test substance applied.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
5 days/week
Dose / conc.:
96 mg/kg bw (total dose)
Remarks:
0.1 ml/kg bw
Dose / conc.:
190 mg/kg bw (total dose)
Remarks:
0.3 ml/kg bw
Dose / conc.:
960 mg/kg bw (total dose)
Remarks:
1 ml/kg bw
No. of animals per sex per dose:
Five
Control animals:
yes
Details on study design:
- Dose selection rationale: No data (might have been missed in translation)
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: To investigate the reversibility of effects.
- Post-exposure recovery period in satellite groups: Two weeks for highest dose group.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on week days, and once daily at weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily, and scored according to the Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: Needs translation

FOOD CONSUMPTION:
- Food consumption was measured once per week, by calculating the amount of food not eaten by the animals. Food consumption per seven days was calculated for each week.


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: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, and at the end of the observation period.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals (except only 4 and 3 males in 0.1 and 0.3 ml/kg groups, respectively)
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, and at the end of the observation period.
- Animals fasted: No
- How many animals: All animals (except only 4 and 3 males in 0.1 and 0.3 ml/kg groups, respectively)
- Parameters checked in table 1 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the exposure period and observation period (16 hour collection period).
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table 1 were examined.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
None reported.
Statistics:
NEEDS TRANSLATION
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
There were no treatment-related effects.
Dose descriptor:
NOAEL
Effect level:
>= 1 other: ml/kg bw
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: NOAEL was ≥ 1 ml/kg bw (equivalent to 960 mg/kg bw/day).
Critical effects observed:
no
Conclusions:
In a three-week dermal exposure study in rabbits conducted using a protocol similar to OECD 410 and GLP (reliability score 2), there were no adverse effects and therefore the dermal NOAEL was ≥ 1 ml/kg bw/day (equivalent to 960 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rabbit

Additional information

No guideline-compliant repeated dose toxicity studies for the oral route are available for D4 although a number of supporting studies exist. Reliable key studies are available for the inhalation and dermal routes.

In the key dermal repeated dose toxicity study (Bayer AG, 1988) D4 was applied to the intact skin of 5 New Zealand white rabbits under open conditions for 3 weeks (5 days/week). The animals were dosed at 0.1, 0.3, 1 ml/kg bw (equivalent to 96, 190, 960 mg/kg bw based on relative density of 0.96). After each daily 6 hour application the site was washed with water. The animals of the high dose group were then observed for 2 weeks. All animals were observed for mortality and signs of toxicity, and their body weight and food consumption recorded. Haematology, clinical chemistry and urinalysis were conducted in all high dose animals and 4 and 3 animals from the low and mid dose groups, respectively. All animals of the control and high dose groups were examined macroscopically and microscopically. There were no adverse systemic or local findings and therefore the NOAEL was ≥ 1 ml/kg bw (equivalent to 960 mg/kg bw/day).

In the key repeated dose inhalation study (Batelle, 2004) Fischer 344 rats were exposed to D4 vapour 6 hours/day, 5 days/week at concentrations of 10, 30, 150, and 700 ppm for up to 24 months. The animals were divided into 4 groups: Subgroup A was scheduled for necropsy after 6 months of exposure (Tissue Level Study; 6 animals/sex/dose); Subgroup B was scheduled for necropsy after 12 months of exposure (Chronic Toxicity Study; 10 animals/sex/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; 20 animals/sex/dose); Subgroup D was scheduled for necropsy after 24 months of exposure (Oncogenicity Study; 60 animals/sex/dose). Exposure to D4 for up to 24 months induced the following notable effects in male and female rats:

·            Reduced two-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 NOAECs for endometrial adenomas and endometrial epithelial hyperplasia were 150 in females (see Section 5.8.3 for carcinogenicity discussion). The NOAEC for general toxicity was 150 ppm, based on chronic nephropathy. The NOAEC for local respiratory effects was also 150 ppm based on findings in the nasal cavity.

In a supporting 90-day repeated dose inhalation toxicity study (RCC Group, 1995a) in Fischer 344 rats, which was conducted using a protocol similar to OECD 413 and to GLP, four females at 883 ppm died. A reversible increase in liver weight occurred at 480 and 883 ppm, and also at 883 ppm, a reversible change in the ovary (hypoactivity) and vagina (mucification) were noted. Evidence of a response to a mild irritant was seen with slight goblet cell proliferation in the nasal cavity in high dose rats and minimal to slight degrees of alveolar macrophage foci and chronic interstitial inflammation of the lung in all treated animals. Following the recovery period these changes showed some resolution. In two further studies Sprague Dawley rats received D4 vapour at 5, 10 or 300 ppm (International Research Development Corporation, 1991) or at 50, 300 or 700 ppm (Dow Corning Corporation, 1989a). In both studies, liver weight increases at 300 ppm and above were almost completely reversible and there were no histopathological correlates. These liver effects were the only findings of relevance; however, they are considered to be adaptive rather than of a toxicological nature. There were no changes noted in ovaries or vagina.

The inhalation repeated-dose studies indicate that the Fischer 344 rat is more sensitive than the Sprague Dawley rat to respiratory and female genital tract changes. An overall NOAEC was not defined in the study in Fischer 344 rats as minimal chronic interstitial inflammation of the lung changes were detected even at the lowest dose level of 34 ppm. However, as this was the only respiratory tract change at this concentration, it is considered a local effect and as it was noted in seven animals compared to two in the controls, the NOAEC for systemic effects can be comfortably set at a higher exposure concentration. The NOAECs in the 90-day studies in Sprague Dawley rats were defined as ≥700 ppm in one study (Dow Corning Corporation, 1989a) and ≥300 ppm in another study (International Research Development Corporation, 1991). These NOAECs are concluded based on the assumption that the hepatic changes are adaptive in nature.


Based on a molecular weight of 296.62 of D4, 150 ppm is equivalent to 1820 mg/m3.

There are studies that support the conclusion that the liver effects following administration of D4 are adaptive rather than toxic. Since these studies are mechanistic rather than standard animal studies, they are included in Section 5.10.1.3 (Specific Investigations).

Justification for classification or non-classification

Based on the available data D4 does not require classification for repeated dose toxicity according to Regulation (EC) No. 1272/2008.