Registration Dossier

Administrative data

Description of key information

In a chronic oral study with rats, the LOAEL was observed at 12 mg/kg bw/day, i.e. at the lowest dose applied (Lilja et al. 1986).

There was no systemic or local toxicity resulted from inhalation to the air saturated vapors of diglycidyl resorcinol in a subchronic study with rats (Hine et al. 1958).

In a chronic dermal study with mice, the LOAEL was observed at 4.8 mg/kg bw/day, i.e. at the mid-dose applied (Holland et al. 1981).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Groups of 10 rats of each sex were administered DGRE in corn oil by gavage, 5 days per week for 13 weeks, at doses of O, 12.5, 25, 50,100, or 200 mg/ kg body weight.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Ciba-Geigy Corporation (Ardsley, NY), Araldite ERE 1359 in a single lot (No. P-60002)
- Purity test date: 81.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the dark at 23 °C in its original container
- Stability under test conditions: stable

FORM AS APPLIED IN THE TEST: suspended in corn oil (dissolved in acetone before being added to corn oil)
Species:
rat
Strain:
Fischer 344
Remarks:
F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: NA
- Fasting period before study: NA
- Housing: five animals/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
Feed: Wayne Laboratory Blox (r), Allied Mills (Chicago, IL)
Water: Edstrom automatic watering system, Edstrom Industries (Waterford, WI)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-32
- Humidity (%): 20-81
- Air changes (per hr): 12 changes of room air per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Corn oil was selected for the gavage vehicle and was analyzed monthly for peroxides.
A quantity of DGRE was melted by warming the chemical in a 40°C water bath and a 2.5 g portion of the clear liquefied chemical was transferred to a 200 mL centrifuge bottle and mixed with 44.2g of corn oil. No acetone was used. The mixture was homogenized using a Brinkman Polytron(R) homogenizer set at low speed for 1 minute. Air bubbles incorporated in the suspension during homogenization were removed by drawing a vacuum on the bottle with an aspirator while agitating the contents periodically for 2-3 minutes. The resulting mixture was visually homogeneous and appeared to remain stable for up to 2 hours. This combination of chemical and corn oil produced 50.0 mL of suspension containing DGRE at a concentration of 50.0 mg/mL.

Maximum storage time: 10 days
Storage conditions: 5 °C

- VEHICLE
- Concentration in vehicle: 4, 8.3, 16.7 mg/mL
- Amount of vehicle: 3 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
One-milliliter aliquots of the sample vials were extracted with 10 mL of methanol containing 0.7 g/mL of dibutyl phthalate as an internal standard. A reference calibration plot was prepared from spiked samples which were extracted in the same manner. The supernatant solutions were analyzed by VPC-FID at 210° on a 6 ft x 1/4 in x 2 mm ID glass column packed with SP2250 on 100/120 Supelcoport.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
first study
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
first study
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
first study
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 females and 10 males
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for signs of morbidity or mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: monthly

BODY WEIGHT: Yes
- Time schedule for examinations: every week for the first 12 weeks and monthly thereafter

HAEMATOLOGY: Not specified

CLINICAL CHEMISTRY: Not specified

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross lesions

HISTOPATHOLOGY: Yes
tissue masses, abnormal lymph nodes, skin, mandibular or mesenteric lymph nodes, mammary gland, salivary gland, sternebrae, bone marrow, thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, small intestine, colon, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate/testes or ovaries/uterus, brain and pituitary.
Statistics:
Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958).
Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) extensions of Cox’s methods for testing for a dose-related trend. All reported P values for the survival analyses are two-sided.
For the statistical analysis of tumor incidence data, two different methods of adjusting for intercurrent mortality were employed. Each used the classical method for combining contingency tables developed by Mantel and Haenszel (1959). Tests of significance included pairwise comparisons of high and low dose groups with controls and tests for overall dose-response trends.


Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male from the 200 mg/kg dosing group died due to undetermined circumstances.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight was depressed 10% or more in males that received 100 mg/ kg and in males and females that received 200 mg/ kg (Table 4).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Compound-related lesions were observed in the forestomach (squamous cell papilloma, hyperkeratosis, and basal cell hyperplasia) and in the liver (minimal to mild centrilobular fatty metamorphosis). Chronic inflammation in the mesenteric lymph nodes was probably secondary to the inflammation or ulceration of the forestomach. Compared with the controls, the three male rats with fatty metamorphosis in the liver had decreased final body weights. However, lower mean body weight gains were also found in other male and female rats administered 200 mg/kg which did not show hepatic fatty metamorphosis.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, the wall of the forestomach of rats was sometimes thickened and the mucosal surface contained small, white papillomatous nodules. When examined microscopically, some nodules were squamous papillomata, having localized acanthosis and papillary projections of the epidermis covered by thick layers of keratinized cells. The basal layer of the epithelium was hyperplastic, sometimes showing finger-like projections into the submucosa. Diffuse hyper keratosis, focal basal cell hyperplasia, or both were usually present in the forestomach of rats without discrete squamous papillomata. In some rats that received 200 mg/ kg, ulceration in the forestomach had completely eroded the epithelium and extended into the muscularis. A few rats without ulcers had circumscribed areas of inflammation in the stomach.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
12.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
12.5 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Under the conditions of the study, the LOAEL may be assigned to 12.5 mg/kg bw/day dose. It is however not excluded, that the real LOAEL value may be lower as under given testing conditions. On the other hand, the chronic toxicity of DGRE via oral exposure is not relevant to humans. There was also an opinion, that the observed forestomach tumors were likely to have resulted from an indirect or local toxic effect of DGRE. Therefore by assessing the substance hazard for humans it is essential to identify the effects of toxicity from the other than oral exposure routes.
Executive summary:

Administration of DGRE to rats and mice caused marked toxicity at the sites of direct con- tact (i.e., the esophagus and stomach). In most cases, the stomach lesions seen in animals dying in the 13-week studies were not severe enough to produce death. The presence of macroscopic lesions in the kidney of rats suggests absorption of DGRE, but the exact cause of death was not apparent.

The LOAEL was set at 12.5 mg/kg

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
12 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
For repeated dose toxicity one 2-year oral (gavage) study in rats was available. It is no guideline and non-GLP study, however, well described and peer reviewed by the National Toxicology Program Board of Scientific Counselors' Technical Reports Review Subcommittee and associated Panel Experts. Therefore, the study was sufficient to assign the reliablity of K2. In the same report there was the study conducted with the test item in mice. The effects observed in mice after 2-year oral (gavage) exposure supported the observations in rats, however they were less toxic. Therefore, rat study was selected for the assessment showing the data of much sensitive species.
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was published in 1958
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The acute toxicity of this compound was assessed as part of a larger study on the toxicity of epoxy resins
Qualifier:
no guideline available
Principles of method if other than guideline:
Groups of rats were exposed for seven hours, five days a week for a total of 50 exposures, with air saturated with diglycidyl resorcinol.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Diglycidyl resorcinol; no other details reported
Species:
rat
Strain:
Long-Evans
Details on species / strain selection:
Male rats: 80 - 104 g body weight. No other details
Sex:
male
Details on test animals and environmental conditions:
No details specified
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Remarks on MMAD:
No details reported
Details on inhalation exposure:
Groups of 10 male rats (80-104g) were exposed five days a week for seven hours, for a total of 50 exposures, to air saturated with the vapor of diglycidyl resorcinol. Similalrly one group of 10 control rats was exposed to uncontaminated air. The animals were exposed in cylindrical steel chambers of 210-litres capacity at 20±1”C.
Air was substantially saturated with DGR by passage through one fritted glass bubbler following warming in a heating jacket to reduce the viscosity. The air flow was held constant at 15 liters per minute. A preliminary saturation period of about an hour insured greater than 95% saturation of the air prior to exposure of the animals. No analytical check was made of the concentration, since it was too low to permit accuracy of the determination.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Five days a week for seven hours, for a total of 50 exposures
Frequency of treatment:
daily
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 10 male rats (80-104g) were exposed five days a week for seven hours, for a total of 50 exposures, to air saturated with the vapor of diglycidyl resorcinol. Similarly one group of 10 control rats was exposed to uncontaminated air. The animals were exposed in cylindrical steel chambers of 210-litres capacity at 20±1”C.
Air was substantially saturated with DGR by passage through one fritted glass bubbler following warming in a heating jacket to reduce the viscosity. The air flow was held constant at 15 liters per minute. A preliminary saturation period of about an hour insured greater than 95% saturation of the air prior to exposure of the animals. No analytical check was made of the concentration, since it was too low to permit accuracy of the determination.

The rats were observed for signs of intoxication during and after each exposure. Individual weights were recorded, and mean weight changes were graphed in the form of growth curves. At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Positive control:
no
Observations and examinations performed and frequency:
The rats were observed for signs of intoxication during and after each exposure. Individual weights were recorded, and mean weight changes were graphed in the form of growth curves. At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Sacrifice and pathology:
At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The series of 50 exposures to the saturated vapours of DGR was free from untoward effects. Aside form a very slight incrustation of the eye-lids of some animals, with red-brown exudate, none of the rats showed any signs of toxicity or irritation attributable to the exposure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rat in the treated group and two in the control group died between the third and fourth weeks. At necropsy these animals were found to have bronchial pneumonia
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Analysis of variance showed no significant difference in mean weight gains or in organ/body weight ratios (p= <0.05).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Analysis of variance showed no significant difference in mean weight gains or in organ/body weight ratios (p= <0.05).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant gross or macroscopic lesions were found in the surviving animals
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Conclusions:
No systemic toxicity has resulted from inhalation to the air saturated vapors of diglycidyl resorcinol.
Executive summary:

The inhalation toxicity of diglycidyl resorciol was examined as part of a study into the toxicity of epoxy resins. Although well described the published data lacks any detailed analysis of the results. There was no analytical data to support the study or to allow effect levels to be determined.

The overall conclusion of the inhalation toxicity described in this study was that no systemic toxicity resulting from the inhalation to the air saturated vapours of diglycidyl resorcinol was seen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was published in 1958
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The acute toxicity of this compound was assessed as part of a larger study on the toxicity of epoxy resins
Qualifier:
no guideline available
Principles of method if other than guideline:
Groups of rats were exposed for seven hours, five days a week for a total of 50 exposures, with air saturated with diglycidyl resorcinol.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Diglycidyl resorcinol; no other details reported
Species:
rat
Strain:
Long-Evans
Details on species / strain selection:
Male rats: 80 - 104 g body weight. No other details
Sex:
male
Details on test animals and environmental conditions:
No details specified
Route of administration:
inhalation: vapour
Type of inhalation exposure:
not specified
Vehicle:
air
Remarks on MMAD:
No details reported
Details on inhalation exposure:
Groups of 10 male rats (80-104g) were exposed five days a week for seven hours, for a total of 50 exposures, to air saturated with the vapor of diglycidyl resorcinol. Similalrly one group of 10 control rats was exposed to uncontaminated air. The animals were exposed in cylindrical steel chambers of 210-litres capacity at 20±1”C.
Air was substantially saturated with DGR by passage through one fritted glass bubbler following warming in a heating jacket to reduce the viscosity. The air flow was held constant at 15 liters per minute. A preliminary saturation period of about an hour insured greater than 95% saturation of the air prior to exposure of the animals. No analytical check was made of the concentration, since it was too low to permit accuracy of the determination.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Five days a week for seven hours, for a total of 50 exposures
Frequency of treatment:
daily
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 10 male rats (80-104g) were exposed five days a week for seven hours, for a total of 50 exposures, to air saturated with the vapor of diglycidyl resorcinol. Similarly one group of 10 control rats was exposed to uncontaminated air. The animals were exposed in cylindrical steel chambers of 210-litres capacity at 20±1”C.
Air was substantially saturated with DGR by passage through one fritted glass bubbler following warming in a heating jacket to reduce the viscosity. The air flow was held constant at 15 liters per minute. A preliminary saturation period of about an hour insured greater than 95% saturation of the air prior to exposure of the animals. No analytical check was made of the concentration, since it was too low to permit accuracy of the determination.

The rats were observed for signs of intoxication during and after each exposure. Individual weights were recorded, and mean weight changes were graphed in the form of growth curves. At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Positive control:
no
Observations and examinations performed and frequency:
The rats were observed for signs of intoxication during and after each exposure. Individual weights were recorded, and mean weight changes were graphed in the form of growth curves. At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Sacrifice and pathology:
At the end of the experimental period, the rats were decapitated under light ether anaesthesia, exanguinated, and examined for gross changes. Representative tissues were taken for histologic study, and the organ/body weight ratios were determined on liver, lungs, and kidneys for statistical analysis.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The series of 50 exposures to the saturated vapours of DGR was free from untoward effects. Aside form a very slight incrustation of the eye-lids of some animals, with red-brown exudate, none of the rats showed any signs of toxicity or irritation attributable to the exposure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rat in the treated group and two in the control group died between the third and fourth weeks. At necropsy these animals were found to have bronchial pneumonia
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Analysis of variance showed no significant difference in mean weight gains or in organ/body weight ratios (p= <0.05).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Analysis of variance showed no significant difference in mean weight gains or in organ/body weight ratios (p= <0.05).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant gross or macroscopic lesions were found in the surviving animals
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Conclusions:
No systemic toxicity has resulted from inhalation to the air saturated vapors of diglycidyl resorcinol.
Executive summary:

The inhalation toxicity of diglycidyl resorciol was examined as part of a study into the toxicity of epoxy resins. Although well described the published data lacks any detailed analysis of the results. There was no analytical data to support the study or to allow effect levels to be determined.

The overall conclusion of the inhalation toxicity described in this study was that no systemic toxicity resulting from the inhalation to the air saturated vapours of diglycidyl resorcinol was seen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
according to
Guideline:
other:
Version / remarks:
No Guideline available.
Deviations:
not applicable
Principles of method if other than guideline:
2 year chronic dermal study conducted in Mice.
Animals were treated three times weekly and observed for systemic toxicity.
Full details available under the methods and results section.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Ciba Geige
- Lot/batch No.of test material: P6602
- Purity test date: 88% (wt)
- Impurities:
total chlorine 1%
toluene 6000 ppm
epichlorohydrin 845 ppm
phenyl glycidyl ether 406 ppm
toluene glycidyl ether present
monochlorohydrin <10 ppm
several aromatic species containing oxygen were present

- Storage condition of test material: at 4°C
Species:
mouse
Strain:
C3H
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NA
- Age at study initiation: 10 weeks
- Weight at study initiation: NA
- Housing: in polycarbonate shoebox cages with hardwood chip bedding
- Diet: ad libitum Purina 5010-C
- Water: ad libitum
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: not specified
- % coverage: not specified
- Time intervals for shavings or clipplings: as required

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.45, 0.9 and 1.8 mg/week (or 0.15, 0.3 and 0.6 mg/day/animal)
- Concentration (if solution): 0.32, 0.63 and 1.25% (wt/vol)

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
Three days per week: on Monday, Wednesday and Friday
Dose / conc.:
0.45 other: mg/week
Remarks:
exposure: 3 days/week
Dose / conc.:
0.9 other: mg/week
Remarks:
exposure: 3 days/week
Dose / conc.:
1.25 other: mg/week
Remarks:
exposure: 3 days/week
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The corrosiveness of the test item limited the MTDD to 1.8 mg/week.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: not specified. Average body weight intervals: 6, 12 and 24 months at the highest dosage

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the experiment
- Anaesthetic used for blood collection: Yes (Metofane (R) of Pitman-Moore)
- Animals fasted: No data
- How many animals: 33
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the experiment
- Animals fasted: No data
- How many animals: 35
- Parameters checked in table 2 were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: yes, but results were not specified in the present report

HISTOPATHOLOGY: yes, but results were not specified in the present report
Statistics:
Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.
Clinical signs:
not specified
Dermal irritation:
no effects observed
Description (incidence and severity):
severe skin irritation above 1.25% (or 1.8 mg/day dose) in the dose finding study. However, at doses selected for the long-term study no skin irritation was observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
see Table 3 Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 4 Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control
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:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
The repeated test item application to the mouse skin during 24 months (3 exposures/week) confirmed the evidence that diglycidyl ether of resorcinol is a systemic toxin at levels that fail to induce either skin irritation or neoplasia.
Key result
Dose descriptor:
LOAEL
Effect level:
4.8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
not specified

Table 3. Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)

Sex

% Survival at 750 days (95% confidence limits)

Χ2for systemic mortalitya

1.8

F

17 (6-36)

66.61***

1.8

M

72 (44-88)

1.47

0.9

F

64 (43-81)

3.93*

0.9

M

72 (53-88)

0.76

0.45

F

84 (66-94)

0.22

0.45

M

84 (66-94)

0.18

Acetone control

F

82 (76-88)

-

Acetone control

M

83 (77-90)

-

*P<0.05, ***P<0.001

Table 4. Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)

Sex

Average body weight (SE) in g at

6 months

12 months

24 months

1.8

F

25.9 (0.3)

25.5 (0.2)

18 (1)*

1.8

M

31.3 (0.5)

30 (0.3)

22 (0.7)*

Acetone control

F

28.2 (0.5)

27.2 (0.3)

27.4 (0.3)

Acetone control

M

33.4 (0.5)

31.8 (0.5)

30 (0.6)

*Mean body weight significantly different form that of vehicle control; P<0.001

Conclusions:
Under the conditions tested, the test item showed for systemic toxicity the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g). For the local effects the test item fail to induce either skin irritation or neoplasia at the highest dose tested (1.8 mg/week or 0.257 mg/day or 6.27 µg/cm2, assuming 27 g bw of mouse).
Executive summary:

Inbred, male and female C3Hf/Bd mice were produced under pathogen-free barrier conditions and held under these conditions for the 24-month duration of the experiment. Mice were weaned at 3-4 weeks of age. At 10 weeks of age the hair was removed from the back with electric clippers and the animals were randomly assigned in groups of five to each treatment dose combination. Mice were housed in polycarbonate shoebox cages with hardwood chip bedding. Food (Purina 5010-C) and water were constantly available.

The dose levels for the test item was selected on the basis of a 2-week, five times weekly application of the test material dissolved in spectra-grade acetone (Matheson-Coleman-Bell). The highest dose for the 2-year exposure was one which could be tolerated without irreversible local skin toxicity or systemic toxicity as reflected by suppression of weight gain or mortality. When no significant local or'systemic toxicity was observed, the viscosity of the material determined the concentration which could be reproducibly applied to the animals. At the start of the experiment the test item was weighed into glass scintillation vials in an amount sufficient to yield the highest concentration after the addition of an appropriate volume of acetone. The vials were kept in the dark at 4°C until used.

The material was applied with a 50 µl micropipette on Monday, Wednesday and Friday, excluding holidays. Mice were reshaved as required. The time of neoplasm appearance was taken as the day on which a raised, circumscribed lesion appeared in the treated area of skin that persisted for the duration of the experiment or until death.

At intervals throughout the experiment body weight was determined, by cage group, at the highest concentration of the test item. Cumulative mortality was noted. Heparinized blood samples taken from a random sample of mice surviving the full 24 month exposure were submitted to the clinical laboratory of the ORNL Health Division. For each sample total red and white cell counts, hematocrit and hemoglobin were determined before the sample was centrifuged to recover the plasma. The plasma was subjected to analysis for total protein, albumin, glutamic-oxalacetic transaminase, alkaline phosphatase, urea nitrogen, glucose and triglycerides.  The viscera were examined in animals that either died or were killed at the end of the study. Lesions noted were recorded on a standard form for each animal and tissues were taken for histology only when the gross diagnosis was questionable.

Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.

Evaluation of chronic toxicity was based upon changes in body weight at the highest dose of the test material, changes in force of non-skin tumor mortality and blood hematologic and chemical alterations at the highest dose level of the test material, relative to the vehicle control.

Body weight was significantly (P < 0.001) suppressed at 24 months in male and female mice exposed to the test item. Mortality was summarized in the result table. Animals that were killed before 750 days were ignored in this calculation. Significant (P < 0.05) systemic mortality was noted at the higher doses, especially in female mice.

Toxic suppression of bone marrow and lymphatic tissues was evaluated by examining the cellular composition of peripheral blood in a randomly selected subset of animals which survived to the end of the experiment. Blood was collected by cardiac puncture, under Metofane(R) (Pitman-Moore) anesthesia, into a syringe which contained heparin to prevent coagulation. The samples were evaluated for total red cell count, total white cell count, hemoglobin and hematocrit by use of conventional clinical laboratory procedures. Treatment-related effects were evaluated by comparison of the data with that from acetone controls, while age- (or acetone-) related changes were evaluated by comparison of vehicle control with 10- to 12-week-old untreated mice (aging control). Only the highest dose of each material was evaluated for which body weight suppression and mortality provided evidence of systemic toxicity.

The data indicate an age- (or acetone-) related decrease in the number of circulating red cells and a proportionate decrease in hemoglobin and hematocrit in all treated animals.

Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.

Plasma total protein levels were increased as a function of age in both sexes, with occasionial high average levels noted in males. Alkaline phosphatase levels varied widely, both within and between groups. It appeared that female C3H mice had significantly higher alkaline phosphatase levels than males, irrespective of age. Glutamic-oxalacetic transaminase levels were relatively uniform across groups and between sexes, but the range of standard errors gives a clear indication of underlying individual heterogeneity. Glucose levels were little affected by sex or age. A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item. Triglyceride levels varied widely, with no clear indication of an effect of treatment or age, although levels were consistently higher in males than in females. Urea nitrogen levels were higher in males than females and also increased as a function of age. Treatment-related differences were not apparent, although standard errors again suggested considerable variation among individual animals. Taken together, these data amplify and confirm the evidence that the test material is a systemic toxin at dose levels that fail to-induce either skin irritation or neoplasia. Since clinical hematologic and chemical analyses were conducted on individual animals and each animal was also subjected to gross and microscopic evaluation, it eventually will be possible to correlate clinical findings with gross and microscopic pathologic changes.

Skin neoplasms were not induced by the test item at the dosage levels applied to C3H mice. The test item also attracts attention as a potential systemic toxicant at levels of exposure that do not induce local irritation. Previously published animal carcinogenicity studies of the test item reveal that it is capable of eliciting skin neoplasms in C57BL, but not in Swiss ICR mice following chronic skin exposure. This is consistent with the present data in which skin tumors were not induced in C3H mice, which have previously been shown to be less sensitive to chemical skin carcinogenesis than C57BL/6.

In opinion of the authors, the most significant occupational risk for the test item would be primary skin irritation and potential systemic toxicity associated with skin absorption. It is unlikely that chronic dermal exposures at concentrations above those used in the present experiment would be unnoticed by workers due to the irritant properties of this material.

In conclusion, under the conditions tested, the test item showed the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
4.8 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
For repeated dose toxicity one chronic dermal study in mouse was available. It is no guideline and non-GLP study, however, well documented to determine the chronic LOAEL in mouse. It is a key study, Klimish 2.
Organ:
blood

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
according to
Guideline:
other:
Version / remarks:
No Guideline available.
Deviations:
not applicable
Principles of method if other than guideline:
2 year chronic dermal study conducted in Mice.
Animals were treated three times weekly and observed for systemic toxicity.
Full details available under the methods and results section.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Ciba Geige
- Lot/batch No.of test material: P6602
- Purity test date: 88% (wt)
- Impurities:
total chlorine 1%
toluene 6000 ppm
epichlorohydrin 845 ppm
phenyl glycidyl ether 406 ppm
toluene glycidyl ether present
monochlorohydrin <10 ppm
several aromatic species containing oxygen were present

- Storage condition of test material: at 4°C
Species:
mouse
Strain:
C3H
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NA
- Age at study initiation: 10 weeks
- Weight at study initiation: NA
- Housing: in polycarbonate shoebox cages with hardwood chip bedding
- Diet: ad libitum Purina 5010-C
- Water: ad libitum
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: not specified
- % coverage: not specified
- Time intervals for shavings or clipplings: as required

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.45, 0.9 and 1.8 mg/week (or 0.15, 0.3 and 0.6 mg/day/animal)
- Concentration (if solution): 0.32, 0.63 and 1.25% (wt/vol)

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
Three days per week: on Monday, Wednesday and Friday
Dose / conc.:
0.45 other: mg/week
Remarks:
exposure: 3 days/week
Dose / conc.:
0.9 other: mg/week
Remarks:
exposure: 3 days/week
Dose / conc.:
1.25 other: mg/week
Remarks:
exposure: 3 days/week
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The corrosiveness of the test item limited the MTDD to 1.8 mg/week.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: not specified. Average body weight intervals: 6, 12 and 24 months at the highest dosage

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the experiment
- Anaesthetic used for blood collection: Yes (Metofane (R) of Pitman-Moore)
- Animals fasted: No data
- How many animals: 33
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the experiment
- Animals fasted: No data
- How many animals: 35
- Parameters checked in table 2 were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: yes, but results were not specified in the present report

HISTOPATHOLOGY: yes, but results were not specified in the present report
Statistics:
Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.
Clinical signs:
not specified
Dermal irritation:
no effects observed
Description (incidence and severity):
severe skin irritation above 1.25% (or 1.8 mg/day dose) in the dose finding study. However, at doses selected for the long-term study no skin irritation was observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
see Table 3 Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 4 Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control
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:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
The repeated test item application to the mouse skin during 24 months (3 exposures/week) confirmed the evidence that diglycidyl ether of resorcinol is a systemic toxin at levels that fail to induce either skin irritation or neoplasia.
Key result
Dose descriptor:
LOAEL
Effect level:
4.8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Critical effects observed:
not specified

Table 3. Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)

Sex

% Survival at 750 days (95% confidence limits)

Χ2for systemic mortalitya

1.8

F

17 (6-36)

66.61***

1.8

M

72 (44-88)

1.47

0.9

F

64 (43-81)

3.93*

0.9

M

72 (53-88)

0.76

0.45

F

84 (66-94)

0.22

0.45

M

84 (66-94)

0.18

Acetone control

F

82 (76-88)

-

Acetone control

M

83 (77-90)

-

*P<0.05, ***P<0.001

Table 4. Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)

Sex

Average body weight (SE) in g at

6 months

12 months

24 months

1.8

F

25.9 (0.3)

25.5 (0.2)

18 (1)*

1.8

M

31.3 (0.5)

30 (0.3)

22 (0.7)*

Acetone control

F

28.2 (0.5)

27.2 (0.3)

27.4 (0.3)

Acetone control

M

33.4 (0.5)

31.8 (0.5)

30 (0.6)

*Mean body weight significantly different form that of vehicle control; P<0.001

Conclusions:
Under the conditions tested, the test item showed for systemic toxicity the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g). For the local effects the test item fail to induce either skin irritation or neoplasia at the highest dose tested (1.8 mg/week or 0.257 mg/day or 6.27 µg/cm2, assuming 27 g bw of mouse).
Executive summary:

Inbred, male and female C3Hf/Bd mice were produced under pathogen-free barrier conditions and held under these conditions for the 24-month duration of the experiment. Mice were weaned at 3-4 weeks of age. At 10 weeks of age the hair was removed from the back with electric clippers and the animals were randomly assigned in groups of five to each treatment dose combination. Mice were housed in polycarbonate shoebox cages with hardwood chip bedding. Food (Purina 5010-C) and water were constantly available.

The dose levels for the test item was selected on the basis of a 2-week, five times weekly application of the test material dissolved in spectra-grade acetone (Matheson-Coleman-Bell). The highest dose for the 2-year exposure was one which could be tolerated without irreversible local skin toxicity or systemic toxicity as reflected by suppression of weight gain or mortality. When no significant local or'systemic toxicity was observed, the viscosity of the material determined the concentration which could be reproducibly applied to the animals. At the start of the experiment the test item was weighed into glass scintillation vials in an amount sufficient to yield the highest concentration after the addition of an appropriate volume of acetone. The vials were kept in the dark at 4°C until used.

The material was applied with a 50 µl micropipette on Monday, Wednesday and Friday, excluding holidays. Mice were reshaved as required. The time of neoplasm appearance was taken as the day on which a raised, circumscribed lesion appeared in the treated area of skin that persisted for the duration of the experiment or until death.

At intervals throughout the experiment body weight was determined, by cage group, at the highest concentration of the test item. Cumulative mortality was noted. Heparinized blood samples taken from a random sample of mice surviving the full 24 month exposure were submitted to the clinical laboratory of the ORNL Health Division. For each sample total red and white cell counts, hematocrit and hemoglobin were determined before the sample was centrifuged to recover the plasma. The plasma was subjected to analysis for total protein, albumin, glutamic-oxalacetic transaminase, alkaline phosphatase, urea nitrogen, glucose and triglycerides.  The viscera were examined in animals that either died or were killed at the end of the study. Lesions noted were recorded on a standard form for each animal and tissues were taken for histology only when the gross diagnosis was questionable.

Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.

Evaluation of chronic toxicity was based upon changes in body weight at the highest dose of the test material, changes in force of non-skin tumor mortality and blood hematologic and chemical alterations at the highest dose level of the test material, relative to the vehicle control.

Body weight was significantly (P < 0.001) suppressed at 24 months in male and female mice exposed to the test item. Mortality was summarized in the result table. Animals that were killed before 750 days were ignored in this calculation. Significant (P < 0.05) systemic mortality was noted at the higher doses, especially in female mice.

Toxic suppression of bone marrow and lymphatic tissues was evaluated by examining the cellular composition of peripheral blood in a randomly selected subset of animals which survived to the end of the experiment. Blood was collected by cardiac puncture, under Metofane(R) (Pitman-Moore) anesthesia, into a syringe which contained heparin to prevent coagulation. The samples were evaluated for total red cell count, total white cell count, hemoglobin and hematocrit by use of conventional clinical laboratory procedures. Treatment-related effects were evaluated by comparison of the data with that from acetone controls, while age- (or acetone-) related changes were evaluated by comparison of vehicle control with 10- to 12-week-old untreated mice (aging control). Only the highest dose of each material was evaluated for which body weight suppression and mortality provided evidence of systemic toxicity.

The data indicate an age- (or acetone-) related decrease in the number of circulating red cells and a proportionate decrease in hemoglobin and hematocrit in all treated animals.

Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.

Plasma total protein levels were increased as a function of age in both sexes, with occasionial high average levels noted in males. Alkaline phosphatase levels varied widely, both within and between groups. It appeared that female C3H mice had significantly higher alkaline phosphatase levels than males, irrespective of age. Glutamic-oxalacetic transaminase levels were relatively uniform across groups and between sexes, but the range of standard errors gives a clear indication of underlying individual heterogeneity. Glucose levels were little affected by sex or age. A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item. Triglyceride levels varied widely, with no clear indication of an effect of treatment or age, although levels were consistently higher in males than in females. Urea nitrogen levels were higher in males than females and also increased as a function of age. Treatment-related differences were not apparent, although standard errors again suggested considerable variation among individual animals. Taken together, these data amplify and confirm the evidence that the test material is a systemic toxin at dose levels that fail to-induce either skin irritation or neoplasia. Since clinical hematologic and chemical analyses were conducted on individual animals and each animal was also subjected to gross and microscopic evaluation, it eventually will be possible to correlate clinical findings with gross and microscopic pathologic changes.

Skin neoplasms were not induced by the test item at the dosage levels applied to C3H mice. The test item also attracts attention as a potential systemic toxicant at levels of exposure that do not induce local irritation. Previously published animal carcinogenicity studies of the test item reveal that it is capable of eliciting skin neoplasms in C57BL, but not in Swiss ICR mice following chronic skin exposure. This is consistent with the present data in which skin tumors were not induced in C3H mice, which have previously been shown to be less sensitive to chemical skin carcinogenesis than C57BL/6.

In opinion of the authors, the most significant occupational risk for the test item would be primary skin irritation and potential systemic toxicity associated with skin absorption. It is unlikely that chronic dermal exposures at concentrations above those used in the present experiment would be unnoticed by workers due to the irritant properties of this material.

In conclusion, under the conditions tested, the test item showed the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
6.27 µg/cm²
Study duration:
chronic
Species:
mouse
Quality of whole database:
For repeated dose toxicity one chronic dermal study in mouse was available. It is no guideline and non-GLP study, however, well documented to determine the chronic NOAEL in mouse. It is a key study, Klimish 2.

Mode of Action Analysis / Human Relevance Framework

In the acute toxicity endpoint was shown that the test item is more toxic when administered intraperitoneally than when administered intragastrically (Hine et al. 1958). It seems that kinetics and metabolism via oral administration makes the test item less toxic comparing to the demal exposure. As result, the chronic oral LOAEL was 12 mg/kg bw/day in rats (Lilja et al. 1986), whereas the chronic dermal LOAEL was 4.8 mg/kg bw/day in mice for the systemic effects (Holland et al. 1981).

The calculated NOAEL at 6.27 µg/cm2 was identified for the chronic dermal local effects of the test item in mouse (Holland et al. 1981). As this effect is also concentration depending, the acute necrosis with sloughing was observed at concentrations greater than 10% after one or two applications or severe skin irritation when applying the test item above 1.25% (calculated 43 µg/cm2) in the repeated 2-week dose finding study in mouse. In another study (Hine et al. 1958), the test item produced severe dermal irritation and three out of the four rabbits died after seven applications, however, the test item concentration range was not indicated.

Based on the exposure relevance to humans, the LOAEL identified in chronic dermal study in mouse may be regarded as better acceptable for the risk assessment in humans.

Additional information

Justification for classification or non-classification

According to chronic (2-year) oral (gavage) study, the LOAEL of the substance was 12 mg/kg bw/day in rats. In the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, the standard guidance value refers to effects based on subchronic study conducted in rats. This interpolation towards shorter duration will however not change the category of classification. Therefore, the test item is suggested to be classified for Category 2 (organ: forestomach).

According to information from the subchronic inhalation study in rats, no adverse effect was observed, therefore no classification is needed.

According to chronic (2-year) dermal study, the systemic LOAEL of the substance was 4.8 mg/kg bw/day in mice. In the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, the standard guidance value refers to effects based on subchronic study conducted in rats. This interpolation towards shorter duration and allometric scaling to rats will however not change the category of classification. Therefore, the test item is suggested to be classified for Category 1 (organ: blood).