Registration Dossier

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Administrative data

Description of key information

Repeated dose toxicity
Dermal NOAEL 20 mg/kg in rats (OECD TG 410)
Inhalation NOAEC 19 mg/m3 in rats (OECD TG 412)
Inhalation BMCL10 36.20 mg/m3

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Conclusions:
A subchronic toxicity study via the oral route in rats is considered not scientifically justified for the following reasons.

The most relevant routes of exposure considering the uses of the test substance are via inhalation and dermal routes. As such conducting such a study would not result in further protection of human health.
Guideline and GLP compliant 28-day repeat dose toxicity studies via the inhalation and dermal routes are available for C10-C14 tert-alkyl amines indicating that the primary toxicity associated with the substance is irritation at the site of dosing. No indications of systemic toxicity were observed in either study in fact NOAELS derived from the study were based on local irritant effects either of the skin or respiratory tract.
In addition to the above information on repeat dose toxicity, a one-generation reproductive toxicity study exists for the substance in which animals were exposed orally via the diet for 10 weeks prior to mating, throughout gestation, lactation and until necropsy.
Four groups of 26 animals per sex per group were administered either 0, 19.1, 55.6, 107.3 mg/kg/day (males) or 0, 21, 62.8, 124.1 mg/kg/day (females). The highest dose was selected since, as noted in the report itself, method development work showed doses above such levels were not well tolerated for extended periods.
As part of the study, in-life observations were conducted including clinical signs, bodyweight and food consumption measurements. At necropsy, a gross pathological examination of all internal tissues was conducted, organ weights were taken and histopathological examination of all reproductive organs plus the pituitary, stomach and any abnormal tissues was conducted.
In the study no signs of systemic toxicity were observed, however significant body weight effects were noted such as reduced bodyweight gains of up to 82% as well as reduced food consumption of up to 23% at the highest dose tested (1500ppm). Such effects indicate issues of palatability with the test material most likely due to its irritancy and that higher doses would not be tolerated. No adverse clinical signs were noted and neither were any gross or histopathological changes.
As further support, In silico modelling information has been added to indicate that the oral absorption is greater than 97% and that the substance is significantly bioavailable via the oral route. Taken together with the results of the one generation reproductive study, these indicate that palatability, due to the corrosiveness of the test material would likely be dose-limiting and if systemic toxicity were to occur it would likely have been observed during the aforementioned reprodevelopmental toxicity study.
Therefore conducting a repeat dose toxicity study via the oral route would not be scientifically justified since;
a) the results would not alter the risk assessment of the substance as only inhalation and dermal routes of exposure are foreseen due to uses of the substance
b) the results would not provide further information concerning hazard of the substance since GLP and guideline compliant repeat dose studies via the dermal and inhalation routes indicate the primary hazards are concerned with local effects of irritation
c) repeat dose oral toxicity information albeit with limited investigations exists from a guideline compliant one generation reproductive toxicity study
d) further in silico information provided indicates significant systemic exposure during the one generation reproductive toxicity study
In conclusion, since it is not possible to expose animals to greater doses systemically it is unlikely that unforeseen hazards would be identified and there would be no adaptation of the current risk assessment since the oral route is not relevant and as such no further protection of human health. As a result, the conducting of such a study would be contrary to Directive 2010/63/EU of the European parliament concerning the protection of animals used for scientific purposes insofar as no discernible benefit to human health would be gained from conducting the study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: Palatability limitations result in reduced food consumption and associated bodyweight effects
Organ:
not specified

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental study period: 25-5-1993 to 22-06-1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test material identified as TAPA, TD 93-030, Lot 5-0027-93
Pale straw coloured liquid
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, New York, USA
- Age at study initiation: 21-23 days, at receipet
- Weight at study initiation: 50-75 g, at receipt
- Fasting period before study: not reported
- Housing: suspended wire mesh cage
- Diet (e.g. ad libitum): Purina certified rodent chow 5002; withheld during exposure
- Water (e.g. ad libitum): ad libitum; withheld during exposure
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.1 - 23.4 degrees C
- Humidity (%): 67.8 - 74.6%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark


IN-LIFE DATES: From: May 25, 1993 To: June 22, 1993
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable- vapor study
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1250 L stainless steel, glass and Plexiglass chambers
- Method of holding animals in test chamber: PVC nose-only restraining tubes
- Source and rate of air: Compressed air at 0.25 to 32 L/min
- Method of conditioning air: no data
- System of generating particulates/aerosols: study conducted with vapor, not aerosol
- Temperature, humidity, pressure in air chamber: 23.1-23.4 degrees C; 67.8 - 74.6%; pressure not reported
- Air flow rate: air flow rate adjutsted to 99% of the maximum vapor concentration
- Air change rate: no reported
- Method of particle size determination: study conducted with vapor, not aerosol
- Treatment of exhaust air: not reported


TEST ATMOSPHERE
- Brief description of analytical method used: an impinger located outside each chamber containing 8-12 mL of propylene glycol placed in an ice bath
- Samples taken from breathing zone: yes


VEHICLE (if applicable) no vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to determine the analytical concentration of the tetst material in the chambers, one impinger located outside wach chamber was used for sampling. The impingers contained 8-12 mL of propylene glycol and were placed in an ice bath. The amount of propylene glycol placed in the impingers was based on the amount of test material (mg) being collected over a 6-hour exposure. The air control chamber was sampled weekly to validate the lack of test material in the control atmosphere.
After collecting a sample with the impingers, the collecting solution was quantitatively transferred to pre-weighted 20 mL scintillation vials and weighed again to determine the total material present. The solution was then sent for gas chromatographic analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Remarks:
Doses / Concentrations:
2, 19, 129, 537 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
In addition, 2 males and 2 females were assigned to each group as health monitoring sentinels
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): computer randomization
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Signs of intoxication, mortality, morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes weekly


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: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) Nembutal
- Animals fasted: Yes ; overnight
- How many animals: all surviving animals
- Parameters checked
hematocrit
hemoglobin
red blood cell
white blood cell count
platelet count
mean cell corpuscular volume
mean cell corpuscular hemoglobin
mean corpuscular hemoglobin conc.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked
triglyceride
cholesterol
blood urea nitrogen
glucose
alkaline phosphatase
total protein
albumin
chloride
sodium
potassium
globulin
albumin:globulin ratio
creatinine
bilirubin (total)
calcium
inorganic phosphorous
glutamic pyruvic transaminase/alanine aminotransferase activity
glutamic oxaloacetic transaminase/asparate aminotransferase activity
gamma-glutamyl transferase activity



URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
- Dose groups that were examined: all animals prior to exposure; all surviving animals on the last day of exposure
- Battery of functions tested: sensory activity / grip strength / motor activity / other: convulsions, tremors, stereotypic or bizzarre behavior


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Macroscopic: Gross examination included examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY: Yes
- Microscopic: Tissues processed and examined microscopically will be collected, saved, weighed and fixed in 10% neutral buffered formalin. Tissues included but not limited to, the nasal cavity, liver, brain, kidney, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals, spleen, trachea, larynx, heart, and gross lesions.
Other examinations:
Neurologic evaluations were performed the day prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of an overall compound effect. Separate one-way analyses were used within the male and female data to assess overall treatment group effects. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals receiving 537 mg/m3 died by exposure Day 11, prior to death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation.
At 129 mg/m3 transient occurences of unstable gait, respiratory noise, salivation and lacrimation were observed.
Animals exposed at 19 mg/m3 showed a single incidence of lacrimation, this was judged not to be treatment related.
No treatment related signs were observed at 2 mg/m3. Red stained fur around eyes and muzzle and frequent struggling in the nose-only restraint tubes were observed throughout the study in all test material exposed groups. A single death in group 1 (Control) was attributable to over restraint in the nose only tube.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals receiving 537 mg/m3 died by exposure Day 11, prior to death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation.
A single death in group 1 (Control) was attributable to over restraint in the nose only tube.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant bodyweight decreases occured in the 537 mg/m3 males during week 1 and in females during Week 1 and 2. Males exposed to 129 mg/m3 showed statistically significant decreases in bodyweight and bodyweight change during Weeks 2, 3 and 4. The females exposed at 129 mg.m3 showed statistically significant decreases in bodyweight and bodyweight changes during Weeks 3 and 4. At 19 mg/m3 statistically significant decreases in bodyweight and bodyweight change were seen in females during Week 2. No other bodyweight effects were observed in this group.Females in the 2 mg/m3 exposure group showed statistically significant decreases in bodyweight and bodyweight change during Weeks 2, 3 and 4. No other bodyweight effects were observed in this group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in feed consumption for the 537 mg/m3 in both sexes was observed during Week 1. A statistically significant decrease i nthe feed consumption for the 129 mg/m3 males was observed during Weeks 2 and 3. No other feed consumption effects were seen i nany other group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals exposed at 19 mg/M3 showed statistically significant increase in platelets for the males and statistically significant decrease in mean cell hemoglobin and mean cell hemoglobin concentrations for the females. No changes in hematology parameters were seen at any other concentration. In the absence of a concentration-response relationship, none of the above changes were judged to be treatment-related.
No exposure related differences in white blood cell differential parameters were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decreases were observed in total protein and globulin in males at 129 mg/M3. At 19 mg/M3 statistically significant decreases were observed in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase in males and triglyceride in females. Males exposed at 2 mg/M3 showed a statistically significant decrease in creatinine and glutamic oxaloacetic transaminase; females in this group showed an increase in glutamic oxaloacetic transaminase. In the absence of a concentration-response relationship, none of the above changes were judged to be exposure-related.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight data showed a statistcally significant increase in the absolute lung weight of the 2 mg/m3 males. No other changes in organ weights were seen in any other group. In the absence of a lung weight effect at higher concentrations, this finding was judgen not to be treatment related.
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The nasal cavity, in particular both respiratory and olfactory epithelium, was the most sensitive target organ.

All animals exposed to 537 mg/M3 died during the study, and at the time of necropsy many animals were observed with distended stomachs.
Difficulty in flushing formalin through the nasopharyngeal duct was noted in many of these animals at the time of necropsy. No exposure-related gross observations were noted on surviving animals.

Microscopic examination of male and female rats exposed to 537 mg/M3 showed changes in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithial necrosis and inflammation of the submucosa. In addition, all meati and primarily the dorsal meati had exudate composed of mucous, fibrin, erythrocytes, exfoliated epithelium and inflammatory cells. The larynx and trachea had epithelial necrosis, submucosal inflammation and exudate in their lumens. Two female rats had foci of inflammation of bronchi, bronchioles and/or alveoli. Specific examination of tissues from the central (brain and spinal cord) and peripheral (sciatic nerve) nervous systems as well as skeletal muscle from animals exposed to 537 mg/M3 revealed no changes
indicative of neurotoxicity.

The nasal cavity was the only target tissue in animals exposed to 129 mg/M3 of the test material. The lesions were primary graded as slight and focal in nature. These lesions consisted of necrosis of respiratory and olfactory epithelium accompanied by inflammation in the mucosa and submucosa, as well as, exudate in meati which usually was overlying necrotic foci. Also observed were foci of respiratory epithelial hyperplasia and squamous metaplasia (respiratory epithelium replaced by squamous epithelium). The olfactory epithelium and underlying Bowman's gland epithelium were atrophied. In the absence of any nervous system lesions at 537 mg/M3, no examination of skeletal muscle or tissues from the central and peripheral nervous systems were performed in the other exposed groups. Rats exposed to 19 or 2 mg/M3 of the test material had no exposure-related microscopic changes.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Time of death: all animals exposed to 537 mg/m3 died by exposure day 11; 1 during week 2 of dosing - Number of deaths at each dose: 10 at 537 mg/m3; 1 accidental death in control group; all animals exposed to 537 mg/m3 died by exposure day 11 and one control animal died during week 2 of exposure.
- Clinical signs: Animals exposed to 537 mg/m3 prior to death exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation, and lacrimation were observed. Animals exposed at 19 and 2 mg/m3 showed no treatment-related signs of toxicity.


BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases in body weight occurred in 537 mg/m3 males during week 1, and in females during weeks 1 and 2. Males exposed to 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 2, 3, 4. The females exposed at 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 3 and 4. At 19 mg/m3 statistically significant decreases in body weight and body weight change were seen in the females during week 2. Females in the 2 mg/m3 exposure group showed statistically significant decreases in body weight and body weight change during weeks 2, 3, and 4.


FOOD CONSUMPTION
A statistically significant decrease in feed consumption was observed in all animals in the 537 mg/m3 exposure groups during week 1. A statistically significant decrease in the feed consumption for the 129 mg/m3 males was observed during weeks 2 and 3.


FOOD EFFICIENCY not examined


WATER CONSUMPTION not examined


OPHTHALMOSCOPIC EXAMINATION not examined


HAEMATOLOGY
No treatment-related effect on any hematologic parameter at any dose level. Animals exposed at 19 mg/m3 showed statistically significant decrease in mean cell hemoglobin and mean cell hemoglobin concentrations for the females. No changes in hematology parameters were seen at any other concentration. In the absence of a dose-response relationship, none of the above changes were judged treatment-related. No exposure-related differences in differential parameters were observed.

CLINICAL CHEMISTRY
- Clinical chemistry: No treatment-related clinical chemistry effects were observed in any group. Statistically significant decreases in total protein and globulin in males at 129 mg/m3. At 19 mg/m3 statistically significant decreases in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase in males and triglyceride in females. Males exposed at 2 mg/m3 showed a statistically significant decrease in creatinine and glutamic oxaloacetic transaminase; females in this group showed an increase in glutamic oxaloacetic transaminase. In the absence of a concentration-response relationship, none of the above changes were judged to be treatment-related.

URINALYSIS not examined


NEUROBEHAVIOUR


ORGAN WEIGHTS
No treatment-related organ weight changes observed at any dose level.


GROSS PATHOLOGY
Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of male and female rats exposed to 537 mg/m3 showed changes in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithial ncerosis and inflammation of the submucosa. In addition, all meati and primarily the dorsal meati had exudates composed of mucous, fibrin, erythrocytes, exfoliated epithelium and inflammatory cells. The larynx and trachea had epithelial necrosis, submucosal inflammation an exudates in their lumens. Two female rats had foci of inflammation of bronchi, bronchioles and/or alveoli. Specific examination of tissues from the central (brain and spinal cord) and peripheral (sciatic nerve) nervous system as well as skeletal muscle from animals exposed to 537 mg/m3 revealed no changes indicative of neurotoxicity. The nasal cavity was the only target tissue in animals exposed to 129 mg/m3. The lesions were primary graded as slight and focal in nature. These lesions consisted of necrosis of respiratory and olfactory epithelium accompanied by inflammation in the mucosa and submucosa, as well as, exudates in meati which usually was overlying necrotic foci. Also observed were foci of respiratory epithelial hyperplasia and squamosus metaplasia (respiratory epithelium replaced by squamous epithelium). The olfactory epithelium and underlying Bowman's gland epithelium were atrophied. Rats exposed to 19 and 2 mg/m3 had not exposure-related microscopic changes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) not applicable

OTHER: The sentinel animal serology results were negative for the presence of adventitious virus and bacteria antibodies.


Dose descriptor:
NOAEL
Effect level:
19 mg/m³ air
Sex:
male/female
Basis for effect level:
other: overall effects histopathology of the nasal cavities
Dose descriptor:
BMCL10
Effect level:
36.2 mg/m³ air
Sex:
male/female
Basis for effect level:
other: BMDL10 based on nasal cavity data, as this was the most sensitive effect (Multistage model)
Critical effects observed:
not specified
Conclusions:
Inhalation exposure of the test material produced 100% mortality at 537 mg/m3. Prior to the death, these animals exhibited labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, lacrimation, and decresed body weight, body weight gain and feed consumption. At 129 mg/m3, transient occurrences of unstable gait, respiratory noise, salivation, lacrimation, and slight decreased body weight gains and feed consumption were observed.

No treatment-related signs were observed at 19 and 2 mg/m3. Organ weights and blood analysis revealed no treatment-related effects. At the end of the four-week exposure period, neurological evalutions of all surviving animals showed no signs of a cumulative toxic effect on the nervous system in any group. On the basis of the most sensitive indictor of toxicity, the histopathological changes seen in the nasal cavities, the NOEL for a four-week nose-only inhalation exposure of the test material was 19 mg/m3.

The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.
Executive summary:

Five groups, designated 1,2,3,4 and 5, each containing 10 male and 10 female Crl:CD BR rats, received nose-only inhalation exposures for 6 hours per day, 5 days a week, for four weeks to air or vapors of the test material. Group 1 animlas were exposed to filtered air only. Groups 2,3,4 and 5 were exposed to 2, 19, 129, and 537 mg/m3 of the test material, respectively. All surviving animals from each group were necropsied at the end of the four week exposure period. Body weight, feed consumption, clinical signs, neurology, clinical chemistry, hematology, organ weight and histopathology evaluations were performed on all surviving animals during this study.

All animlas exposed to 537 mg/m3 died by exposure day 11. Prior ro death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation and lacrimation were observed. Animals exposed to 19 mg/m3 showed no treatment related signs of toxicity, with the exception of one incident of lacrimation. No treatment-related signs were observed at 2 mg/m3. At the end of the four-week exposure period, neurological evaluations of all surviving animals showed no effect on the nervous system in any group.

Treatment -related decreases in body weight and feed consumption was observed in both sexes at 537 mg/m3. Animlas exposed to 129 mg/m3 showed a decrease in body weights for both sexes, and a decrease in feed consumption in males. Animlas exposed to 19 and 2 mg/m3 showed no treatment-related changes in body weight or feed consumption.

No treatment-related effects were seen in any group for clinical chemistry, hematology or organ weights.

Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs. Microscopic examination of male and female rats exposed to 537 mg/m3 showed chnages in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithelial necrosis and inflammation. The larynx and trachea had epithelial necrosis and inflammation. The lungs of two females showed foci of inflammation in the bronchi, bronchioloes and/or alveoli. Male and female rats exposed to 129 mg/m3 showed slight focal lesions of the nasal cavity. Rats exposed to 19 or 2 mg/m3 had no exposure related nasal cavity microscopic changes.

On the basis of the most sensitive indicator of toxicity, the histopathological changes seen in the nasal cavities, the no observed effect level (NOEL) following four weeks of nose-only inhalation exposure to the test material was 19 mg/m3.

A benchmark dose (BMD) analysis of relevant toxicity data for primene was conducted in order to establish BMD and BMDL (lower limit of the 95% confidence interval on the BMD) values. The U.S. EPA’s Benchmark Dose Software (BMDS) was used for the analysis (U.S. EPA 2009). Data from a four week vapor inhalation study in rats was used (Hagan et al., 1994). The exposure doses were 0, 2, 19, 129, and 537 mg/cubic meter of primene.

The key endpoints evaluated were body weight at week four (dose 537 not used due to deaths before the end of the study) and the nasal cavity with both the respiratory and olfactory epithelium. The study report indicated the nasal cavity was the most sensitive target organ with histopathologic changes, particularly desquamation, necrosis and inflammation.

The results for models for a particular response were all within a factor of 3 so the model with the lowest AIC is the most desirable. The plots show that the fits for the models for incidence 0 0 0 2 10 were all very similar except the quantal linear model which had a poorer fit than the others. The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMCL10
36.2 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
30
Species:
rat
System:
respiratory system: upper respiratory tract
Organ:
lungs

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental study period: 25-5-1993 to 22-06-1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test material identified as TAPA, TD 93-030, Lot 5-0027-93
Pale straw coloured liquid
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, New York, USA
- Age at study initiation: 21-23 days, at receipet
- Weight at study initiation: 50-75 g, at receipt
- Fasting period before study: not reported
- Housing: suspended wire mesh cage
- Diet (e.g. ad libitum): Purina certified rodent chow 5002; withheld during exposure
- Water (e.g. ad libitum): ad libitum; withheld during exposure
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.1 - 23.4 degrees C
- Humidity (%): 67.8 - 74.6%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark


IN-LIFE DATES: From: May 25, 1993 To: June 22, 1993
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable- vapor study
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1250 L stainless steel, glass and Plexiglass chambers
- Method of holding animals in test chamber: PVC nose-only restraining tubes
- Source and rate of air: Compressed air at 0.25 to 32 L/min
- Method of conditioning air: no data
- System of generating particulates/aerosols: study conducted with vapor, not aerosol
- Temperature, humidity, pressure in air chamber: 23.1-23.4 degrees C; 67.8 - 74.6%; pressure not reported
- Air flow rate: air flow rate adjutsted to 99% of the maximum vapor concentration
- Air change rate: no reported
- Method of particle size determination: study conducted with vapor, not aerosol
- Treatment of exhaust air: not reported


TEST ATMOSPHERE
- Brief description of analytical method used: an impinger located outside each chamber containing 8-12 mL of propylene glycol placed in an ice bath
- Samples taken from breathing zone: yes


VEHICLE (if applicable) no vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to determine the analytical concentration of the tetst material in the chambers, one impinger located outside wach chamber was used for sampling. The impingers contained 8-12 mL of propylene glycol and were placed in an ice bath. The amount of propylene glycol placed in the impingers was based on the amount of test material (mg) being collected over a 6-hour exposure. The air control chamber was sampled weekly to validate the lack of test material in the control atmosphere.
After collecting a sample with the impingers, the collecting solution was quantitatively transferred to pre-weighted 20 mL scintillation vials and weighed again to determine the total material present. The solution was then sent for gas chromatographic analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Remarks:
Doses / Concentrations:
2, 19, 129, 537 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
In addition, 2 males and 2 females were assigned to each group as health monitoring sentinels
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): computer randomization
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Signs of intoxication, mortality, morbidity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined : Yes weekly


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: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) Nembutal
- Animals fasted: Yes ; overnight
- How many animals: all surviving animals
- Parameters checked
hematocrit
hemoglobin
red blood cell
white blood cell count
platelet count
mean cell corpuscular volume
mean cell corpuscular hemoglobin
mean corpuscular hemoglobin conc.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked
triglyceride
cholesterol
blood urea nitrogen
glucose
alkaline phosphatase
total protein
albumin
chloride
sodium
potassium
globulin
albumin:globulin ratio
creatinine
bilirubin (total)
calcium
inorganic phosphorous
glutamic pyruvic transaminase/alanine aminotransferase activity
glutamic oxaloacetic transaminase/asparate aminotransferase activity
gamma-glutamyl transferase activity



URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
- Dose groups that were examined: all animals prior to exposure; all surviving animals on the last day of exposure
- Battery of functions tested: sensory activity / grip strength / motor activity / other: convulsions, tremors, stereotypic or bizzarre behavior


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Macroscopic: Gross examination included examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents.

HISTOPATHOLOGY: Yes
- Microscopic: Tissues processed and examined microscopically will be collected, saved, weighed and fixed in 10% neutral buffered formalin. Tissues included but not limited to, the nasal cavity, liver, brain, kidney, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals, spleen, trachea, larynx, heart, and gross lesions.
Other examinations:
Neurologic evaluations were performed the day prior to the first day of exposure, and shortly after animals were removed from the chambers on the last day of exposure.
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of an overall compound effect. Separate one-way analyses were used within the male and female data to assess overall treatment group effects. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals receiving 537 mg/m3 died by exposure Day 11, prior to death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation.
At 129 mg/m3 transient occurences of unstable gait, respiratory noise, salivation and lacrimation were observed.
Animals exposed at 19 mg/m3 showed a single incidence of lacrimation, this was judged not to be treatment related.
No treatment related signs were observed at 2 mg/m3. Red stained fur around eyes and muzzle and frequent struggling in the nose-only restraint tubes were observed throughout the study in all test material exposed groups. A single death in group 1 (Control) was attributable to over restraint in the nose only tube.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals receiving 537 mg/m3 died by exposure Day 11, prior to death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation.
A single death in group 1 (Control) was attributable to over restraint in the nose only tube.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant bodyweight decreases occured in the 537 mg/m3 males during week 1 and in females during Week 1 and 2. Males exposed to 129 mg/m3 showed statistically significant decreases in bodyweight and bodyweight change during Weeks 2, 3 and 4. The females exposed at 129 mg.m3 showed statistically significant decreases in bodyweight and bodyweight changes during Weeks 3 and 4. At 19 mg/m3 statistically significant decreases in bodyweight and bodyweight change were seen in females during Week 2. No other bodyweight effects were observed in this group.Females in the 2 mg/m3 exposure group showed statistically significant decreases in bodyweight and bodyweight change during Weeks 2, 3 and 4. No other bodyweight effects were observed in this group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in feed consumption for the 537 mg/m3 in both sexes was observed during Week 1. A statistically significant decrease i nthe feed consumption for the 129 mg/m3 males was observed during Weeks 2 and 3. No other feed consumption effects were seen i nany other group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals exposed at 19 mg/M3 showed statistically significant increase in platelets for the males and statistically significant decrease in mean cell hemoglobin and mean cell hemoglobin concentrations for the females. No changes in hematology parameters were seen at any other concentration. In the absence of a concentration-response relationship, none of the above changes were judged to be treatment-related.
No exposure related differences in white blood cell differential parameters were observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decreases were observed in total protein and globulin in males at 129 mg/M3. At 19 mg/M3 statistically significant decreases were observed in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase in males and triglyceride in females. Males exposed at 2 mg/M3 showed a statistically significant decrease in creatinine and glutamic oxaloacetic transaminase; females in this group showed an increase in glutamic oxaloacetic transaminase. In the absence of a concentration-response relationship, none of the above changes were judged to be exposure-related.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight data showed a statistcally significant increase in the absolute lung weight of the 2 mg/m3 males. No other changes in organ weights were seen in any other group. In the absence of a lung weight effect at higher concentrations, this finding was judgen not to be treatment related.
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The nasal cavity, in particular both respiratory and olfactory epithelium, was the most sensitive target organ.

All animals exposed to 537 mg/M3 died during the study, and at the time of necropsy many animals were observed with distended stomachs.
Difficulty in flushing formalin through the nasopharyngeal duct was noted in many of these animals at the time of necropsy. No exposure-related gross observations were noted on surviving animals.

Microscopic examination of male and female rats exposed to 537 mg/M3 showed changes in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithial necrosis and inflammation of the submucosa. In addition, all meati and primarily the dorsal meati had exudate composed of mucous, fibrin, erythrocytes, exfoliated epithelium and inflammatory cells. The larynx and trachea had epithelial necrosis, submucosal inflammation and exudate in their lumens. Two female rats had foci of inflammation of bronchi, bronchioles and/or alveoli. Specific examination of tissues from the central (brain and spinal cord) and peripheral (sciatic nerve) nervous systems as well as skeletal muscle from animals exposed to 537 mg/M3 revealed no changes
indicative of neurotoxicity.

The nasal cavity was the only target tissue in animals exposed to 129 mg/M3 of the test material. The lesions were primary graded as slight and focal in nature. These lesions consisted of necrosis of respiratory and olfactory epithelium accompanied by inflammation in the mucosa and submucosa, as well as, exudate in meati which usually was overlying necrotic foci. Also observed were foci of respiratory epithelial hyperplasia and squamous metaplasia (respiratory epithelium replaced by squamous epithelium). The olfactory epithelium and underlying Bowman's gland epithelium were atrophied. In the absence of any nervous system lesions at 537 mg/M3, no examination of skeletal muscle or tissues from the central and peripheral nervous systems were performed in the other exposed groups. Rats exposed to 19 or 2 mg/M3 of the test material had no exposure-related microscopic changes.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Time of death: all animals exposed to 537 mg/m3 died by exposure day 11; 1 during week 2 of dosing - Number of deaths at each dose: 10 at 537 mg/m3; 1 accidental death in control group; all animals exposed to 537 mg/m3 died by exposure day 11 and one control animal died during week 2 of exposure.
- Clinical signs: Animals exposed to 537 mg/m3 prior to death exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation, and lacrimation were observed. Animals exposed at 19 and 2 mg/m3 showed no treatment-related signs of toxicity.


BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases in body weight occurred in 537 mg/m3 males during week 1, and in females during weeks 1 and 2. Males exposed to 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 2, 3, 4. The females exposed at 129 mg/m3 showed statistically significant decreases in body weight and body weight change during weeks 3 and 4. At 19 mg/m3 statistically significant decreases in body weight and body weight change were seen in the females during week 2. Females in the 2 mg/m3 exposure group showed statistically significant decreases in body weight and body weight change during weeks 2, 3, and 4.


FOOD CONSUMPTION
A statistically significant decrease in feed consumption was observed in all animals in the 537 mg/m3 exposure groups during week 1. A statistically significant decrease in the feed consumption for the 129 mg/m3 males was observed during weeks 2 and 3.


FOOD EFFICIENCY not examined


WATER CONSUMPTION not examined


OPHTHALMOSCOPIC EXAMINATION not examined


HAEMATOLOGY
No treatment-related effect on any hematologic parameter at any dose level. Animals exposed at 19 mg/m3 showed statistically significant decrease in mean cell hemoglobin and mean cell hemoglobin concentrations for the females. No changes in hematology parameters were seen at any other concentration. In the absence of a dose-response relationship, none of the above changes were judged treatment-related. No exposure-related differences in differential parameters were observed.

CLINICAL CHEMISTRY
- Clinical chemistry: No treatment-related clinical chemistry effects were observed in any group. Statistically significant decreases in total protein and globulin in males at 129 mg/m3. At 19 mg/m3 statistically significant decreases in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase in males and triglyceride in females. Males exposed at 2 mg/m3 showed a statistically significant decrease in creatinine and glutamic oxaloacetic transaminase; females in this group showed an increase in glutamic oxaloacetic transaminase. In the absence of a concentration-response relationship, none of the above changes were judged to be treatment-related.

URINALYSIS not examined


NEUROBEHAVIOUR


ORGAN WEIGHTS
No treatment-related organ weight changes observed at any dose level.


GROSS PATHOLOGY
Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of male and female rats exposed to 537 mg/m3 showed changes in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithial ncerosis and inflammation of the submucosa. In addition, all meati and primarily the dorsal meati had exudates composed of mucous, fibrin, erythrocytes, exfoliated epithelium and inflammatory cells. The larynx and trachea had epithelial necrosis, submucosal inflammation an exudates in their lumens. Two female rats had foci of inflammation of bronchi, bronchioles and/or alveoli. Specific examination of tissues from the central (brain and spinal cord) and peripheral (sciatic nerve) nervous system as well as skeletal muscle from animals exposed to 537 mg/m3 revealed no changes indicative of neurotoxicity. The nasal cavity was the only target tissue in animals exposed to 129 mg/m3. The lesions were primary graded as slight and focal in nature. These lesions consisted of necrosis of respiratory and olfactory epithelium accompanied by inflammation in the mucosa and submucosa, as well as, exudates in meati which usually was overlying necrotic foci. Also observed were foci of respiratory epithelial hyperplasia and squamosus metaplasia (respiratory epithelium replaced by squamous epithelium). The olfactory epithelium and underlying Bowman's gland epithelium were atrophied. Rats exposed to 19 and 2 mg/m3 had not exposure-related microscopic changes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) not applicable

OTHER: The sentinel animal serology results were negative for the presence of adventitious virus and bacteria antibodies.


Dose descriptor:
NOAEL
Effect level:
19 mg/m³ air
Sex:
male/female
Basis for effect level:
other: overall effects histopathology of the nasal cavities
Dose descriptor:
BMCL10
Effect level:
36.2 mg/m³ air
Sex:
male/female
Basis for effect level:
other: BMDL10 based on nasal cavity data, as this was the most sensitive effect (Multistage model)
Critical effects observed:
not specified
Conclusions:
Inhalation exposure of the test material produced 100% mortality at 537 mg/m3. Prior to the death, these animals exhibited labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation, lacrimation, and decresed body weight, body weight gain and feed consumption. At 129 mg/m3, transient occurrences of unstable gait, respiratory noise, salivation, lacrimation, and slight decreased body weight gains and feed consumption were observed.

No treatment-related signs were observed at 19 and 2 mg/m3. Organ weights and blood analysis revealed no treatment-related effects. At the end of the four-week exposure period, neurological evalutions of all surviving animals showed no signs of a cumulative toxic effect on the nervous system in any group. On the basis of the most sensitive indictor of toxicity, the histopathological changes seen in the nasal cavities, the NOEL for a four-week nose-only inhalation exposure of the test material was 19 mg/m3.

The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.
Executive summary:

Five groups, designated 1,2,3,4 and 5, each containing 10 male and 10 female Crl:CD BR rats, received nose-only inhalation exposures for 6 hours per day, 5 days a week, for four weeks to air or vapors of the test material. Group 1 animlas were exposed to filtered air only. Groups 2,3,4 and 5 were exposed to 2, 19, 129, and 537 mg/m3 of the test material, respectively. All surviving animals from each group were necropsied at the end of the four week exposure period. Body weight, feed consumption, clinical signs, neurology, clinical chemistry, hematology, organ weight and histopathology evaluations were performed on all surviving animals during this study.

All animlas exposed to 537 mg/m3 died by exposure day 11. Prior ro death these animals exhibited treatment-related labored breathing, respiratory noise, gasping, unstable gait, tremors, salivation and lacrimation. At 129 mg/m3 transient occurrences of unstable gait, respiratory noise, salivation and lacrimation were observed. Animals exposed to 19 mg/m3 showed no treatment related signs of toxicity, with the exception of one incident of lacrimation. No treatment-related signs were observed at 2 mg/m3. At the end of the four-week exposure period, neurological evaluations of all surviving animals showed no effect on the nervous system in any group.

Treatment -related decreases in body weight and feed consumption was observed in both sexes at 537 mg/m3. Animlas exposed to 129 mg/m3 showed a decrease in body weights for both sexes, and a decrease in feed consumption in males. Animlas exposed to 19 and 2 mg/m3 showed no treatment-related changes in body weight or feed consumption.

No treatment-related effects were seen in any group for clinical chemistry, hematology or organ weights.

Gross pathologic changes were observed only in animals exposed to 537 mg/m3. These changes consisted of partially or fully blocked nasal cavities and gas-filled stomachs. Microscopic examination of male and female rats exposed to 537 mg/m3 showed chnages in the nasal cavity, larynx, trachea, and lung. The nasal cavity changes were moderate to severe desquamation of the respiratory and olfactory epithelium, epithelial necrosis and inflammation. The larynx and trachea had epithelial necrosis and inflammation. The lungs of two females showed foci of inflammation in the bronchi, bronchioloes and/or alveoli. Male and female rats exposed to 129 mg/m3 showed slight focal lesions of the nasal cavity. Rats exposed to 19 or 2 mg/m3 had no exposure related nasal cavity microscopic changes.

On the basis of the most sensitive indicator of toxicity, the histopathological changes seen in the nasal cavities, the no observed effect level (NOEL) following four weeks of nose-only inhalation exposure to the test material was 19 mg/m3.

A benchmark dose (BMD) analysis of relevant toxicity data for primene was conducted in order to establish BMD and BMDL (lower limit of the 95% confidence interval on the BMD) values. The U.S. EPA’s Benchmark Dose Software (BMDS) was used for the analysis (U.S. EPA 2009). Data from a four week vapor inhalation study in rats was used (Hagan et al., 1994). The exposure doses were 0, 2, 19, 129, and 537 mg/cubic meter of primene.

The key endpoints evaluated were body weight at week four (dose 537 not used due to deaths before the end of the study) and the nasal cavity with both the respiratory and olfactory epithelium. The study report indicated the nasal cavity was the most sensitive target organ with histopathologic changes, particularly desquamation, necrosis and inflammation.

The results for models for a particular response were all within a factor of 3 so the model with the lowest AIC is the most desirable. The plots show that the fits for the models for incidence 0 0 0 2 10 were all very similar except the quantal linear model which had a poorer fit than the others. The response of 0 0 0 2 10 was the most sensitive therefore the BMD value of 80.96 mg/m3 and the BMDL value of 36.20 mg/m3 are chosen corresponding to the multistage model with the lowest AIC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMCL10
36.2 mg/m³
Study duration:
subacute
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Stoneridge, Kingston, New York, USA
- Age at study initiation: 35 days
- Weight at study initiation: 104-156 g (males); 116-153 g (females)
- Fasting period before study: not reported
- Housing: 1/cage in suspended stainless steel cages
- Diet (e.g. ad libitum): Purina certified rodent chow 5002M
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 degrees C
- Humidity (%): 28-79%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: September 16, 1992 To: October 15, 1992
Type of coverage:
open
Vehicle:
other: Neutral Oil 100 N
Details on exposure:
Route of Administration: dermal
TEST SITE
- Area of exposure: the hair around the entire trunk between the flank and shoulders of each animal was closely clipped
- % coverage: 10% of the bosy surface area
- Type of wrap if used: none
- Time intervals for shavings or clipplings: repeated as required


REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with paper towels saturated with 1% aqueous soap solution. The exposure sight was then rinsed with paper towels saturated with tap water and gently blotted dry with paper towels.
- Time after start of exposure:6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg/day; 0.0 ml/kg/day- Sham control
- Concentration (if solution): 0.0025, 0.010, 0.030 g/mL
- Constant volume or concentration used: yes
- For solids, paste formed: not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance was not soluble in water but in the Neutral Oil 100N
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg/day
- Concentration (if solution): suspension: 0.0025, 0.010, 0.030 g mL
- Lot/batch no. (if required): TD92-145 (number assigned internal to company)
- Purity: 100% of a refined paraffinic distillate


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; cardboard collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (1 mL) of each dosing solution, 0.0025, 0.010 and 0.030 g/mL, were submitted weekly for analysis in order to verify proximity to target concentration and stability. Gas chromatography was used to measure the amount of test substance in the samples. Before analysis, samples were stored at room temperature and kept at that temperature throughout the study. The first and last week's samples were analyed. Each sample was diluted 2 parts sample to 1 part MDC. Duplicate injections of each sample were run and the levels were calculated using a four-level calibration curve generated before sample analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Remarks:
Doses / Concentrations:
0 (Sham Control), 0 (Solvent Control), 5, 20, 60 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
6
Control animals:
other: Sham Control and Solvent Control (Neutral Oil 100N)
Details on study design:
- Dose selection rationale: based on a 5-day pilot dermal study
- Rationale for animal assignment (if not random): computer randomiation
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): males follwed by females next day
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality, signs of ill health or reaction to treatment


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: each treatment day prior to the application and prior to necropsy


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes; weekly


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: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) pentobarbital
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Hematocrit
Erythrocytes
Hemoglobin
Total white blood cell count and differential
Platelets
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Serum glutamic pyruvic transaminase
Serum glutamic oxaloacetic transaminase
Cholesterol
Triglycerides
Total Bilirubin
Alkaline Phosphatase
Glucose
Gamma glutamyl transpeptidase
Blood urea nitrogen
Creatinine
Total protein
Albumin
Albumin/globulin ratio
Calcium
Sodium
Potassium
Chloride
Inorganic Phosphorus


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: performed on each animal
- Battery of functions tested: sensory activity / grip strength / motor activity / other: appearance, posture, gait,


OTHER: external structures, body orifices, palpation for body masses weekly
Other examinations:
none
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of a treatment effect for body weight, cumulative body weight gain and feed consumption. Separate analyses were conducted at each sampling time for each sex. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
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):
no effects observed
Food efficiency:
not examined
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:
not examined
Behaviour (functional findings):
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: no treatment-related mortalities; - Time of death: 2 rats sacrificed for humane reasons on day 11 - Number of deaths at each dose: 2 females at 60 mg/kg/day;
Red-stained muzzle and red-stained eyes were observed in all treatment groups and were judged to be caused by the collar. Alopecia of the forelegs and scabs on the head or neck were observed and judged unrelated to test substance exposure.


BODY WEIGHT AND WEIGHT GAIN: No treatment-related effect on body weight or cumulative body weight gain was evident in females at any dose level. In males, both body weight and cumulative body weight gain were statistically significantly less than the Sham control in Groups 4 (20 mg/kg/day) and 5 (60 mg/kg/day) throughout the treatment period, and in Group 3 (5 mg/kg/day) at the end of weeks 1 and 4. Body weight was also consistently less in Group 2 (Solvent Control) males throughout the treatment period. Though this difference was statistically significant only at the end of week 4. When compared to Group 2 (Solvent Control), body weight and cumulative body weight gain were significantly lower only in Group 5 males at the end of weeks 1 and 2. These results indicate that body weight effects observed in Groups 3 and 4 (5 and 20 mg/kg/day) males were attributable to Neutral Oil 100N exposure, and only in Group 5 were effects on body weights directly related to test material exposure.


FOOD CONSUMPTION: There was no treatment-related effect on feed consumption in male or female rats at any dose level. A statistically significant reduction in feed consumption was noted in male rats at 60 mg/kg/day during week two of exposure; but this reduction was judged incidental and not test substance-related.


FOOD EFFICIENCY: not conducted


WATER CONSUMPTION: not conducted


OPHTHALMOSCOPIC EXAMINATION: not conducted


HAEMATOLOGY: No treatment-related effect on any hematologic parameter at any dose level. A significant increase in hemoglobin concentration was observed in Group 2 (Solvent Control) females and a significant reduction in lymphocyte count was observed in females at 60 mg/kg/day dose group. These observations were judged incidental due to the minimal change observed and the lack of dose response.


CLINICAL CHEMISTRY: No treatment-related clinical chemistry effects were observed in any group. However, numerous statistically significant changes in clinical chemistry parameters were noted but were judged to be incidental since there was a general lack of dose response or consistent pattern between endpoints and sexes, and absence of correlative histopathologic changes. Alkaline phosphatase levels were significantly increased in males at 60 mg/kg/day. Triglyceride levels were significantly reduced in males at 5 and 60 mg/kg/day groups and chloride was significantly increased in males at 20 mg/kg/day. In females, blood urea nitrogen levels were significantly increased in 60 mg/kg/day dose group, and total protein was significantly increased in Solvent Control and 20 mg/kg/day dose groups.


URINALYSIS: not conducted


NEUROBEHAVIOUR: no effects


ORGAN WEIGHTS: The only treatment-related organ weight changes observed were significantly increased absolute and relative adrenal weights in both sexes at the 60 mg/kg/day dose group.


GROSS PATHOLOGY: Treatment-related gross pathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Gross observations included scales, erosions, scabs and/or cracking fissures of the epidermis.


HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related histopathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Microscopic observations included multifocal to diffuse, and moderate to severe hyperkeratosis, acanthosis and dermal inflammation. In addition, inflammation of the subcutaneous tissue underlying the skin at the application site was observed with low incidence.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): not applicable


Key result
Dose descriptor:
NOAEL
Effect level:
20 other: mg/kg
Sex:
male/female
Basis for effect level:
other: There were no test substance related effects observed at this concentration
Key result
Dose descriptor:
LOAEL
Effect level:
60 other: mg/kg
Sex:
male/female
Basis for effect level:
dermal irritation
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
Daily dermal application of the test material to the skin of rats for four weeks (5 days/week) resulted in a No Observed Effect Level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.
Executive summary:

The dermal toxicity of the test material was assessed after topical application of the test substancec to the shaved intact skin of Crl:CD BR rats. The test material was dispersed in Neutral Oil 100 N for application. Five groups of six male and six female rats were administered the test substancce or Neutral Oil 100 N (solvent control) dermally, to non-occluded sites for 6 hrs/day, 5 days/week for 4 weeks. Group 1 (sham control) animals were treated in a manner identical to animals in other groups with th exception that they did not receive any test material. group 2 (solvent control) received Neutral Oil 100 N at a constant volume of 2.0 mg/kg/day. Groups 3, 4 and 5 received the test material, dispersed in Neutral Oil 100 N at doses of 5, 20 and 60 mg/kg/day, respectively, using a constant dosing volume of 2.0 ml/kg/day.

All animals were observed at least once daily for mortality, signs of ill health or reaction to treatment. Application sites were evaluated for skin irrittation prior to each daily dose and before terminal sacrifice. Body weight and feed consumption were measured weekly; physical examinations were perfomred weekly at the time of weighing. At the end of the 4 - week treatment period, all rats were killed and necropsied. Blood samples for hematology and clinical chemistry were collected and analyzed. Adrenals, brain, kidneys, liver and testes were weighed in all groups.

Histopathologic evaluations were performed on all gross lesions as well as liver, treated and untreated skin, kidney, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals and tested collected from all animals in Group 5 ( Test material; 60 mg/kg/day), Groups 1 and 2 (Sham and Solvent controls, respectively).

There were no treatment-related mortalities, clinical signs or effects on feed consumption. Two females of Group 5 (60 mg/kg/day) were sacrificed following the 11th day of test material application due to severe skin irritation and signs of extreme discomfort.

Body weight and cumulative body weight gain were not affected in femlaes of any treatment group. Treatment-related effects on body weight and cumulative body weight gain were observed in Group 5 males at the end of weeks 1 and 2. Other statistically significant changes in body weight and body weight gain were attributed to solvent exposure.

In both males and females, local skin irritation at the application site was observed at all dose levels of the test material, the duration and severity of which were dose-dependent. Signs of slight application site irritation were alos observed in animals of Group 2 (solvent control).

There was no treatment-related effect on any hematologic or clinical chemistry parameter.

Test material exposure resulted in increased absolute and relative adrenal weights in both males and females of Group 5 (60 mg/kg). Other statistically significant changes in absolute and relative organ weights were attributed to solvent exposure or judged incidental.

Treatment-related gross and histopathologic effects were confined to the skin (epidermis and dermis) and underlying subcutaneous tissues at the treatment site. Doses of 60 mg/kg/day proved severely irritating to the skin of rats.

Daily application of the test material to the skin of rats for four weeks at doses up to and including 60 mg/kg/day had a no observed effect level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
42
Species:
rat
Organ:
skin

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Stoneridge, Kingston, New York, USA
- Age at study initiation: 35 days
- Weight at study initiation: 104-156 g (males); 116-153 g (females)
- Fasting period before study: not reported
- Housing: 1/cage in suspended stainless steel cages
- Diet (e.g. ad libitum): Purina certified rodent chow 5002M
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 degrees C
- Humidity (%): 28-79%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: September 16, 1992 To: October 15, 1992
Type of coverage:
open
Vehicle:
other: Neutral Oil 100 N
Details on exposure:
Route of Administration: dermal
TEST SITE
- Area of exposure: the hair around the entire trunk between the flank and shoulders of each animal was closely clipped
- % coverage: 10% of the bosy surface area
- Type of wrap if used: none
- Time intervals for shavings or clipplings: repeated as required


REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with paper towels saturated with 1% aqueous soap solution. The exposure sight was then rinsed with paper towels saturated with tap water and gently blotted dry with paper towels.
- Time after start of exposure:6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg/day; 0.0 ml/kg/day- Sham control
- Concentration (if solution): 0.0025, 0.010, 0.030 g/mL
- Constant volume or concentration used: yes
- For solids, paste formed: not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance was not soluble in water but in the Neutral Oil 100N
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg/day
- Concentration (if solution): suspension: 0.0025, 0.010, 0.030 g mL
- Lot/batch no. (if required): TD92-145 (number assigned internal to company)
- Purity: 100% of a refined paraffinic distillate


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; cardboard collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (1 mL) of each dosing solution, 0.0025, 0.010 and 0.030 g/mL, were submitted weekly for analysis in order to verify proximity to target concentration and stability. Gas chromatography was used to measure the amount of test substance in the samples. Before analysis, samples were stored at room temperature and kept at that temperature throughout the study. The first and last week's samples were analyed. Each sample was diluted 2 parts sample to 1 part MDC. Duplicate injections of each sample were run and the levels were calculated using a four-level calibration curve generated before sample analysis.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 4 weeks
Remarks:
Doses / Concentrations:
0 (Sham Control), 0 (Solvent Control), 5, 20, 60 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
6
Control animals:
other: Sham Control and Solvent Control (Neutral Oil 100N)
Details on study design:
- Dose selection rationale: based on a 5-day pilot dermal study
- Rationale for animal assignment (if not random): computer randomiation
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): males follwed by females next day
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
Mortality, signs of ill health or reaction to treatment


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: each treatment day prior to the application and prior to necropsy


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes; weekly


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: on the day of necropsy
- Anaesthetic used for blood collection: Yes (identity) pentobarbital
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Hematocrit
Erythrocytes
Hemoglobin
Total white blood cell count and differential
Platelets
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes
- How many animals: all surviving animals on the day of necropsy
- Serum glutamic pyruvic transaminase
Serum glutamic oxaloacetic transaminase
Cholesterol
Triglycerides
Total Bilirubin
Alkaline Phosphatase
Glucose
Gamma glutamyl transpeptidase
Blood urea nitrogen
Creatinine
Total protein
Albumin
Albumin/globulin ratio
Calcium
Sodium
Potassium
Chloride
Inorganic Phosphorus


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: performed on each animal
- Battery of functions tested: sensory activity / grip strength / motor activity / other: appearance, posture, gait,


OTHER: external structures, body orifices, palpation for body masses weekly
Other examinations:
none
Statistics:
One-way analysis of covariance models were used to assess the presence or absence of a treatment effect for body weight, cumulative body weight gain and feed consumption. Separate analyses were conducted at each sampling time for each sex. Pairwise comparisons of least square means between control and each treatment level were evaluated using Dunnett's t-test. The criterion for statistical significance for all comparisons was p-value of 0.05 or less.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
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):
no effects observed
Food efficiency:
not examined
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:
not examined
Behaviour (functional findings):
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: no treatment-related mortalities; - Time of death: 2 rats sacrificed for humane reasons on day 11 - Number of deaths at each dose: 2 females at 60 mg/kg/day;
Red-stained muzzle and red-stained eyes were observed in all treatment groups and were judged to be caused by the collar. Alopecia of the forelegs and scabs on the head or neck were observed and judged unrelated to test substance exposure.


BODY WEIGHT AND WEIGHT GAIN: No treatment-related effect on body weight or cumulative body weight gain was evident in females at any dose level. In males, both body weight and cumulative body weight gain were statistically significantly less than the Sham control in Groups 4 (20 mg/kg/day) and 5 (60 mg/kg/day) throughout the treatment period, and in Group 3 (5 mg/kg/day) at the end of weeks 1 and 4. Body weight was also consistently less in Group 2 (Solvent Control) males throughout the treatment period. Though this difference was statistically significant only at the end of week 4. When compared to Group 2 (Solvent Control), body weight and cumulative body weight gain were significantly lower only in Group 5 males at the end of weeks 1 and 2. These results indicate that body weight effects observed in Groups 3 and 4 (5 and 20 mg/kg/day) males were attributable to Neutral Oil 100N exposure, and only in Group 5 were effects on body weights directly related to test material exposure.


FOOD CONSUMPTION: There was no treatment-related effect on feed consumption in male or female rats at any dose level. A statistically significant reduction in feed consumption was noted in male rats at 60 mg/kg/day during week two of exposure; but this reduction was judged incidental and not test substance-related.


FOOD EFFICIENCY: not conducted


WATER CONSUMPTION: not conducted


OPHTHALMOSCOPIC EXAMINATION: not conducted


HAEMATOLOGY: No treatment-related effect on any hematologic parameter at any dose level. A significant increase in hemoglobin concentration was observed in Group 2 (Solvent Control) females and a significant reduction in lymphocyte count was observed in females at 60 mg/kg/day dose group. These observations were judged incidental due to the minimal change observed and the lack of dose response.


CLINICAL CHEMISTRY: No treatment-related clinical chemistry effects were observed in any group. However, numerous statistically significant changes in clinical chemistry parameters were noted but were judged to be incidental since there was a general lack of dose response or consistent pattern between endpoints and sexes, and absence of correlative histopathologic changes. Alkaline phosphatase levels were significantly increased in males at 60 mg/kg/day. Triglyceride levels were significantly reduced in males at 5 and 60 mg/kg/day groups and chloride was significantly increased in males at 20 mg/kg/day. In females, blood urea nitrogen levels were significantly increased in 60 mg/kg/day dose group, and total protein was significantly increased in Solvent Control and 20 mg/kg/day dose groups.


URINALYSIS: not conducted


NEUROBEHAVIOUR: no effects


ORGAN WEIGHTS: The only treatment-related organ weight changes observed were significantly increased absolute and relative adrenal weights in both sexes at the 60 mg/kg/day dose group.


GROSS PATHOLOGY: Treatment-related gross pathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Gross observations included scales, erosions, scabs and/or cracking fissures of the epidermis.


HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related histopathologic observations were confined to the dermal application site (treated skin) of the test material. The severity and distribution as well as the number of lesions at the application site were dose-dependent. Microscopic observations included multifocal to diffuse, and moderate to severe hyperkeratosis, acanthosis and dermal inflammation. In addition, inflammation of the subcutaneous tissue underlying the skin at the application site was observed with low incidence.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): not applicable


Key result
Dose descriptor:
NOAEL
Effect level:
20 other: mg/kg
Sex:
male/female
Basis for effect level:
other: There were no test substance related effects observed at this concentration
Key result
Dose descriptor:
LOAEL
Effect level:
60 other: mg/kg
Sex:
male/female
Basis for effect level:
dermal irritation
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
Daily dermal application of the test material to the skin of rats for four weeks (5 days/week) resulted in a No Observed Effect Level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.
Executive summary:

The dermal toxicity of the test material was assessed after topical application of the test substancec to the shaved intact skin of Crl:CD BR rats. The test material was dispersed in Neutral Oil 100 N for application. Five groups of six male and six female rats were administered the test substancce or Neutral Oil 100 N (solvent control) dermally, to non-occluded sites for 6 hrs/day, 5 days/week for 4 weeks. Group 1 (sham control) animals were treated in a manner identical to animals in other groups with th exception that they did not receive any test material. group 2 (solvent control) received Neutral Oil 100 N at a constant volume of 2.0 mg/kg/day. Groups 3, 4 and 5 received the test material, dispersed in Neutral Oil 100 N at doses of 5, 20 and 60 mg/kg/day, respectively, using a constant dosing volume of 2.0 ml/kg/day.

All animals were observed at least once daily for mortality, signs of ill health or reaction to treatment. Application sites were evaluated for skin irrittation prior to each daily dose and before terminal sacrifice. Body weight and feed consumption were measured weekly; physical examinations were perfomred weekly at the time of weighing. At the end of the 4 - week treatment period, all rats were killed and necropsied. Blood samples for hematology and clinical chemistry were collected and analyzed. Adrenals, brain, kidneys, liver and testes were weighed in all groups.

Histopathologic evaluations were performed on all gross lesions as well as liver, treated and untreated skin, kidney, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, skeletal muscle, adrenals and tested collected from all animals in Group 5 ( Test material; 60 mg/kg/day), Groups 1 and 2 (Sham and Solvent controls, respectively).

There were no treatment-related mortalities, clinical signs or effects on feed consumption. Two females of Group 5 (60 mg/kg/day) were sacrificed following the 11th day of test material application due to severe skin irritation and signs of extreme discomfort.

Body weight and cumulative body weight gain were not affected in femlaes of any treatment group. Treatment-related effects on body weight and cumulative body weight gain were observed in Group 5 males at the end of weeks 1 and 2. Other statistically significant changes in body weight and body weight gain were attributed to solvent exposure.

In both males and females, local skin irritation at the application site was observed at all dose levels of the test material, the duration and severity of which were dose-dependent. Signs of slight application site irritation were alos observed in animals of Group 2 (solvent control).

There was no treatment-related effect on any hematologic or clinical chemistry parameter.

Test material exposure resulted in increased absolute and relative adrenal weights in both males and females of Group 5 (60 mg/kg). Other statistically significant changes in absolute and relative organ weights were attributed to solvent exposure or judged incidental.

Treatment-related gross and histopathologic effects were confined to the skin (epidermis and dermis) and underlying subcutaneous tissues at the treatment site. Doses of 60 mg/kg/day proved severely irritating to the skin of rats.

Daily application of the test material to the skin of rats for four weeks at doses up to and including 60 mg/kg/day had a no observed effect level (NOEL) for systemic toxicity of 20 mg/kg/day. The minimum effect level was 60 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/cm²
Study duration:
subacute
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Substance causes local irritation/corrosivity at the site of dosing, these are considered concentration dependent effects

Additional information

The dermal toxicity of the test material was assessed after topical application of the test substance to the shaved intact skin of Crl:CD BR rats. The test material was dispersed in Neutral Oil 100 N for non-occluded application for 6 hrs/day, 5 days/week for 4 weeks.

Daily application of the test material to the skin of rats for four weeks at doses up to and including 60 mg/kg/day had a No-Observed Effect Level (NOEL) for systemic toxicity of 20 mg/kg/day. The test material exposure resulted in increased absolute and relative adrenal weights in both males and females of Group 5 (60 mg/kg/day). The minimum effect level was 60 mg/kg/day. In both males and females, local skin irritation at the application site was observed at all dose levels of the test material, the duration and severity of which were dose-dependent. Treatment-related gross and histopathologic effects were confined to the skin (epidermis and dermis) and underlying subcutaneous tissues at the treatment site. Doses of 60 mg/kg/day proved severely irritating to the skin of rats.   

Crl: CD BR rats, received nose-only inhalation exposures for 6 hours per day, 5 days a week, for four weeks to air or vapors of the test material. On the basis of the most sensitive indicator of toxicity, the histopathological changes seen in the nasal cavities, the no-observed effect-level (NOEL) following four-weeks of nose-only inhalation exposure to the test material was 19 mg/M3. The LOAEL in the 28 day rat vapor inhalation study was 129 mg/m3.


Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: nose

Repeated dose toxicity: dermal - systemic effects (target organ) glandular: adrenal gland; other: skin

Repeated dose toxicity: oral

A subchronic toxicity study via the oral route in rats is considered not scientifically justified for the following reasons.

 The most relevant routes of exposure considering the uses of the test substance are via inhalation and dermal routes. As such conducting such a study would not result in further protection of human health.

Guideline and GLP compliant 28-day repeat dose toxicity studies via the inhalation and dermal routes are available for C10-C14 tert-alkyl amines indicating that the primary toxicity associated with the substance is irritation at the site of dosing. No indications of systemic toxicity were observed in either study in fact NOAELS derived from the study were based on local irritant effects either of the skin or respiratory tract.

In addition to the above information on repeat dose toxicity, a one-generation reproductive toxicity study exists for the substance in which animals were exposed orally via the diet for 10 weeks prior to mating, throughout gestation, lactation and until necropsy.

Four groups of 26 animals per sex per group were administered either 0, 19.1, 55.6, 107.3 mg/kg/day (males) or 0, 21, 62.8, 124.1 mg/kg/day (females). The highest dose was selected since, as noted in the report itself, method development work showed doses above such levels were not well tolerated for extended periods. 

As part of the study, in-life observations were conducted including clinical signs, bodyweight and food consumption measurements. At necropsy, a gross pathological examination of all internal tissues was conducted, organ weights were taken and histopathological examination of all reproductive organs plus the pituitary, stomach and any abnormal tissues was conducted.

In the study no signs of systemic toxicity were observed, however significant body weight effects were noted such as reduced bodyweight gains of up to 82% as well as reduced food consumption of up to 23% at the highest dose tested (1500ppm). Such effects indicate issues of palatability with the test material most likely due to its irritancy and that higher doses would not be tolerated. No adverse clinical signs were noted and neither were any gross or histopathological changes.

As further support, In silico modelling information has been added to indicate that the oral absorption is greater than 97% and that the substance is significantly bioavailable via the oral route. Taken together with the results of the one generation reproductive study, these indicate that palatability, due to the corrosiveness of the test material would likely be dose-limiting and if systemic toxicity were to occur it would likely have been observed during the aforementioned reprodevelopmental toxicity study.

Therefore conducting a repeat dose toxicity study via the oral route would not be scientifically justified since;

a)      the results would not alter the risk assessment of the substance as only inhalation and dermal routes of exposure are foreseen due to uses of the substance

b)     the results would not provide further information concerning hazard of the substance since GLP and guideline compliant repeat dose studies via the dermal and inhalation routes indicate the primary hazards are concerned with local effects of irritation

c)      repeat dose oral toxicity information albeit with limited investigations exists from a guideline compliant one generation reproductive toxicity study

d)     further in silico information provided indicates significant systemic exposure during the one generation reproductive toxicity study

In conclusion, since it is not possible to expose animals to greater doses systemically it is unlikely that unforeseen hazards would be identified and there would be no adaptation of the current risk assessment since the oral route is not relevant and as such no further protection of human health. As a result, the conducting of such a study would be contrary to Directive 2010/63/EU of the European parliament concerning the protection of animals used for scientific purposes insofar as no discernible benefit to human health would be gained from conducting the study.

 

 

Justification for classification or non-classification

The test material is not classified based on the results of the dermal toxicity. Although, a classification for inhalation appears to be warranted based on a LOAEL of 129 mg/m3 seen in the 28 day rat, no further classification is needed based on the corrositivity of the material. Effects observed in the upper respiratory tract in 28 day study rat inhalation study were determined to be point of contact (local) effects.

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