Registration Dossier

Administrative data

Description of key information

ORAL
A 28 day repeated dose/reprotoxicity study was carried out in rats in accordance with OECD 422 on a structural analogue of the registered material. The NOAEL was deemed to be greater than or equal to 200 mg/kg/day.
INHALATION
A 14 day repeated dose inhalation study was carried out in rats. The NOAEC was deemed to be 200 mg/m³.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed per OECD 422 and following GLP. A subset of the animals tested were evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. A justification for the read-across is provided.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: P = 63 days old
- Housing: singly in polycarbonate cages with bedding except during mating.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77 degrees F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light cycle
IN-LIFE DATES: From: 2003-09-2 To: 2004-01-06
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dosing formulations were analyzed for concentration verification. Standards for acceptable accuracy of mixing were that the means of the analyzed samples was within +/- 10% of the nominal concentration and the relative standard deviation for triplicate samples did not exceed 10%.
Duration of treatment / exposure:
F0 females were dosed premating through the day prior to necropsy.
Recovery males, females and 28 days females were dosed for 28 days.
F1 animals were dosed post-weaning until the day before scheduled necropsy, at least 7 weeks duration.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 10, 100, 200 mg/kg/day
Basis:
other: nominal concentration at a dose volume of 5 mL/kg/day
No. of animals per sex per dose:
F0 generation: 10 males and 10 females plus 5 addition males and females each in the control and high dose groups
designated recovery animals.
In addition, 10 females (5 control and 5 in the high dose group) designated as the 28 day females were dosed for 28
days and necropsied.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a dose range finding study, the test substance was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore, 300 mg/kg/day was considered too toxic for the OECD 422 study design.
- Rationale for animal assignment (if not random): Randomization stratified by body weight
Observations and examinations performed and frequency:
PARENTAL OBSERVATIONS:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and included changes to skin and fur, eyes, mucous membranes, respiratory and circulatory sytems, autonomic and central nervous systems, somatomotor activity, and behavior patterns.
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreeding, for F0 females during gestation (gd 0,7,14, and 21) and lactation (pnd 0, 4, 7, 14, and 21). Body weights of the 28 day females and and recovery males and females were recorded on a weekly basis until termination. Body weight gains were computed.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

LITTER OBSERVATIONS:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, a maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, euthanized,
necropsied and completed external and visceral examinations performed. Survival indices were calculated at least weekly through weaning (pnd21).
GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or
found dead.
OTHER:
At weaning at least one male and one female from each litter (when possible) were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for retained F1 females included examination of vaginal patency and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last three weeks of the postwean exposure period prior to scheduled sacrifice. For each retained male offspring, observations or cleavage of the balanopreputial gland began at day 35 and continued until preputial separation. Androgenic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected F1 adult males and females per dose group. Body weights for selected F1 offspring from weaning through scheduled sacrifice were taken. Functional Observation Battery (FOB), including cage side observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations was performed on 5 F1 females and 5 F1 males once midway during the postwean exposure period. Grip strength was also assessed for the 5 F1 males and 5 F1 females per group selected for FOB during the last week of the post weaning exposure.
Sacrifice and pathology:
-All F0 parental animals, 28-day females, recovery males and females, adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of FO females were examined for the number of nidation (implantation) scars.

A full histopathology was performed on all retained organs from 5 randomly selected F0 males and females in the control and high-dose groups as well as for the 28-day females. Since no treatment-related findings were identified, no histopathology on lower dose groups or recovery groups was performed.

All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. Retained F1 adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of F0 females were examined for the number of nidation (implantation) scars. All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. The retained F1 offspring were sacrificed at 70 days of age and subjected to the same assessments as the F0 parents.

Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females in the control and high-dose groups. Since no treatment-related findings were identified, no histopathology on lower dose groups was performed.
Other examinations:
OTHER:
Urinalysis: Prior to necropsy, 5 F0 males per group, randomly selected at the end of the mating period, and the 28-day females were singly housed overnight in metabolism cages. The total amount of time the animal was in the chamber and the amount of urine collected were recorded. The urine was evaluated for appearance and by dipstick analysis for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes.

Hematology: Blood was collected for hematology determinations from 5 randomly selected F0 females per group on the last day of the prebreed exposure period (sd 13). Blood was collected for hematology prior to necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. The parameters measured included evaluation of hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, RBC indices, and prothrombin time (PT), a measure of blood clotting time/potential (Becton Dickinson Fibrometer).

Clinical chemistry: Blood was collected for clinical chemistry at necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. Evaluations in serum included sodium, potassium, chloride, glucose, total cholesterol, blood urea nitrogen (BUN), creatinine, total protein and albumin, and 2 enzymes indicative of hepatocellular effects (alanine aminotransferase and aspartate aminotransferase).

Functional Observation Battery (FOB), including cage side observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations was performed on all initial animals once during quarantine and at least once per week for F0 animals during prebreed, mating (both sexes), gestation, lactation (F0 females) treatment period.



Five F0 males and 5 F0 females per dose group were were evaluated for for auditory functions, motor activity, and assessment of grip strength prior to necropsy.

In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected parental F0 males and females per dose group.
Statistics:
Appropriate statistical analyses were used throughout the assay.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
post dose rooting - consistent with taste aversion
Mortality:
mortality observed, treatment-related
Description (incidence):
post dose rooting - consistent with taste aversion
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Treatment-related clinical observations of F0 males included rooting post-dosing in 5 males at 200 mg/kg/day. No other findings exhibited a treatment- or dose-related pattern of incidence or severity.
All 10 F0 females/group survived to scheduled sacrifice

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No treatment-related effects on F0 male body weights were observed. Significantly higher weights for sd 7, 14, 21, and 28 (but not sd 0) occurred at 10 mg/kg/day, with no effects 100 or 200 mg/kg/day. F0 male body weight change was significantly increased at 10 mg/kg/day for sd 0-7 and unaffected for all other intervals at this dose and for all intervals at 100 and 200 mg/kg/day. No treatment-related effects on feed consumption were observed. Feed consumption values, expressed as g/day, were significantly increased at 10 mg/kg/day for sd 0 to 7, 7 to 14, and 0 to 14, with no effects at 100 or 200 mg/kg/day. There were no significant effects of treatment on F0 male feed consumption.

There were no significant differences among groups for F0 female body weights or body weight changes during the prebreed period (sd 0-14) or for the few females in the control and low-dose groups (10 mg/kg/day) that were not identified as sperm positive during the mating (sd 0-28) or postmating (sd 14-42) periods. There were no significant effects on F0 female feed consumption, expressed as g/day or g/kg/day, in any group during the 2 weeks of the prebreed period.

HEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS (PARENTAL ANIMALS):
No clinical chemistry or hematology parameters exhibited treatment- or dose-related changes for F0 males. Blood urea nitrogen, creatinine, glucose, total cholesterol, aspartate aminotransterase, alanine aminotransferase, sodium, and chloride were unaffected across groups. Total protein and albumin were significantly reduced at 100 and 200 mg/kg/day. Potassium concentrations were significantly increased at 10, 100, and 200 mg/kg/day. There were no statistically significant or biologically relevant differences across groups for any blood parameters, including absolute or corrected white blood cell count, absolute or nucleated red blood cell count, hemoglobin,'hematocrit, mean corpuscular volume, hemoglobin or hemoglobin concentrations, red blood cell distribution width, platelet count or volume, percentages of segmented neutrophils, lymphocytes, monocytes, eosinophils, or prothrombin clotting time. The specific gravity and pH of urine were also equivalent across groups.

For F0 females there were no treatment-related changes on any hematology measurements following the 2-week prebreed exposure. A significant increase in hematocrit at 200 mg/kg/day was observed, which was not considered treatment-related based on a lack of effects on correlating parameters or similar findings in the males at this dose. Also, mean corpuscular hemoglobin was significantly reduced at 100 mg/kg/day but unaffected at 10 and 200 mg/kg/day.

ORGAN WEIGHTS (PARENTAL ANIMALS):
F0 males (10/group) were necropsied after 28 days of dosing (2 weeks prebreed + 2 weeks mating). There were no treatment-related effects on organ weights. Sacrifice body weights and absolute weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles with coagulating glands were all unaffected. Absolute prostate weight was significantly reduced at 100 mg/kg/day but statistically equivalent at 10 and 200 mg/kg/day. Organ weights relative to terminal body weights were unaffected for the thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles plus coagulating glands. Relative brain weight was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day. Relative prostate weight was significantly reduced at 10 and 100 mg/kg/day but unaffected at 200 mg/kg/day. Organ weights relative to brain weights were unaffected for the thymus, heart, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, prostate, and seminal vesicles with coagulating glands. Relative liver weight was significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS):
At scheduled necropsy of F0 females, there were no differences among groups for terminal body weights and organ weights (absolute, relative to terminal body weights, or relative terminal brain weights) for the brain, heart, liver, spleen, paired kidneys, paired adrenal ,lands, uterus with cervix and vagina, and paired ovaries. Absolute and relative (to both body and brain weights) weights of the thymus were significantly increased at 100 mg/kg/day and unaffected at 10 or 200 mg/kg/day.
No treatment-related gross necropsy findings were observed. No treatment related microscopic findings in any females (of 5/group) at 200 mg/kg/day were observed.

OTHER FINDINGS (PARENTAL ANIMALS):
FOB: No parameters evaluated in the FOB exhibited any statistically significant or biologically relevant difference across male F0 groups. There was also no effect of treatment on motor activity, auditory startle, or grip strength which were performed in week 4 prior to scheduled termination.
For female F0 groups the week 1 pupil size score was significantly reduced in the 200 mg/kg/day dose group and in week 3 the tail pinch score was also reduced in this dose group. No other parameters were affecteed at any time period in any dose group.


RESULTS for F1 Offspring:
CLINICAL SIGNS (OFFSPRING):
Treatment related clinical observations in F1 males included rooting postdosing, occurred in 2,2,7 and 9 F1 males at 0,10, 100 and 200 mg/kg/day, respectively. Salivating pre-/post dosing was observed in 4 males only at 100 mg/kg/day.
In F1 females, 7 females each in the 100 and 200 mg/kg/day exhibited rooting and 1,4 and 2 females in the 10, 100 and 200 mg/kg/day exhibited salivation post dosing, respectively.

BODY WEIGHT (OFFSPRING):
Body weight change values were unaffected for all F1 male groups for all intervals from pnd 22 through 78. There were no differences in feed consumption for any postwean interal in any group.
On pnd 22, the mean F1 female body weight at 200 mg/kg/day was significantly reduced with no effects at any later time points at this dose or any time points at any other doses.

SEXUAL MATURATION (OFFSPRING):
F1 male age at PPS was unaffected across all dose groups. F1 female age acquisition of vaginal patency was equivalent across all groups. Estrous cycle length monitored for the last 3 weeks of the post wean period for the F1 females, were equivalent across all the dose groups.

CLINICAL CHEMISTRY and HEMATOLOGY (OFFSPRING):
In F1 females blood urea nitrogen, creatinine, total protein, albumin, total cholesterol, aspartate aminotransferase, alanine aminotransferase, sodium, potassium, and chloride were unaffected by treatment. Blood glucose was significantly elevated at 200 mg/kg/day which was not considered treatment related due to the magnitude of the change, lack of effects on other parameters, and lack of similar effects in FO females and males. White blood cell count, nucleated red blood cell count, corrected white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet count, mean platelet volume, segmented neutrophils, lymphocytes, monocytes, eosinophils and prothombin time were alos unaffected by treatment.

OTHER FINDINGS (OFFSPRING):
FOB which was performed midway through the postweaning period for 5 of the F1 males and females/group showed no significant difference for home cage observations, handling observations, sensory and neuromuscular observations, or open field observations.Grip strength was also evaluated. Forelimb grip strength was unaffected across groups. Hindlimb grip strength was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no indications of toxicity (systemic, reproductive, developmental, or neurological) in either the paraental or F1 generation, the recovery males or females or the 28 day female dose groups evaluated in this study.
Critical effects observed:
not specified

RECOVERY MALES:

FOB analysis for the recovery males (5/group at 0 and 200 mg/kg/day) during quarantine indicated no effects on body weight or on any parameters evaluated as part of the FOB assessment. High-dose recovery males exhibited no effects on body weights on sd 0, 7, 14, 21, and 27 during the 4-week treatment period or on sd 34 or 41 during the 2-week recovery period. Body weight gains were similarly unaffected in the high-dose recovery males for all intervals during the 28-day dosing period and the 2-week recovery period. The only treatment-related clinical sign during the dosing phase was rooting postdosing in 3 males at 200 mg/kg/day. FOB evaluations during weeks 1 through 4 and week 7 of the recovery males indicated no differences in body weights or in any parameters evaluated as part of the FOB assessment between males at 0 and 200 mg/kg/day. Necropsy of recovery males (5/group at 0 and 200 mg/kg/day) occurred after the 2-week recovery period. No treatment-related effects were observed. Terminal body weights and all organ weights (absolute and relative to terminal body weight and to terminal brain weight) were equivalent between the 2 groups except for paired adrenal glands; absolute weight and weight relative to terminal body weight were significantly reduced relative to the control values. Paired adrenal weight relative to brain weight was equivalent between the 2 groups. Since there was no effect on adrenal gland weight following 28 days of dosing, this difference in the recovery group was considered due to random biological variation. No gross lesions were observed in the organs examined from the recovery males. No histopathology was performed.

RECOVERY FEMALES:

The FOB performed during the quarantine period on the F0 recovery females (at 0 and 200 mg/kg/day, 5/group) indicated no differences between the 2 groups for body weights or any of the parameters evaluated in the FOB assessment.

The body weights of the recovery females during exposure (sd 0 through 27) and in the recovery period (sd 28 through 41) were equivalent between the 2 groups for all time points examined (sd 0, 7, 14, 21, 27, 34, and 41). Body weight changes for all intervals were equivalent between the 2 groups except for sd 34-41 when the recovery female mean body weight change at 200 mg/kg/day was significantly higher than the value at 0 mg/kg/day.

Treatment-related clinical observations of the recovery females were limited to rooting postdosing in all 5 females at 200 mg/kg/day. FOB evaluations were performed on the recovery females once per week during the 4 -week exposure period and once (week 7) during the 2-week recovery period. For all 5 of these FOB evaluations, there were no differences between groups for mean body weights or for any of the parameters evaluated in the FOB assessment.

At scheduled necropsy of the recovery females at the end of the 2-week recovery period, there were no differences between groups (0 and 200 mg/kg/day, 5/group) for terminal body weights or for the weights of any organs, absolute, relative to terminal body weights, or relative to terminal brain weights. There were no treatment-related gross findings at necropsy. No histopathology was performed.

Conclusions:
In conclusion, the test substance, administered by gavage once daily at 0, 10, 100, and 200 mg/kg/day to parental rats, from prebreed through mating, gestation, and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice, resulted in no adult F0 parental toxicity and no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals.
Therefore, systemic NOAEL was at least 200 mg/kg/day.
A justification for the use of read-across has been provided.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
With the key study being of high quality, the overall quality of the database is considered to be good. In addition to the key study, 3 supporting studies are provided. All were awarded a reliability score of 3 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997) due to being non-guideline studies with limited information provided.

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:
June 16, 1989 to May 7, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented non-guideline study performed under GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
14 day inhalation study with exposure 5 days/week.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc., Kingston, NY
- Age at study initiation: 5-6 weeks (at receipt)
- Weight at study initiation: 134.4-160.5 g (males at randomization); 109.0-122.8 g (females at randomization)
- Housing: Animals were individually housed in stainless steel wire mesh cages which were located in a Hazleton 1000, whole-body, inhalation exposure chamber. One cage unit was used to house all animals assigned to a particular exposure group.
- Diet: ad libitum (during non-exposure periods)
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67-77 degrees F
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 16, 1989 To: June 30, 1989
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Pre-animal exposure characterization: The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD were 0.72, 1.06 and 1.42, respectively.
System performance during study: Count median aerodynamic diameters ranged between 0.56 um and 0.68 um, and GSD values were between 1.22 and 1.41. The corresponding calculated mass median aerodynamic diameters ranged between 0.64 um and 0.90 um.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H1000, one cubic meter stainless steel and glass chambers were used.
- Method of holding animals in test chamber: Animal cages were housed within the exposure chambers.
- System of generating particulates/aerosols: The primary component of the generation system was a Sonimist spray nozzle. A fine stream of heated liquid test substance was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of pressed air producing sonic disturbance. The resulting spray of fine droplets vaporized. As the vapor cooled to room temperature, a fine condensation aerosol formed.
The secondary component of the generation system consisted of a test substance reservoir that was heated and pressurized for the delivery of the test substance to the Sonimist Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and manual pressure relief valve (in addition to the factory installed automatic pressure relief valve). Both the reservoir and the liquid feed line were wrapped with heat tape to maintain the necessary temperature of the test substance in a liquid state until it was vaporized. In addition, the carrier air line and the Sonimist nozzle were wrapped with heat tape. The temperature at several locations of the generation was controlled by variacs and was measured by type 'T' thermocouples at the following locations: reservoir core, reservoir skin, nozzle skin and carrier air line skin. The Sonimist nozzle discharged directly into a 1.3 cubic meter plenum chamber. This plenum chamber allowed the Sonicated aerosol to vaporize and achieve equilibrium at room temperature.
The test substance atmosphere within the plenum chamber was transported into a single manifold system that supplied the three exposure chambers. The total concentration of test substance aerosol in the plenum chamber and manifold air was approximately the same as that used for the high exposure level. The target concentrations for the 2 lower exposure levels were achieved by diverting a metered fraction of manifold air into the exposure chamber and diluting it with HEPA/activated charcoal filtered room air.
- Temperature, humidity, pressure in air chamber: 72 +/- 5 degrees F; 55 +/- 15 % humidity
- Air flow rate: 15+/-2
- Method of particle size determination: Particle size distributions were measured and calculated using an APS 3310 Aerodynamic Particle Sizer with a 100:1 dilutor. During the pre-study development, particle size distributions were determined twice in each exposure chamber and twice in the plenum chamber. During the animal exposure, particle size distributions were measured once per exposure chamber per day.
- Treatment of exhaust air: Exhaust air passed through Cambridge Sidlock HEPA and Prefilter Assemblies to remove particulate before combining the outflow into a Mystaire scrubber utilizing a 50:50 mixture of chlorine bleach and water. The scrubbed air was then vented outside the building.

TEST ATMOSPHERE
- Brief description of analytical method used: Because generation of test substance atmospheres produced both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow control was used to draw air samples from each chamber. These samples were first drawn through a 25 mm glassfiber filter mounted in an open faced Delrin filter holder attached to a sample line inserted into the chamber near the breathing zone of the animals. The glass fiber filtered the particulate material while the vapor phase material remained in the sampling air stream and was directed into a Miran 1A Infrared Analyzer where the total vapor concentration was measured. The sample flow rate was set at 5 L per minute while sample duration varied according to chamber test substance concentration. Gravimetric mass was calculated from the filter weight gain and the sample volume. Vapor phase concentration was determined by measuring the absorbance of each sample and mathematically regressing the absorbance value from a calibration curve to calculate the mass vapor concentration. The two values, gravimetric and vapor concentrations, were combined to give the total mass concentration.
During the pre-study development, techniques were defined to verify the chamber test substance concentrations by chemical analysis. Gas chromatography of impinger samples was the chosen technique. One hundred twenty five mL gas washing bottle with glass fronts were filled with 100 mL of reagent grade methanol. Two impingers in series were placed into a wet ice bath and a 5 liter per minute sample was drawn from the exposure chamber. Simultaneous samples were collected with the gravimetric/Miran system. After the completion of the impinger sampling, the sample lines were back flushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph. A Varian Model 6000 gas chromatography was used with 30m x 0.75mm Supelco SPB-1 column with a 1.5 micron film thickness. The oven was set at 70 degrees C with the injector and detector at 90 degrees C. The carrier flow was at 20 mL/min. Injection volumes were constant at ~0.6 uL. Actual impinger samples were obtained from each exposure level during the second exposure day of the animal study to verify the results of the gravimetric/Miran atmosphere measurement system.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pre-animal exposure characterization: The total exposure chamber mass concentrations were within an average of 16.2% of target values and reproducible on a daily basis, with a <25% RSD. The mean concentration values for the 67, 200 and 670 mg/m3 groups were 84.4, 227 and 731, respectively, averaged over the three separate 6-hour trial generation periods. The greatest RSD was 22.6%, measured in the 200 mg/m3 chamber, while the greatest deviation from target concentration values was 26% which occurred in the 67 mg/m3.
System performance during study: All mean total concentration values had relative standard deviations of less than 6 %. Deviations from targeted concentrations were all less than or equal to 5 %. Within each chamber all mean vapor phase concentrations had RDSs of less than 6%. Results showed there was good agreement between gravimetric/vapor determination and GC determination methods. The largest variance between the two was 17%.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Animals were treated 5 days/week for a total of 10 exposures, conducted within 14 days.
Remarks:
Doses / Concentrations:
67, 200 and 670 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Concentrations were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for clinical evidence of toxicity or other abnormalities. Evaluations included a thorough examination of the animal’s general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behaviour, coordination and general neuro-muscular function.

BODY WEIGHT: Yes
- Time schedule for examinations: Study Day 1, prior to the first exposure, and again on Study Days 8, 14 and prior to necropsy.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes; propylene glycol-free sodium pentobarbital
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood samples was analyzed for the following: red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, and platelet count. Smears for differential cells counts were made. The absolute number of each cell type per microliter was calculated. The number of nucleated red blood cells per 100 white blood cell was determined. The total reticulocyte count was determined after red blood cells were pre-stained and a smear prepared. In addition, the number of reticulocytes per 100 red blood cells was counted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood sample was analyzed for the following: blood urea nitrogen, creatinine, fasting glucose, total protein, albumin, globulin, cholesterol, lactate dehydrogenase, serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, bilirubin, calcium, sodium, potassium, chloride, and phosphorus.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Each animal was given a complete gross examination, with special attention given to the lungs and upper respiratory tract. Organs weighed from all animals were: liver, lung, kidneys (pair), and heart (excluding major vessel). Organ/body weight ratios were calculated. The following tissues were carefully examined, dissected from the carcass and preserved: liver, ling, kidney, trachea, heart, nasal cavity and mainstem bronchi.
HISTOPATHOLOGY: Yes; The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (2 cross-sections), and trachea (cross and longitudinal sections) and all gross lesions suspected to be exposure related were examined. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
Statistics:
Normally distributed data (parametric) was analyzed for treatment effects by analysis of variance and pairwise comparisons made between groups using Dunnett’s multiple range t-test. Non-parametric data was analyzed by the Kruskal Wallis test and by the Mann-Whitney U Test for pairwise group comparisons.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY-Clinical signs of toxicity were confined to male and female rats in the high dose group. A red nasal discharge was observed in all animals in the high dose group, beginning on Study Day 1. This condition was apparent at the end of the 6-hour exposure period and would abate overnight, with the animals appearing nearly normal the next morning. This was noted by the authors to be a sign of upper respiratory tract irritation and ulceration. No other findings were observed.

BODY WEIGHT AND WEIGHT GAIN-The mid and high dose males had group mean body weight values that were significantly decreased relative to control; -5.6 % and -10.2 %, respectively on Study Day 8 and -6.2 % and /13.1 %, respectively on study Day 14. The high dose females had a group mean body weight value that was significantly less than the female control group (-8.8% on Study Day 8 and -11.2 % on Study Day 14). No clinical signs indicative of emaciation or thinness were apparent in animals showing decreased weight gains relative to those of control animals.
The terminal body weights of males in the mid and high dose groups were significantly less (p
HAEMATOLOGY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

CLINICAL CHEMISTRY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

ORGAN WEIGHTS- Absolute kidney weight value of male and female rats in the high dose group were significantly greater (pThere was a significant increase (pThe high dose female group had a significantly elevated (pThe male and female high dose groups had significantly increased liver to body weight ratios.

GROSS PATHOLOGY-There were few and only incidental gross tissue changes present in the study animals. The gross lesions that occurred were sporadic and were not considered exposure related. These lesions were common spontaneous lesions that were expected to occur in a group of Fischer 344 rats of this age range.

HISTOPATHOLOGY: NON-NEOPLASTIC-Lesions considered to be related to the treatment were confined to the nasal cavity of the high dose males and females and were present in the same anatomical location and at essentially the same degree of severity from both sexes. These changes were indentified in levels II, III, and IV of the nasal cavity with level IV showing the most extensive changes. Specifically, these lesions involved the olfactory epithelium lining the dorsal meatus, dorsal portion of the nasal septum, and the adjacent ethmoid turbinates. Morphologically, the lesions consisted of necrosis/degeneration of the affected olfactory epithelium. This was most severe and extensive in the area of the dorsal meatus and nasal septum. The lesions were more focal to multifocal where it involved the epithelium covering the adjacent ethmoid turbinates. In conjunction with the olfactory epithelial necrosis/degeneration was a moderate degree of a serofibrinous/suppurative inflammation which was also most severe and extensive in the immediate area of the dorsal meatus. The overall thickness of the affected regions of olfactory epithelium decreased in proportion to the amount of involvement of the inflammatory, necrotic and degenerative lesions and ranged from a slight change in the less affected regions, to a gradual thinning into a single layer of cuboidal cells, and finally to complete denudation in the most severely affected areas involving the dorsal meatus.
In some areas, the affected mucosa had changes consistent with a diagnosis of squamous metaplasia. The submucosa in the region of the dorsal meatus was thickened, secondary to the presence of inflammatory exudates, edema fluid, and proliferating loose fibrous connective tissue. In many areas the involved olfactory epithelium had inverted or became trapped in the underlying submucosa where it formed pseudorosettes or pseudoacinar/glandular structures which were lined by columnar, cuboidal, or squamoid appearing olfactory epithelium. Many of these structures were ectatic to varying degrees and contained inflammatory debris and protein aceous fluid within the lumen. Six of the high dose females and three of the high dose males had bilateral adhesions between the adjacent ethmoid turbinates and the dorsal portion of the nasal septum and or the wall of the dorsal meatus. These adhesions were becoming organized through the proliferation of dense fibrous connective tissue and most probably would have remained as a permanent malformation in the nasal cavity if exposure was discontinued and resolution of the necroinflammatory process was allowed to take place.
Although not diagnosed separately, olfactory nerve fibers were occasionally noted to be somewhat vacuolated in appearance, usually in association with areas of significant inflammation. Whether this represented fixation artifact, or early signs of neural degeneration, either as a direct result of treatment or secondarily as a result of the inflammation, could not be definitely ascertained.
After examining the respiratory tract tissues from the high dose and air control rats, these same tissue sections collected from the low and mid dose rats of both sexes were trimmed, processed, stained and examined in an attempt to establish a NOEL. All tissues required to be examined histopathologically were present from the low and mid dose rats with the exception of the larynx from one mid dose female, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high dose animals were not present in the low and mid dose rats. Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used was determined to be 200 mg/m3.
Small, focal, subpleural aggregations of lymphocytes/macrophages were also seen in the lungs of treated rats of both sexes. However, these changes were also seen in control male and female rats and therefore, were considered to be an incidental finding and not related to treatment. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract were observed.



Dose descriptor:
NOAEC
Effect level:
200 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEC
Effect level:
670 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced body weight and red nasal discharge upon exposure which was deemed indicative of upper respiratory tract irritation and ulceration. This was supported by histopathology.
Critical effects observed:
not specified
Conclusions:
Male and female rats were treated with 2,6-xylenol (67, 200, 670 mg/m3) by whole body inhalation for 6 hours/day, 5 days/week, for a total of ten exposure, conducted within 14 days. The NOAEC was determined to be 200 mg/m3. The LOEC was determined to be 670 mg/m3.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was not conducted in accordance with a standardised guideline though it was well reported and carried out under GLP conditions. The duration was only 14 days. The study was awarded a reliability sore of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

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:
June 16, 1989 to May 7, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented non-guideline study performed under GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
14 day inhalation study with exposure 5 days/week.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc., Kingston, NY
- Age at study initiation: 5-6 weeks (at receipt)
- Weight at study initiation: 134.4-160.5 g (males at randomization); 109.0-122.8 g (females at randomization)
- Housing: Animals were individually housed in stainless steel wire mesh cages which were located in a Hazleton 1000, whole-body, inhalation exposure chamber. One cage unit was used to house all animals assigned to a particular exposure group.
- Diet: ad libitum (during non-exposure periods)
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67-77 degrees F
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 16, 1989 To: June 30, 1989
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Pre-animal exposure characterization: The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD were 0.72, 1.06 and 1.42, respectively.
System performance during study: Count median aerodynamic diameters ranged between 0.56 um and 0.68 um, and GSD values were between 1.22 and 1.41. The corresponding calculated mass median aerodynamic diameters ranged between 0.64 um and 0.90 um.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H1000, one cubic meter stainless steel and glass chambers were used.
- Method of holding animals in test chamber: Animal cages were housed within the exposure chambers.
- System of generating particulates/aerosols: The primary component of the generation system was a Sonimist spray nozzle. A fine stream of heated liquid test substance was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of pressed air producing sonic disturbance. The resulting spray of fine droplets vaporized. As the vapor cooled to room temperature, a fine condensation aerosol formed.
The secondary component of the generation system consisted of a test substance reservoir that was heated and pressurized for the delivery of the test substance to the Sonimist Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and manual pressure relief valve (in addition to the factory installed automatic pressure relief valve). Both the reservoir and the liquid feed line were wrapped with heat tape to maintain the necessary temperature of the test substance in a liquid state until it was vaporized. In addition, the carrier air line and the Sonimist nozzle were wrapped with heat tape. The temperature at several locations of the generation was controlled by variacs and was measured by type 'T' thermocouples at the following locations: reservoir core, reservoir skin, nozzle skin and carrier air line skin. The Sonimist nozzle discharged directly into a 1.3 cubic meter plenum chamber. This plenum chamber allowed the Sonicated aerosol to vaporize and achieve equilibrium at room temperature.
The test substance atmosphere within the plenum chamber was transported into a single manifold system that supplied the three exposure chambers. The total concentration of test substance aerosol in the plenum chamber and manifold air was approximately the same as that used for the high exposure level. The target concentrations for the 2 lower exposure levels were achieved by diverting a metered fraction of manifold air into the exposure chamber and diluting it with HEPA/activated charcoal filtered room air.
- Temperature, humidity, pressure in air chamber: 72 +/- 5 degrees F; 55 +/- 15 % humidity
- Air flow rate: 15+/-2
- Method of particle size determination: Particle size distributions were measured and calculated using an APS 3310 Aerodynamic Particle Sizer with a 100:1 dilutor. During the pre-study development, particle size distributions were determined twice in each exposure chamber and twice in the plenum chamber. During the animal exposure, particle size distributions were measured once per exposure chamber per day.
- Treatment of exhaust air: Exhaust air passed through Cambridge Sidlock HEPA and Prefilter Assemblies to remove particulate before combining the outflow into a Mystaire scrubber utilizing a 50:50 mixture of chlorine bleach and water. The scrubbed air was then vented outside the building.

TEST ATMOSPHERE
- Brief description of analytical method used: Because generation of test substance atmospheres produced both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow control was used to draw air samples from each chamber. These samples were first drawn through a 25 mm glassfiber filter mounted in an open faced Delrin filter holder attached to a sample line inserted into the chamber near the breathing zone of the animals. The glass fiber filtered the particulate material while the vapor phase material remained in the sampling air stream and was directed into a Miran 1A Infrared Analyzer where the total vapor concentration was measured. The sample flow rate was set at 5 L per minute while sample duration varied according to chamber test substance concentration. Gravimetric mass was calculated from the filter weight gain and the sample volume. Vapor phase concentration was determined by measuring the absorbance of each sample and mathematically regressing the absorbance value from a calibration curve to calculate the mass vapor concentration. The two values, gravimetric and vapor concentrations, were combined to give the total mass concentration.
During the pre-study development, techniques were defined to verify the chamber test substance concentrations by chemical analysis. Gas chromatography of impinger samples was the chosen technique. One hundred twenty five mL gas washing bottle with glass fronts were filled with 100 mL of reagent grade methanol. Two impingers in series were placed into a wet ice bath and a 5 liter per minute sample was drawn from the exposure chamber. Simultaneous samples were collected with the gravimetric/Miran system. After the completion of the impinger sampling, the sample lines were back flushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph. A Varian Model 6000 gas chromatography was used with 30m x 0.75mm Supelco SPB-1 column with a 1.5 micron film thickness. The oven was set at 70 degrees C with the injector and detector at 90 degrees C. The carrier flow was at 20 mL/min. Injection volumes were constant at ~0.6 uL. Actual impinger samples were obtained from each exposure level during the second exposure day of the animal study to verify the results of the gravimetric/Miran atmosphere measurement system.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pre-animal exposure characterization: The total exposure chamber mass concentrations were within an average of 16.2% of target values and reproducible on a daily basis, with a <25% RSD. The mean concentration values for the 67, 200 and 670 mg/m3 groups were 84.4, 227 and 731, respectively, averaged over the three separate 6-hour trial generation periods. The greatest RSD was 22.6%, measured in the 200 mg/m3 chamber, while the greatest deviation from target concentration values was 26% which occurred in the 67 mg/m3.
System performance during study: All mean total concentration values had relative standard deviations of less than 6 %. Deviations from targeted concentrations were all less than or equal to 5 %. Within each chamber all mean vapor phase concentrations had RDSs of less than 6%. Results showed there was good agreement between gravimetric/vapor determination and GC determination methods. The largest variance between the two was 17%.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Animals were treated 5 days/week for a total of 10 exposures, conducted within 14 days.
Remarks:
Doses / Concentrations:
67, 200 and 670 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Concentrations were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for clinical evidence of toxicity or other abnormalities. Evaluations included a thorough examination of the animal’s general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behaviour, coordination and general neuro-muscular function.

BODY WEIGHT: Yes
- Time schedule for examinations: Study Day 1, prior to the first exposure, and again on Study Days 8, 14 and prior to necropsy.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes; propylene glycol-free sodium pentobarbital
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood samples was analyzed for the following: red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, and platelet count. Smears for differential cells counts were made. The absolute number of each cell type per microliter was calculated. The number of nucleated red blood cells per 100 white blood cell was determined. The total reticulocyte count was determined after red blood cells were pre-stained and a smear prepared. In addition, the number of reticulocytes per 100 red blood cells was counted.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes
- How many animals: all
- Parameters examined: Each blood sample was analyzed for the following: blood urea nitrogen, creatinine, fasting glucose, total protein, albumin, globulin, cholesterol, lactate dehydrogenase, serum alanine aminotransferase, serum aspartate aminotransferase, alkaline phosphatase, bilirubin, calcium, sodium, potassium, chloride, and phosphorus.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Each animal was given a complete gross examination, with special attention given to the lungs and upper respiratory tract. Organs weighed from all animals were: liver, lung, kidneys (pair), and heart (excluding major vessel). Organ/body weight ratios were calculated. The following tissues were carefully examined, dissected from the carcass and preserved: liver, ling, kidney, trachea, heart, nasal cavity and mainstem bronchi.
HISTOPATHOLOGY: Yes; The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (2 cross-sections), and trachea (cross and longitudinal sections) and all gross lesions suspected to be exposure related were examined. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
Statistics:
Normally distributed data (parametric) was analyzed for treatment effects by analysis of variance and pairwise comparisons made between groups using Dunnett’s multiple range t-test. Non-parametric data was analyzed by the Kruskal Wallis test and by the Mann-Whitney U Test for pairwise group comparisons.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY-Clinical signs of toxicity were confined to male and female rats in the high dose group. A red nasal discharge was observed in all animals in the high dose group, beginning on Study Day 1. This condition was apparent at the end of the 6-hour exposure period and would abate overnight, with the animals appearing nearly normal the next morning. This was noted by the authors to be a sign of upper respiratory tract irritation and ulceration. No other findings were observed.

BODY WEIGHT AND WEIGHT GAIN-The mid and high dose males had group mean body weight values that were significantly decreased relative to control; -5.6 % and -10.2 %, respectively on Study Day 8 and -6.2 % and /13.1 %, respectively on study Day 14. The high dose females had a group mean body weight value that was significantly less than the female control group (-8.8% on Study Day 8 and -11.2 % on Study Day 14). No clinical signs indicative of emaciation or thinness were apparent in animals showing decreased weight gains relative to those of control animals.
The terminal body weights of males in the mid and high dose groups were significantly less (p
HAEMATOLOGY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

CLINICAL CHEMISTRY- While there were some statistically significant changes in the measured parameters, these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rat. Therefore, none of the variations were considered to be test substance related.

ORGAN WEIGHTS- Absolute kidney weight value of male and female rats in the high dose group were significantly greater (pThere was a significant increase (pThe high dose female group had a significantly elevated (pThe male and female high dose groups had significantly increased liver to body weight ratios.

GROSS PATHOLOGY-There were few and only incidental gross tissue changes present in the study animals. The gross lesions that occurred were sporadic and were not considered exposure related. These lesions were common spontaneous lesions that were expected to occur in a group of Fischer 344 rats of this age range.

HISTOPATHOLOGY: NON-NEOPLASTIC-Lesions considered to be related to the treatment were confined to the nasal cavity of the high dose males and females and were present in the same anatomical location and at essentially the same degree of severity from both sexes. These changes were indentified in levels II, III, and IV of the nasal cavity with level IV showing the most extensive changes. Specifically, these lesions involved the olfactory epithelium lining the dorsal meatus, dorsal portion of the nasal septum, and the adjacent ethmoid turbinates. Morphologically, the lesions consisted of necrosis/degeneration of the affected olfactory epithelium. This was most severe and extensive in the area of the dorsal meatus and nasal septum. The lesions were more focal to multifocal where it involved the epithelium covering the adjacent ethmoid turbinates. In conjunction with the olfactory epithelial necrosis/degeneration was a moderate degree of a serofibrinous/suppurative inflammation which was also most severe and extensive in the immediate area of the dorsal meatus. The overall thickness of the affected regions of olfactory epithelium decreased in proportion to the amount of involvement of the inflammatory, necrotic and degenerative lesions and ranged from a slight change in the less affected regions, to a gradual thinning into a single layer of cuboidal cells, and finally to complete denudation in the most severely affected areas involving the dorsal meatus.
In some areas, the affected mucosa had changes consistent with a diagnosis of squamous metaplasia. The submucosa in the region of the dorsal meatus was thickened, secondary to the presence of inflammatory exudates, edema fluid, and proliferating loose fibrous connective tissue. In many areas the involved olfactory epithelium had inverted or became trapped in the underlying submucosa where it formed pseudorosettes or pseudoacinar/glandular structures which were lined by columnar, cuboidal, or squamoid appearing olfactory epithelium. Many of these structures were ectatic to varying degrees and contained inflammatory debris and protein aceous fluid within the lumen. Six of the high dose females and three of the high dose males had bilateral adhesions between the adjacent ethmoid turbinates and the dorsal portion of the nasal septum and or the wall of the dorsal meatus. These adhesions were becoming organized through the proliferation of dense fibrous connective tissue and most probably would have remained as a permanent malformation in the nasal cavity if exposure was discontinued and resolution of the necroinflammatory process was allowed to take place.
Although not diagnosed separately, olfactory nerve fibers were occasionally noted to be somewhat vacuolated in appearance, usually in association with areas of significant inflammation. Whether this represented fixation artifact, or early signs of neural degeneration, either as a direct result of treatment or secondarily as a result of the inflammation, could not be definitely ascertained.
After examining the respiratory tract tissues from the high dose and air control rats, these same tissue sections collected from the low and mid dose rats of both sexes were trimmed, processed, stained and examined in an attempt to establish a NOEL. All tissues required to be examined histopathologically were present from the low and mid dose rats with the exception of the larynx from one mid dose female, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high dose animals were not present in the low and mid dose rats. Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used was determined to be 200 mg/m3.
Small, focal, subpleural aggregations of lymphocytes/macrophages were also seen in the lungs of treated rats of both sexes. However, these changes were also seen in control male and female rats and therefore, were considered to be an incidental finding and not related to treatment. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract were observed.



Dose descriptor:
NOAEC
Effect level:
200 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOEC
Effect level:
670 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced body weight and red nasal discharge upon exposure which was deemed indicative of upper respiratory tract irritation and ulceration. This was supported by histopathology.
Critical effects observed:
not specified
Conclusions:
Male and female rats were treated with 2,6-xylenol (67, 200, 670 mg/m3) by whole body inhalation for 6 hours/day, 5 days/week, for a total of ten exposure, conducted within 14 days. The NOAEC was determined to be 200 mg/m3. The LOEC was determined to be 670 mg/m3.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was not conducted in accordance with a standardised guideline though it was well reported and carried out under GLP conditions. The duration was only 14 days. The study was awarded a reliability sore of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be good.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

In the key study, the repeated dose toxicity of 2,4,6 trimethyl phenol, a structural analogue of the registered material was investigated in a study conducted in accordance with the standardised guideline OECD 422 under GLP conditions. A subset of the animals tested was evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). 2,6-Xylenol and the structural analogue were determined to have sufficiently similar properties, such that available data on the structural analogue is considered to be suitable to address this endpoint. Due to the use of read across, the study was awarded a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Male and female Sprague-Dawley rats were exposed to the test material at concentrations of 0, 10, 100, 200 mg/kg/day in corn oil via oral gavage. F0 females were dosed premating through to the day prior to necropsy. Recovery males, females and 28 days females were dosed for 28 days. F1 animals were dosed post-weaning until the day before scheduled necropsy (duration of at least 7 weeks).

In the F0 generation, 10 males and 10 females were dosed, plus 5 additional animals per sex in the control and high dose groups which were designated recovery animals. In addition, 10 females (5 control and 5 in the high dose group) were designated as the 28 day females and were dosed for 28 days.

Parental animals were subjected to cage-side observations and detailed clinical observations. Body weight and food consumption were measured.

There were no indications of toxicity (systemic, reproductive, developmental, or neurological) in the parental or F1 generation, the recovery males or females or the 28 day female dose groups evaluated in this study. The only effects were a post-dose rooting seen at the highest dose group. 

Under the conditions of this study the NOAEL was therefore determined to be at least 200 mg/kg bw.

 

The first supporting study provided is an 8 month oral study evaluating the toxicological effects of 2,6 xylenol in White rats at 2% and 0.2 % of the LD50 6 and 0.6 mg/kg).

Body weight, peripheral blood analyses, blood pressure (before and after adrenalin injection), SH groups of blood serum, proteins fractions in blood serum, antitoxic activity of the liver, excretion of free conjugated phenols in urine, effects on CNS, ability to summarize subthreshold impulses, SH groups in internal organs and Vitamin C in adrenals were evaluated.

Rats administered 6 mg/kg showed significant changes in body weight, pre-adrenalin injection blood pressure, SH groups in the blood serum and internal organs, and pathomorphological changes of internal organs. No changes were observed in white rats administered 0.6 mg/kg.

 

In the second supporting study, a dose 100 times the allowable daily intake was evaluated for toxic effects after oral administration in albino rats over 90 days when administered in the feed. The average daily intake was 5.99 and 6.85 mg/kg males and females, respectively.

The body weights of individual rats, food consumption and efficiency were recorded. Haematological examination and blood urea determinations were carried out. At autopsy, the liver and kidneys were weighed and gross and histological examinations were carried out on a wide range of organs.

2,6 xylenol fed to rats for 90 days at more than 100 times the equivalent of the human daily intake had no adverse effect on growth, food intake, haematological and clinical chemistry parameters, organ weights or organ pathology. The NOEL was determined to be greater than or equal to 6.85 mg/kg-bw/day for females and greater than or equal to 5.99 mg/kg-bw/day for males.

 

The third supporting study evaluated general toxicity when the test material was administered for 8 months to albino male rats at dose levels of 6 and 0.06 mg/kg bw/day.

Parameters and data points that were examined within this study included animal behavior and weight, the morphologial composition of the blood, the content of SH groups in the blood serum, blood pressure with and without an adrenalin load, oxygen consumption and the concentration of phenols in the urine, as well as conditioned reflexes. In addition, post mortem investigations were performed of SH groups in the tissues, ascorbic acid in the adrenals, and the weight coefficients and histological appearance of the internal organs.

Weight loss, blood pressure increase, pathological changes in the liver, kidney and spleen were seen at 6 mg/kg/day, therefore the reported NOAEL was 0.06 mg/kg bw.

It is considered to be scientifically unjustified to conduct the 90 day repeated dose toxicity study via the oral route required under section 8.6.2 of Annex IX and the further long-term repeated toxicity study specified under section 8.6.3 of Annex X.

Adequate data to address the 28 day endpoint is provided on an extremely close structural analogue of the substance. It is therefore considered by the registrant unlikely that further vertebrate animal studies would provide significant new information, given that it has been possible to set a precautionary DNEL based upon the results of the 28 day read across study. The calculated RCR values show a large margin of safety when derived using a Tier 1 model with minimal refinement.

Furthermore, the substance is used as an intermediate. Although the material is not registered as an intermediate used under strictly controlled conditions, it is nonetheless used under highly controlled conditions and this is considered to mitigate the vast majority of potential exposure during the life cycle of the substance.

It is therefore considered that in accordance with section 3.2 of Annex XI, point a, the RCR values determined for the substance are always sufficiently low to mitigate the testing.

Inhalation

The toxicity of the test material via the inhalation route was investigated in a well-documented, non-guideline study performed under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Male and female Fischer 344 rats were exposed to dose levels of 67, 200 and 670 mg/m³ (10 rats per sex per dose) in a 14 day inhalation study with exposure 5 days per week. The test material was administered as an aerosol in air. The rats were subjected to the test material 6 hours a day using a whole body exposure technique.

The rats were subjected to detailed clinical observations. Body weight, haematology and clinical chemistry parameters were measured. Following sacrifice, gross and histopathological examinations took place. 

Clinical signs of toxicity were confined to rats in the high dose group. A red nasal discharge was observed in all animals, noted to be a sign of upper respiratory tract irritation and ulceration. Mid and high dose males and high dose females showed reduced body weight. No effects were noted on haematology and clinical chemistry parameters.

Absolute kidney weight was increased in the high dose group. Increased organ to body weight ratios were also increased for the kidney, heart and liver. The lung to body weight ratio was increased for high dose females only.

No gross pathology findings were noted. Lesions considered to be related to the treatment were confined to the nasal cavity of the high dose males and females and were present in the same anatomical location and at essentially the same degree of severity from both sexes. Histopathologically, the lesions involving the nasal cavities seen in the high dose animals were not present in the low and mid dose rats.

Under the conditions of this study the NOEL for rats of both sexes was determined to be 200 mg/m³. The LOEC was determined to be 670 mg/m³.

It is considered to be scientifically unjustified to conduct the 28 and 90 day repeated dose toxicity studies via the inhalation route required under section 8.6 of Annexes VIII and IX.

The available data provided for the oral route of exposure and the 14 day inhalation study are considered to be adequate to allow robust risk assessment by the inhalation route. Therefore further long-term testing for the inhalation route is not considered to be necessary.

Furthermore, the substance is used as an intermediate. Although the material is not registered as an intermediate used under strictly controlled conditions, it is nonetheless used under highly controlled conditions and this is considered to mitigate the vast majority of potential exposure during the life cycle of the substance.

It is therefore considered that in accordance with section 3.2 of Annex XI, point a, the RCR values determined for the substance are always sufficiently low to mitigate the testing.

Dermal

It is considered to be scientifically unjustified to conduct the 28 and 90 day repeated dose toxicity studies via the dermal route required under section 8.6 of Annexes VIII and IX.

The available data provided for the oral route of exposure and the 14 day inhalation study are considered to be adequate to allow robust risk assessment by the dermal route. Therefore further long-term testing for the dermal route is not considered to be necessary.

Furthermore, the substance is used as an intermediate. Although the material is not registered as an intermediate used under strictly controlled conditions, it is nonetheless used under highly controlled conditions and this is considered to mitigate the vast majority of potential exposure during the life cycle of the substance.

It is therefore considered that in accordance with section 3.2 of Annex XI, point a, the RCR values determined for the substance are always sufficiently low to mitigate the testing.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study was conducted in accordance with a standardised guideline under GLP conditions.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one study available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Only one study available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to respiratory tract irritation. This is detailed in the irritation/corrosion section.