2,3,6-trimethylphenol

Endpoint summary

Description of key information

oral
subacute, rat, oral gavage, NOAEL male/female: 100 mg/kg bw/day (nominal); LOAEL 300 mg/kg bw/day (nominal), GLP, similar to OECD 407 (Ohara 1999)
subacute, rat, oral feeding, NOAEL male: ca. 273 mg/kg bw/day; NOAEL female: ca. 55 mg/kg bw/day; LOAEL male: ca. 1346 mg/kg bw/day; LOAEL female: ca. 273 mg/kg bw/day, GLP, OECD 407 (BASF 1996)
Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, structural analogue of 2,3,6-trimethylphenol rat, oral gavage, NOAEL male/female: ≥ 200 mg/kg bw/day (actual ingested);

GLP, OECD 422 (RTI 2005)

inhalation
Study conducted with structural analogue of 2,3,6-trimethylphenol: 10 working days, rat, inhalation, NOEC male/female: ≥ 200 mg/m³ due to nasal lesions in the higher dose; LOEC male/female: ca. 670 mg/m³, GLP, no guideline (Battelle Memorial Institute 1991)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-10-25 to 1993-11-09 (In-life)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 12, 1981

Qualifier:

according to guideline

Guideline:

other: EC Commission directive 92/69/EEC; July 31, 1992

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 35 days (arrival); 42 days (start of dosing)
- Weight at study initiation: males: 204 (191-223)g; females: 155 (142-164) g
- Fasting period before study: no data
- Housing: individually in mesh wire cages
- Diet: ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 04, 1993 (arrival of the animals) To: November 09, 1993 (necropsy)

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the diet was determined analytically before the start of the study.
The homogeneous distribution of the test substance in the diet was proven at the start of the administration period in samples of the high and low concentration. These homogeneity analyses served also as concentration controls. Additionally, concentrational control analyses were performed in samples of the mid concentration at the beginning of the administration period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously in the diet

Remarks:

Doses / Concentrations:
ca. 55, 273, 1346 mg/kg bw/d (600, 3000, 15000 ppm in the diet)
Basis:
actual ingested

No. of animals per sex per dose:

5 rats/sex/group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
In a palatability study, the test substance was administered to groups of 3 male and 3 female Wistar rats at dietary concentrations of 0; 5000 and 15000 ppm for 2 weeks. Following substance-related findings were obtained:
- slight impairment of food consumption at both concentrations
- slight impaired of body weight at both concentrations: the values on day 14 were about 6%/7% (males/females) below control at the high concentration and about 5%/6% below control at the low concentration.
Therefore, the following concentrations were chosen for the present study:
15000 ppm: as highest concentration, corresponding to a test substance intake of at least 1000 mg/kg body weight
3000 ppm: as intermediate concentration
600 ppm: as expected "no observed adverse effect level "
- Rationale for animal assignment: randomly; identification by ear tattoo number
- Post-exposure recovery period: none

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
Food consumption was determined weekly over a period of 7 days and calculated as mean food consumption in grams per animal and day .
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: determined weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: no data
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined:
leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: no data
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined:
alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
- Time schedule for collection of urine: no data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined:
volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The data were evaluated statistically on the computer systems of the Department of Toxicology of BASF Aktiengesellschaft

Means and standard deviations were calculated for variables.
Methods used (depending on the variable): KRUSKAL-WALLIS test, MANN-WHITNEY U-test.
Significance labels: p<0.05, p> 0.02, p<0.01, p<0.002; depending on method and variable.

Remark: Due to the limited maximum size of this field, no more details on statistics are given herein.

Details on results:

CLINICAL SIGNS AND MORTALITY
no substance-related changes

BODY WEIGHT AND WEIGHT GAIN
substance-related changes in high-dose males and females; see below

FOOD CONSUMPTION
substance-related changes in high-dose males and females; see below

WATER CONSUMPTION
no substance-related changes

HAEMATOLOGY
substance-related changes in high-dose males and females and in mid dose females; see below

CLINICAL CHEMISTRY
no substance-related changes

URINALYSIS
no substance-related changes

ORGAN WEIGHTS
substance-related changes in high-dose males and females; see below

GROSS PATHOLOGY
no substance-related changes

HISTOPATHOLOGY: NON-NEOPLASTIC
substance-related changes in high-dose males and females; see below

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
no substance-related changes

Dose descriptor:

NOAEL

Effect level:

ca. 273 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

haematology

Dose descriptor:

NOAEL

Effect level:

ca. 55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

haematology

Dose descriptor:

LOAEL

Effect level:

ca. 1 346 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Dose descriptor:

LOAEL

Effect level:

ca. 273 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: Decrease in hematocrit in females

Critical effects observed:

not specified

According to the authors, the following significant, substance-related findings were obtained in the respective dose groups:

15000 ppm (about 1,346 mg/kg body weight):

• slightly decreased food consumption in males and females, especially during the first week of the study
• impairment of body weight in males and females; the values on day 28 were about 4% and 12% below controls, respectively
• impairment of body weight change in males and females ; the values on day 28 were about 9% and 41% below controls, respectively
• decrease in red blood cells, hemoglobin and hematocrit in both sexes
• decrease in mean corpuscular hemoglobin concentration in females
• increase in polychromasia and cholesterol in both sexes
• increase in mean corpuscular volume and mean corpuscular hemoglobin in females
• increase of absolute spleen weight in females and of relative spleen weights in males and females
• increase of extramedullary erythropoiesis and hemosiderosis of the spleen in males and females
• slight increase of erythropoiesis in the bone marrow of two males and two females

3000 ppm (about 273 mg/kg body weight):

• decrease in hematocrit in females

600 ppm (about 55 mg/kg body weight) :

• no substance-related changes

Thus, substance-related effects were seen at 15000 ppm in both sexes and at 3000 ppm in females. Target organs were spleen, bone marrow and erythron. The only toxic effects observed were signs of marked anemia at the high dose (> 1000 mg/kg body weight) with subsequent enhancement of hematopoiesis in bone marrow and spleen. Only a marginal sign of this effect was seen at the mid dose in females.

The no observed adverse effect level (NOAEL) under the conditions of this study was 3000 ppm (about 273 mg/kg body weight) in males and 600 ppm (about 55 mg/kg body weight) in females.

Executive summary:

The test substance was administered to rats for 4 weeks at concentrations of 600, 3000 and 15000 ppm for 4 weeks. The concentrations used provided dose levels of approximately 55, 273, and 1346 mg/kg bw/d, respectively. Control animals were administered plain diet.

Administration of the test substance at 15000 ppm adversely affected food consumption, body weight, body weight gain, hematological parameters, spleen weight and induced histological changes in the spleen and bone marrow of males and females. The mid dose level affected a single hematological parameter in females only. No adverse effects were seen in the rats administered 600 ppm.

Under the conditions of this study, the NOAEL was 3000 ppm in the diet (ca. 273 mg/kg bw/d) for males and 600 ppm in the diet (ca. 55 mg/kg bw/d) for females.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

japanese guideline

Principles of method if other than guideline:

Guidelines for 28-Day Repeat Dose Toxicity Testing of Chemicals (Japan)

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
male and female Sprague-Dawley rats (Crj:CD;SPF
- Source: CHARLES RIVER LABORATORIES JAPAN
- Age at study initiation: 4 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually in a metallic cage with a metallic net floor
- Diet (ad libitum): solid feed (CE-2, CLEA Japan, Inc.)
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 26 °C
- Humidity (%): 46 - 63 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was solved in corn oil so as to prepare an administered specimen with the concentration of 20 w/v%, 6 w/v% and 2 w/v%. Stability and content of the test substance in the administered specimen were measured. As a result, it was confirmed that the test substance in the corn oil solution of 0.2w/v% and 20w/v% was stable for eight days under the conditions of room temperature and light interception. It was also confirmed that the content of the test substance lies in the range of 99.5w/v% to 103w/v% of the predetermined concentration value.

VEHICLE
corn oil, Nakarai Tesque, Inc.
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 6, 2 % (w/v)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): V7R2020
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
no data

No. of animals per sex per dose:

10 males, 10 females (control, 1000 mg/kg bw/d)
5 males, 5 females (100, 300 mg/kg bw/d)

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 14 days (control, 1000 mg/kg bw/d)
- Dose selection rationale:
In preliminary tests performed prior to the main tests, the test substance of 40 mg/kg, 200 mg/kg and 1000 mg/kg were repeatedly administered orally to female and male rats for seven days. As a result, for male rats in the dose group of 1000 mg/kg, transient total debilitation was observed after the first administration. In addition, for the female and male rats in the same dose group, suppression of body weight gain was observed. As findings at necropsy at the end of the administration period, ulcers in the forestomach and changes suggesting their repairing were occasionally found. In the dose group of 40 mg/kg and 200 mg/kg, no obvious toxicity was recognized both for the female and male rats. Therefore, in the main tests, 1000mg/kg, which is the certainly toxic dose, is set to be the maximum dose with respect to both female and male rats. Then dividing the value by common ratio of about 3, dose groups of 300 mg/kg and 100 mg/kg were set.

- Rationale for animal assignment : random sampling method stratified by body weight measured on the previous day before starting the administration
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily during dosing period, once daily during recovery

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each rat was measured immediately before the administration and on the fourth day during the first administration week, and then twice per week since the second week during the administration period and during the recovery week. It was also measured on the last day of the administration period and of the recovery period, on a necropsy day, and at the death time of the rats.

FOOD CONSUMPTION:
The amount of feed consumption for each rat per day was measured on the first administration day during the administration starting week, and then once per week since the second week during the administration period and during the recovery period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day prior to necropsy (i.e. the last day of treatment or recovery)
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes, 18 to 24 h prior to collection
- How many animals: all animals
- Parameters examined:
red blood cell count, white blood cell count, hemoglobin content, mean corpuscular volume, platelet count, hematocrit, hemoglobin mean
corpuscular and concentration of hemoglobin mean corpuscular, leukocyte classification, prothrombin time, active partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after collection for hematological examinations
- Animals fasted: Yes
- How many animals: all animals except decedents
- Parameters examined: total protein concentration, albumin concentration, total cholesterol concentration, glucose concentration, urea nitrogen concentration, creatinine concentration, alkaline phosphatase activity, GOT activity, GPT activity, LDH activity, potassium concentration, inorganic phosphorus concentration, triglyceride concentration, γ-GTP activity, A/G ratio, and sodium, potassium and chlorine concentrations

URINALYSIS: Yes
- Time schedule for collection of urine: during the first and fourth week of the administration period and during the last week of the recovery period, 5 animals per group
- Metabolism cages used for collection of urine: Yes urine collection for 4 and 24 hours
- Animals fasted: No data
- Parameters examined: pH, occult blood, protein, urinary sugar, ketone, bilirubin, urobilinogen and sediment (4-hour urine)
urine amount (the urine weight measured by scale was divided by the specific gravity), color, turbidity (visual inspection) and specific gravidity (urine weight measured per unit volume (24-hour urine

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

ORGAN WEIGHTS: Yes

Following blood collection, rats were killed through exsanguination by cutting axillary artery if necessary. Then, organs and tissues of the rats were observed on gross. In addition, the weight of brain, thymus, heart, liver, kidneys, spleen, adrenal gland, ovaries or testes and epididymides for each animal was measured, so that relative weight of each organ of the rats was calculated. Furthermore, brain, spinal marrow, pituitary gland, eye ball, harderian gland, thyroid gland (including parathyroid gland), submandibular gland (including sublingual gland), thymus, heart, lung, liver, kidneys, spleen, pancreas, adrenal gland, stomach, intestinum duodenum, intestinum jejunum, intestinum ileum, colon, intestinum rectum, ovaries or testes, seminal vesicle, epididymides, bladder, prostate gland, fermur bone marrow, ischiadic nerve and skeletal muscles of leg region were fixed in 0.1M phosphate buffered 10% formalin solution. (pH 7.2) Besides brain, thymus, heart, liver, kidneys, spleen, adrenal gland, stomach, testes, epididymides, ovaries and bone marrow (femur), affected regions (lung and skin) were subject to a histopathological examination. Next, thymus, liver, kidneys, spleen and stomach for which affection due to the administered test substance was suspected, were examined in all cases. As brown pigmentation was found in spleen during the histological examination, specimens with Berlin blue stain were prepared for the spleens of all the rats. For dead animals, all examination items for the histopathological examination were examined except for measurement of body weight. For rats subject to necropsy at the end of the recovery period, histological examination with respect to liver, spleen, stomach and an affected region (lung) was performed.

Statistics:

With respect to the result values of the measurements of body weight, feed consumption and water intake, of the urinalysis except for semi-quantitative test, and of the hematological examination, the blood chemical examination and the measurement of organ weight for the rats subject to periodical dissection, a mean value and standard deviation for each group was calculated. In the case that more than two groups including the control group were formed as test groups, multiple comparison was performed by way of Bartlett’s dispersion uniformity test, one-way analysis of variance or Kruskal-Wallis’ rank test and Dunnet or Dunnett type test. In the case that two groups were formed, Student’s t-test or Aspin-Welch’s t-test was performed. As for the findings in the histopathological examination, graded data was subject to Mann-Whitney U test (two-sided test), and the total number of cases showing positive grade was subject to one-sided Fisher's exact test. Significance level in a significant difference test between the value of the control group and that of the dose group of the test substance was set to be 5% in all these cases.

Details on results:

CLINICAL SIGNS AND MORTALITY
Two female rats in the dose group of 1000mg/kg died on the third and the ninth administration day, respectively. As a result of necropsy, oily liquid, which was supposed to be the test substance, was found in the chest cavity of the two rats. Therefore, they were determined to have died due to mistaken administration.
As changes in general status, some female and male rats in the dose groups of 1000mg/kg showed prone position and ataxia of gait during the administration period. All these changes were occasionally found during the observation after administration, and were eliminated on the next day before the next administration except for the dead cases, so that they were transient changes. These changes were repeatedly observed, in total six cases for the male rats and nine cases for the female rats.
With respect to female and male rats in the dose groups of 300mg/kg and 1000mg/kg, transient salivation was recognized immediately after administration. This change was recognized earlier and more frequently for both female and male rats in the dose group of 1000mg/kg. In this group, all rats except for one female rat (died on the third administration day) showed this change. Some showed salivation even before the administration, i.e., at the time of being retained.
As another change, dehairing on the surface of the body was recognized with respect to one female rat in the control group and to one female rat in the dose group of 300mg/kg, respectively, from the third administration week to the time of being killed at the end of the administration.
No changes were observed at all in all cases during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
During the administration period, suppression tendency of body weight gain was observed for female and male rats in the dose group of 1000mg/kg in comparison with the female and male rats in the control group, respectively. For the male rats, significant low values were recognized on the 25th and 28th day, while for the female rats, it was recognized on the fourth and eighth administration day. With respect to the male rats in the dose group of 300mg/kg, suppression tendency of body weight gain was observed on the fourth and eighth day. During the recovery period, all measured values with respect to female rats in the dose group of 1000mg/kg were significantly low, while the values with respect to the male rats in this group did not show the difference.

FOOD CONSUMPTION
Significant decrease in feed consumption was recognized on the first administration day with respect to female and male rats in the dose groups of 1000mg/kg. In addition, significant decrease in feed consumption was recognized on the eighth administration day with respect to the female rats in the dose group of 300mg/kg.
HAEMATOLOGY
In the hematological examination at the end of the administration period, significantly decrease in red blood cell count, hemoglobin content, and hematocrit value with respect to female rats in the dose group of 1000mg/kg was recognized. In two rats of each sex in the same group, increase in polychromatic erythrocyte was observed. With respect to the female rats in this group, blood reticulocyte ratio tended to increase.
In the examination at the end of the recovery period, it was recognized that hemoglobin mean corpuscular and mean corpuscular volume significantly decreased with respect to male rats in the dose group of 1000mg/kg.

CLINICAL CHEMISTRY
In blood chemical examination at the end of the administration period, concentration of inorganic phosphorus significantly increased with respect to male rats in the dose groups of 300mg/kg and 1000mg/kg, while albumin concentration significantly decreased in the dose group of 1000mg/kg. There are other items showing a significant difference, but their changes were not dose-dependent.
In the examination at the end of the recovery period, all the above-mentioned changes were eliminated. With respect to some items with no changes shown at the end of the administration period, significant difference was observed occasionally.

URINALYSIS
In the urinalysis in the last week during the administration period, three male rats and all female rats in the dose group of 1000mg/kg showed yellow urine. Male rats in this group showed significant increase in urine amount and significant decrease in urinary specific gravity. The significant decrease in urinary specific gravity was recognized with respect to male rats in the dose group of 300mg/kg, too. In the case of female rats in the dose group of 1000mg/kg, urine protein, bilirubin and urobilinogen in urine tended to increase.
In the urinalysis in the last week during the recovery period, increase in urine protein with respect to the female rats in the dose group of 1000mg/kg was alleviated. Other changes were mostly eliminated.

ORGAN WEIGHTS
In the case of the rats killed at the end of the administration period, absolute weights of adrenal gland significantly decreased with respect to male rats in the dose groups of 300mg/kg and 1000mg/kg. For female rats in the dose group of 1000mg/kg, on the other hand, absolute weight and relative weight of the spleen and relative weight of the liver significantly increased. With respect to male rats in the dose group of 1000mg/kg showing low body weight values, relative weight of the brain and the testes increased significantly.
In the case of the rats killed at the end of the recovery period, though relative weight of the spleen with respect to female rats in the dose group of 1000mg/kg increased significantly, other changes were mostly eliminated. There were other changes having shown statistically significant differences, which were occasionally observed, but neither of them had been recognized at the end of the administration period.

GROSS PATHOLOGY
In the case of the killed rats at the end of the administration period, hydrops was observed in the forestomach mucosa with respect to one male rat in the dose group of 1000mg/kg. With respect to one male rat in the dose group of 300mg/kg, white substance which was supposed to be the test substance was recognized in the chest cavity. In addition, dark color dots, dark red dots and white regions were observed in the lung and bronchial tubes, light-color dots in the liver, dark color dots in the adrenal gland, dilatation of bursa ovarica, and hypotrichosis as well as crust formation on the skin were observed in the dose groups of the test substance. But it was not apparent whether these changes were relevant to the administration of the test substance.
In the case of the killed rats at the end of the recovery period, no abnormal findings other than dark-color dots in the lung of male rats in the control group and in the dose group of 1000mg/kg were observed.

HISTOPATHOLOGY
i) Animals Killed at the End of Administration Period
(Heart)
With respect to two male rats and one female rat in the control group, myocardial degeneration was observed, but no abnormal symptoms were found in the dose group of 1000mg/kg.
(Liver)
With respect to four male rats and one female rat in the dose group of 1000mg/kg, centrolobular hypertrophy of hepatocyte was observed, and fogged-glass-like status change was found slightly in the cell cytoplasm. With respect to male rats in the dose group of 1000mg/kg, expression frequency of hypertrophy of hepatocyte increased significantly. In addition, periportal adiposity was observed, too. But the expression frequency and the extent of this change of the control group and the dose groups of the test substance were not different each other. Localized adiposity was observed with respect to one female rat in the dose group of 100mg/kg. This change was not recognized in other groups.
(Spleen)
With respect to female and male rats in the dose group of 1000mg/kg, hemosiderosis increased and sinus venosus dilatation was observed compared to those in the control group. Statistically, with respect to female and male rats in this group, the extent of the hemosiderosis and the extent and the expression frequency of the sinus venosus dilatation significantly increased compared to those of the control group. With respect to the female rats in this group, the extent of extramedullary hematopoiesis increased significantly compared to that of the control group.
(Kidneys)
With respect to female and male rats in the control group and in the dose group of 1000mg/kg, basophilic renal tubule was observed in the cortex. With respect to male rats in these groups, eosinophilic body was observed. But its expression frequency and its extent were not different in the two groups. Besides, cyst was observed with respect to one male rat in the control group. With respect to one female rat in the control group, mineral deposit was recognized in the cortico-medullary junction.
(Stomach)
With respect all male and female rats in the dose group of 1000mg/kg, diffuse hyperplasia was recognized in squamous epithelium of the forestomach. Its extent and expression frequency showed significant difference. In addition, one male rat in this group showed production of erosion in the glandular stomach.
(Affected Regions confirmed on Gross)
In the dark dots of the lung, bleeding was confirmed in all cases, but this change was slight or very slight. In the light-color region of the lung, aggregation of foam cells was observed. With respect to the above-mentioned rats showing white substance in the chest cavity of the lung, foreign-body granuloma was recognized in the pleura. In the skin regions with dehairing, hypotrichosis or crust, ulcers, crusts and hyperplasia of epithelial cell, which was supposed to be induced for the regeneration of the skin, were observed.
The brain, spinal marrow, thymus, bladder, adrenal gland, intestinum ileum, ischiadic nerve and fermur bone marrow, no abnormal symptoms were recognized with respect to female and male rats in the dose group of 1000mg/kg.

ii) Animals Killed at the End of Recovery Period
(Liver)
The hypertrophy of hepatocyte which was observed in the case of the killed animals at the end of the administration period was not recognized here. Periportal adiposity was observed. But no difference in the extent and the expression frequency exists between the control group and the dose group of 1000mg/kg.
(Spleen)
With respect to female and male rats in the dose group of 1000mg/kg, hemosiderosis increased. In the male cases, its extent showed significant increase. With respect to one male rat in this group, sinus venosus dilatation was observed. In all rats of this group, extramedullary hematopoiesis was recognized, but its extent was not different from that of the control group for both female and male rats.
(Stomach)
Except for diffuse hyperplasia in the squamous epithelium of the forestomach observed with respect to one female rat in the dose group of 1000mg/kg, no abnormal symptoms were observed.
(Lung)
With respect to two male rats in the dose group of 1000mg/kg and one male rat in the control group, slight bleeding was observed.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no treatment related effects observed

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Critical effects observed:

not specified

Executive summary:

Abstract

Twenty-eight-day repeat oral dose toxicity test of 2,3,6-trimethylphenol (Recovery for 14 days) was conducted using Sprague-Dawley (Crj: CD) female and male rats. Dose was set to be 0 mg/kg (control group), 100 mg/kg, 300 mg/kg and 1000 mg/kg both for female and male rats. To the dose groups of 0 mg/kg and 1000 mg/kg, 10 animals per group including five animals for recovery tests were allocated for a toxicity test, respectively, while to the dose groups of 100 mg/kg and 300 mg/kg, five animals per group were allocated. The following result was obtained.

During the administration period, two female rats in the dose group of 1000 mg/kg died due to mistaken administration.

As changes suggesting the relationship to the administration of the test substance, the following findings were recognized in the dose group of 1000 mg/kg: Immediately after administration, besides prone position and ataxia of gait which were transient changes, salivation was occasionally and repeatedly found. These changes were all eliminated by the next day when the next administration was performed were recognized. The salivation was determined to be no toxicity change but reflex response due to administration stimulus. During the administration period, suppression of body weight gain and reduction in feed consumption were shown. Urinalysis for male rats revealed significant increase in urine amount, significant decrease in urinary specific gravity and yellow urine. Urinalysis for male rats revealed, besides yellow urine, protein, bilirubin, urobilinogen in urine tended to increase. In a hematological examination, red blood cell count, hemoglobin content, hematocrit value significantly decreased for female rats. In blood chemical examination, with respect to male rats, concentration of inorganic phosphorus significantly increased, while albumin concentration significantly decreased. In the measurement of organ weight with respect to female rats, absolute weights of adrenal gland for male rats significantly decreased, while absolute weight and relative weight of spleen, and relative weight of liver significantly increased. In a pathological examination, centrolobular hypertrophy of hepatocyte together with fogged-glass-like status change was found occasionally for the liver of female and male rats, of which expression frequency was significantly higher for the male rats. In female and male rats’ spleen, sinus venosus dilatation and hemosiderosis were remarkable. In male rats, increase in extramedullary hematopoiesis was recognized. Diffuse hyperplasia was the most part after two-week cessation of the drug. At the end of the recovery period, significant decrease in mean corpuscular volume and hemoglobin mean corpuscular were shown with respect to the male rats, while significant increase in relative weight of the spleen weight as seen at the end of the administration period was observed with respect to the female rats. In a histopathological examination, the above-mentioned changes in the liver and the stomach as well as increase in extramedullary hematopoiesis for the female rats were eliminated or alleviated. The hemosiderosis in the spleen was remarkable with respect to both female and male rats compared to those in the control group. As for the dose group of 300 mg/kg, among the above-mentioned changes recognized in the dose group of 1000 mg/kg, transient salivation was recognized for the male and female rats, suppression of body weight gain and decrease in the feed consumption were recognized for the female rats, decrease in urinary specific gravity, increase in the concentration of inorganic phosphorus in the blood as well as decrease in the absolute weight of the adrenal grand were recognized for the male rats, respectively. As for the dose group of 100 mg/kg, no changes suggesting the relevancy to the administration of the test substance were recognized for both female and male rats.

It is determined from the results above, that no-observed-adverse-effect level of 2,3,6-trimethylphenol under the conditions of this test is 100 mg/kg/day in both female and male rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: 10 day treatment on working days

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: for justification of read-across see endpoint study summary

Qualifier:

no guideline followed

Principles of method if other than guideline:

10-day-inhalation study: Ten rats per sex per dose were exposed to the test compound (67, 200 and 670 mg/m³), or an air control (0 mg/m³) on 10 consecutive working days over a 14 day period in rats. Animals were evaluated based on clinical condition, body weight change, clnical pathology profiles and anatomical pathology assessments.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc., Kingston, New York
- Age at study initiation: 5-6 weeks
- Weight range at study initiation: males: 134.4 to 160.5 g; females: 109.0 to 122.8 g
- Fasting period before study: No data
- Housing: individually in stainless steel wire mesh cages, (manufactured by Lab Products, Inc., Maywood, New Jersey) which were located in a Hazleton 1000, whole-body, Inhalation exposure chamber.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow® (pellets) was provided ad libitum during non-exposure periods
- Water (e.g. ad libitum): Water was provided ad libitum (except during the daily exposure periods)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.44 - 25.00 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h /12 h

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Remarks on MMAD:

MMAD / GSD: The count median aerodynamic diameters (CMAD) and the geometric standard deviations (GSD) were recorded from the APS results and the mass median aerodynamic diameters (MMAD) were calculated from the CMAD and GSD values using the Hatch-Choate equation. The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD during the pre-animal exposure period were 0.72, 1.06, and 1.42, respectively. During animal study, CMAD ranged between 0.56 gm and 0.68 gm, and GSD values were between 1.22 and 1.41. The corresponding calculated MMAD ranged between 0.64 gm 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 for the test article and air control exposures.
- Method of holding animals in test chamber: The cages with the animales were located in a Hazleton 1000, whole-body, inhalation exposure chamber. There were 24 compartments (12 per side) in each cage unit. One cage unit was used to house all animals assigned to a particular exposure group for the study.
- System of generating particulates/aerosols:
• The test article generation system was designed to simulate a possible manufacturing process failure which could result in release of a pressurized stream of liquified structural analogue into the atmosphere of the work place, where likely exposure would be inhalation of vaporized structural analogue and/or condensation aerosols of the test article.
• This generation system was designed to produce the highest reasonably or likely achievable concentration of structural analogue generated from its liquid state and allowed to achieve room temperature equilibrium conditions.
• The primary component of the generation system was a Sonimist® spray nozzle (Model HS600-2, Heat Systems Ultrasonics, Inc., Farmingdale, New York). A fine stream of heated liquid test article was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of compressed air producing a 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 chemical reservoir that was heated and pressurized for the delivery of the structural analogue to the Sonimist ® Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and a 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 article 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 (reservoir core, reservoir skin, nozzle skin and carrier air line
skin) of the generation was controlled and measured.
• 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.
- Exposure System
• The test article atmosphere within the plenum chamber was transported into a single manifold system that supplied the three animal exposure chambers.
• The total concentration of structural analogue 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.
• Control rats were exposed in a chamber that received HEPA and activated charcoal-filtered room air only. The air handling system for the control chamber was separate from the test article generation and delivery system.
- Temperature, humidity, pressure in air chamber: 22.22 ± 2.78 °C, 55 ± 15 % recorded three times per day during the 1st, 3rd and 6th hour of animal exposure.
- Air flow rate and air change rate: Chamber air flow rates were controlled by orifice plate tube flowmeters and valves. Flow rates through the chambers were controlled to 15 ± 2 air changes per hour.
- Method of particle size determination:
Particles were assumed to be normally distributed with respect to the logarithm of their diameters for calculation of aerosol mass median aerodynamic diameters (MMAD) and geometric standard deviations (GSD). The aerodynamic diameters were defined as the diameter of unit density spheres which have the same settling velocity as particles of the test aerosol. 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 exposures, particle size distributions were measured once per exposure chamber (including air control) per day.
- Treatment of exhaust air: The exhaust air from all the chambers passed through Cambridge Sidelock® HEPA and Prefilter Assemblies (Cambridge Filter Corp., Syracuse, NY) to remove particulate structural analogue before combining the outflow into a Scrubber. The 'scrubbed' air was then vented outside the building.

TEST ATMOSPHERE
- Aerosol Concentration
• Because generation of structural analogue atmospheres under the test conditions produce both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow controller was used to draw an air sample from each chamber or sample location. The sample was first drawn through a 25 mm glass fiber filter mounted in an openfaced 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 entrained
in the sampling air stream and was directed into a Infrared Analyzer where the total vapor concentration was measured.
• The sample flow rate was set at 5 liter per minute while sample duration varied according to chamber test article 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 of structural analogue and were also expressed as the percent of total mass.
The Miran 1A Infrared Analyzer was equipped with a 20 meter variable pathlength gas sample cell. A standard calibration loop consisting of an injection port and circulation pump was added for calibration purposes. In this way, by knowing the volume of the sample cell and the volume of the injected amount of test material, the concentration in the cell was calculated. The absorbance (or in this case the analog output in VDC) was observed for a given injection or calibration point. The data were plotted and regressed such that input at an absorbance valve was derived for each known mass concentration value.
- Calibration
• The Miran 1A was calibrated during the pre-study development. First, a suitable wavelength was selected by completing a full infrared spectrum scan of structural analogue. Because of the physical properties of structural analogue at room temperature (solid phase), preventing injection of the test compound in liquid form, the structural analogue was dissolved in reagent grade methanol. A methanol scan was also performed. By comparing the scans, a wavelength of 8.25 gm at a detection cell pathlength of 20.25 meters was selected to be the most specific for structural analogue without interference from methanol and water vapor.
• Using known amounts of structural analogue standard prepared in methanol the absorbance value were recorded. A standard was prepared in reagent grade ethanol at a concentration of 0.204502 g/mL. A series of injections into the Miran closed-loop calibration system were conducted in triplicate. The Miran detection cell was maintained at 70°C to prevent condensation of structural analogue. The calibration was conducted over a concentration range of 4 ppm to 167 ppm (18 mg/m³ to 724 mg/m³).
- Samples taken: During the animal studies, all locations (as described above) were sampled once per hour with the gravimetric aerosol/Miran vapor sampling system, except that gravimetric samples were not collected from the room air, the air control chamber, or the Mystaire ® scrubber exhaust.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography (GC) of impinger samples was the chosen technique. One hundred twenty-five mL gas washing bottles 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 backflushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph.
For the GC a 30 m x 0.75 mm Supelco SPB-1 column with a 1.5 micron film thickness was used. The oven was set at 70°C with the injector and detector at 90°C. The carrier flow was at 20 mL/min. Injection volumes were constant at 0.6 L. To establish a standard calibration curve, feference test material was dissolved in reagent grade methanol between the ranges of 25 gg/mL to 764 gg/mL. The correlation coefficient for the curve was 0.999.
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. The results show that there was good agreement between gravimetric/vapor determination and GC determination methods.

Duration of treatment / exposure:

14 days

Frequency of treatment:

6 hours/day, 5 days/week for a total of 10 exposures

Remarks:

Doses / Concentrations:
0, 67, 200, 670 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 84.4, 227, and 731 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

other: filtered air alone

Details on study design:

- Dose selection rationale: The exposure concentrations for the study were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response. The Inhalation exposure system was designed to simulate release of heated, liquified structural analogue into ambient atmosphere and to deliver a near-saturated atmosphere of respirable test article to the study animals. Preliminary studies showed that 670 mg/m³ represented the approximate-saturation concentration of the respirable fraction (comprised of both vapor and aerosol phases) of structural analogue in absence of air under dynamic air-flow conditions. The middle and low concentrations represented approximated half-log interval decreases from the maximum achievable concentration. The lowest concentration level examined, was at least 2 to 3 orders of magnitude greater than the average concentration of the test article measured in the workplace.
- Post-exposure recovery period in satellite groups: None, animals were killed one day after the last exposure.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes,
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes,
- Time schedule: twice daily (in the morning, at least 6 hours apart, before 10:00 a.m. and after 2:00 p.m., 7 days per week) before and after each daily exposure or at similar intervals on nonexposure days for clinical evidence of toxicity or other abnormalities.
The clinical evaluations included a thorough examination of the animals general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behavior, coordination and general neuro-muscular function. All
observations were recorded on a Data Management System.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight values of all animals were recorded on Study Day -8 (within 48 hours of receipt) and again on Study Day -2 for randomization and assignment to study groups. The body weights of animals assigned to the study were determined on Study Day 1, prior to the first exposure and again on Study Days 8, 14, and prior to scheduled necropsy.

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes (identity), propylene glycolfree sodium pentobarbital
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: all animals
- The following parameters were examined:
• red blood cell count (RBC, 10e6 cells per microliter)
• hematocrit (HCT, percent)
• hemoglobin (Hgb, g/dL)
• mean corpuscular volume (MCV, femtoliters)
• mean corpuscular hemoglobin (MCH, picograms)
• mean corpuscular hemoglobin concentration (MCHC, percent)
• white blood cell count (WBC, 10³ per microliter)
• platelet count (PLAT, 103 platelets per microliter)
Smears for differential cell counts were also made from the blood samples and were stained using a modified Wright-Giemsa stain.
• The relative number of nonsegmented neutrophils (BAN),
• segmented neutrophils (SEG),
• eosinophils (EOS),
• basophils (BAS),
• lymphocytes (LYM), and
• monocytes (MON) was determined for each animal.
• The absolute number of each cell type per microliter was also calculated.
• The number of nucleated red blood cells per 100 white blood cell (nRBC/100 WBC) was also determined.
• The total reticulocyte count (RETIC) was determined after red blood cells were pre-stained with new methylene blue and a smear prepared.
• The number of reticulocytes per 100 red blood cells was counted microscopically and the results reported as the percentage of red cells which were
reticulocytes.
CLINICAL CHEMISTRY: Yes, from the retro-orbital Sinus
- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: All animals
- The following parameters were examined:
• blond urea nitrogen (BUN, mg/dL)
• creatinine (CRET, mg/dL)
• fasting glucose (GLU, mg/dL)
• total protein (TP, g/dL)
• albumin (ALB, g/dL)
• globulin (GLO, A:G ratio)
• cholesterol (CHOL, mg/dL)
• lactate dehydrogenase (LDH, IU/L)
• serum alanine aminotransferase (ALT, IU/L)
• serum aspartate aminotransferase (AST, IU/L)
• alkaline phosphatase (ALP, IU/L)
• bilirubin (Bil, mg/dL)
• calcium (CA, meg/L)
• sodium (NA, meg/L)
• potassium (K, meg/L)
• chloride (CL, meg/L)
• phosphorus (IP, mg/dL)
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes,
- Each animal was killed by exsanguination
- 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.

HISTOPATHOLOGY: Yes, liver, kidney, heart, mainstem bronchi, lung trachea, nasal cavity
- The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (two cross-sections), and trachea (crossand longitudinal sections), and all gross lesions suspected to be exposure-related, from all animals of each exposure group were blocked for slides, sectioned, stained with hematoxylin and eosin (H&E), and submitted for light microscopic examination. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
- The nasal cavity was prepared in four sections using the landmarks described by Young (Fundam. Appl. Toxicol., 1: 309-312, 1981).
- All other tissues collected from all other animals were preserved and held in formalin for possible future histopathological evaluation.

Details on results:

CLINICAL SIGNS AND MORTALITY
- All animals survived to study termination.
- Clinical signs of toxicity were confined to male and female rats exposed to the high-concentration (670 mg/m³) of structural analogue. A red nasal discharge was observed in all animals in the high-concentration 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.
- Nasal discharge, red: Males: day 1: 10/10, day 2 : 2/10, day 4 - 8: 10/10 and day 12 - 14: 10/10; Females: day 1: 10/10, day 4 - 8: 10/10 and day 12 - 14: 10/10;
- There were no other clinical or behavioral changes observed in the high concentration group that were thought to be related to structural analogue exposure.
- In addition, all other rats at the middle and low exposure concentration group were similar to control animals in behavior and in general health and clinical appearance.

BODY WEIGHT AND WEIGHT GAIN
- At randomization and on Study Day 1, all exposure groups had group mean body weights that were statistically similar to that of their respective control group. The 200 and 670 mg/m³ male exposure groups has 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 670 mg/m³ female group 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.
- All other male and female groups had group mean body weight values that were similar (p > 0.05) to those of their respective control group.

- The terminal body weights of male rats exposed to 200 and 670 mg/m³ of structural analogue were significantly less (p < 0.05 and 0.01, respectively) than the control group (after 8 days: control: 178.8 g; 200 mg/m³: 168.7 (5.6 %) and 670 mg/m³: 160.8 g (10.1 %); after 14 days: control: 185.4 g; 200 mg/m³: 173.9 (6.2 %) and 670 mg/m³: 181.2 g (2.3 %)). The group mean terminal body weights of females (the 670 mg/m³ exposure group) were also significantly decreased (p < 0.01) compared to control animals (after 8 days: control: 131.7 g; 670 mg/m³: 120.1 g (8.8 %); after 14 days: control: 132.3 g; 670 mg/m³: 117.5 g (11.2 %)).
FOOD CONSUMPTION
- no data

FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no data

HAEMATOLOGY
- Platelet Count was significantly changed in males of the mid and high dose group: control: 749.70 x 10³/µL; 200 mg/m³: 646.90 x 10³/µL; 670 mg/m³: 704.90 x 10³/µL.
- Hemoglobin was significantly changed in males of the high dose group: control: 16.71 g/dL; 200 mg/m³: 16.78 g/dL; 670 mg/m³: 16.19 g/dL.
- WBC Differential Count / Monocyte was significantly changed in females of the mid dose group: control: 0.70 x 10³/µL; 200 mg/m³: 1.80 x 10³/µL; 670 mg/m³: 1.11 x 10³/µL.
- These changes were small in magnitude and not considered to be related to structural analogue exposure.

CLINICAL CHEMISTRY
While there were some statistically significant changes (Creatinine: 670 mg/m³ females (control: 0.63 mg/dL; 670 mg/m³: 0.556 mg/dL); Total Protein: 200 mg/m³ females (control: 6.48 g/dL; 67 mg/m³: 6.22 g/dL); Albumin 67 mg/m³ females (control: 4.43 g/dL; 67 mg/m³: 4.28 g/dL); Globulin 670 mg/m³ males (control: 2.10; 670 mg/m³: 1.94); A/G Ratio 670 mg/m³ males (control: 2.11; 670 mg/m³: 2.36); Alanine Aminotransferase 670 mg/m³ females (control: 41.90 IU/L; 670 mg/m³: 53.44 IU/L); Alkaline Phosphatase 670 mg/m³ males (control: 413.80 IU/L; 670 mg/m³: 363.30 IU/L)), these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rats. Therefore, none of the variations in clinical pathology parameters were considered to be due to structural analogue exposure.

URINALYSIS
- no data

NEUROBEHAVIOUR
- no data

ORGAN WEIGHTS
• Kidney: Absolute kidney weight value of male (7.5 %) and female (12.4 %) rats in the 670 mg/m³ group were significantly greater (p < 0.05 and 0.01, respectively) than control animals. Kidney to body weight ratios were also significantly increased (p < 0.01) in the male (22.6 %) and female (25.4 %) 670 mg/m³ exposure groups.
• Heart: There were no significant differences between the exposure and control animals in the absolute mean heart weights. However, there was a significant increase (p < 0.01) in organ-to-body weight ratios for the male (11.8 %) and female (9.2 %) high exposure (670 mg/m³ ) groups
compared to control animals.
• Lung: There were no significant differences (p > 0.05) between the exposure and control groups in absolute mean lung weights.
However, the female high concentration (670 mg/m³) group, had significantly elevated (p < 0.05) lung-to-body weight ratios (9.5 %) relative to control rats.
• Liver: There were no significant differences in group mean absolute liver weights between any groups. However, the male (21.8%) and female (12.4 %) high-concentration (670 mg/m³) 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. These lesions consisted of an apparent cystic left ovary in one air control female, and 3 low-concentration group females, and a red, discolored mediastinal lymph node in one middle-concentration (200 mg/m³) male.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Lesions considered to be related to the inhalation exposure of structural analogue were confined to the nasal cavity of the high-concentration group animals of both sexes and were present in the same anatomical location and at essentially the same degree of severity from both sexes.
- These changes were identified 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 lesion was 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 exudate, 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 proteinaceous fluid within the Lumen. Six of the high-concentration group females
and three of the high- concentration group males (670 mg/m³) 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 exposure to the test article or secondarily as a result of the inflammation, could not be definitively ascertained.
- Exposure-related lesions involving the nasal cavity in the high-concentration group (670 mg/m³) male and female rats are summarized in the following
table:
• Moderate, necrosis/degeneration of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, suppurative, inflammation of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, squamous, metaplasia, of the olfactory epithelium. Males: 10/10, Females: 10/10
• Adhesions, of the turbinate-dorsal wall: Males: 1/10, Females: 1/10
• Adhesions, of the turbinate-septum: Males: 2/10, Females: 5/10
- After examining the respiratory tract tissues from the high concentration and air-control rats, these same tissue sections collected from the low- and mid-concentration rats of both sexes were trimmed, processed, stained with hematoxylin and eosin and examined microscopically in an attempt to establish a no effect level (NOEL) for this particular compound. All tissues required to be examined histopathologically were present from the low-and middle-concentration rats of both sexes with the exception of the larynx from one middle-concentration (200 mg/m³) female rat, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high-concentration animals were not present in the low- and middle-concentration rats of both sexes.
- Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used in this study 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 the exposure to structural analogue. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract (nasopharynx, larynx, trachea) were observed in control or exposed rats of either sex.
- In conclusion, the whole-body inhalation exposure of rats to structural analogue for 10 days at a concentration of 670 mg/m³ resulted in a moderate degree of necrosis/degeneration of the olfactory epithelium lining of the nasal cavity in areas covering the dorsal meatus, adjacent nasal septum, and nasal turbinates. Associated changes consisted of a serofibrinous/suppurative inflammatory response, squamous metaplasia of affected epithelium and adhesions between the ethmoid turbinates and the nasal septum or the wall of the dorsal meatus. The lesions were of approximately the same extent and severity in both sexes with the exception of the adhesions which were more prevalent in the high-concentration group female rats. Microscopic lesions were identified in levels II, III, and IV of the nasal cavity with those involving level IV being the most extensive.
- Histopathologic examination of the respiratory tract tissues collected from the lower concentration group rats of both sexes did not reveal the nasal lesions seen in the high-concentration group rats. No changes in the lower respiratory tract (nasopharynx, larynx, trachea, and lungs) considered to be related to the inhalation of structural analogue were found. These irritation-related effects, seen only at the highest exposure level, were not unexpected for a respiratory irritant material, and could be explored further through additional evaluations, if warranted. A NOEC for this effect was determined to be 200 mg/m³.

Dose descriptor:

NOEC

Remarks:

local

Effect level:

>= 200 mg/m³ air

Sex:

male/female

Basis for effect level:

other: At highest dose, 670 mg/m3, microscopic findings included necrosis/degeneration, inflammation, and squamous cell metaplasia of the olfactory epithelium and nasal septum adhesions.

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

>= 200 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Decreased group mean body weight values (18 %).

Dose descriptor:

LOEC

Remarks:

local and systemic

Effect level:

670 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Decreased group mean body weight values (18%); red nasal discharges; necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions.

Critical effects observed:

not specified

The test atmosphere generation and delivery system used in this study functioned well and as designed. Heated, pressurized liquid structural analogue was delivered into a plenum of air held at room temperature, where the material vaporized and condensed, achieving equilibrium (under dynamic air flow conditions). The atmosphere within the plenum was effectively saturated at the nominal air flow of 350 L/min used to entrain and deliver the test atmosphere. The liquid structural analogue delivered to the plenum stabilized into a biphasic atmosphere, consisting of vapor and a fine condensation solid aerosol. The percentage of the total mass concentration present in aerosol phase within the animal exposure chambers was directly proportional to the total structural analogue concentration. Therefore, as portions of the saturated manifold atmosphere were diluted in the middle- and low-concentration chambers, a lower percentage of the chamber atmosphere was in particle phase.

Observation of toxic effects during this repeated exposure inhalation study was generally limited to the highest exposure level of structural analogue. Male and female rats exposed to 670 mg/m³ structural analogue had decreased group mean body weight values relative to controls of approximately 10 percent. The reduced body weights were apparent on Study Day 8 and continued until study termination. Rats exposed to 670 mg/m³ also showed red nasal discharges that were apparent after the end of each daily exposure, which was indicative of upper respiratory tract irritation and ulceration. Microscopic examinations showed that rats exposed to 670 mg/m³ of structural analogue had necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions. Squamous cell metaplasia is a dedifferentiation change that may become progressive and more proliferative in nature with continued exposure and is often considered to be a pre-neoplastic lesion.

While male and female rats exposed at the 670 mg/m³ level showed significant increases in absolute kidney weight relative to control and increases in several organ-to-body weight ratios, the toxicological significance of these findings is unclear in the absence of clinical chemistry and microscopic evaluations. Male rats exposed to 200 mg/m³ structural analogue had smaller body weight decreases of about 6 percent on Study Day 8.

No clinical signs of toxicity or treatment-related microscopic findings were seen in rats exposed to 67 or 200 ppm structural analogue. There were no concentration-related changes in clinical pathology studies that were considered to be biologically significant or exposure-related.

The observed lesions are considered to be typical of a respiratory irritant and are not unexpected following exposure to such a compound. The changes were noted only after repeated exposure to the highest concentration (670 mg/m³) used in this study, which would classify this test material as a mild to moderate acute respiratory irritant.

In conclusion, based on the results of this study the no-observable adverse effect concentration for structural analogue following repeated, 6 -hour exposures is 200 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Remarks:

other: 10 day treatment on working days

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: for justification of read-across see endpoint study summary

Qualifier:

no guideline followed

Principles of method if other than guideline:

10-day-inhalation study: Ten rats per sex per dose were exposed to the test compound (67, 200 and 670 mg/m³), or an air control (0 mg/m³) on 10 consecutive working days over a 14 day period in rats. Animals were evaluated based on clinical condition, body weight change, clnical pathology profiles and anatomical pathology assessments.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc., Kingston, New York
- Age at study initiation: 5-6 weeks
- Weight range at study initiation: males: 134.4 to 160.5 g; females: 109.0 to 122.8 g
- Fasting period before study: No data
- Housing: individually in stainless steel wire mesh cages, (manufactured by Lab Products, Inc., Maywood, New Jersey) which were located in a Hazleton 1000, whole-body, Inhalation exposure chamber.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow® (pellets) was provided ad libitum during non-exposure periods
- Water (e.g. ad libitum): Water was provided ad libitum (except during the daily exposure periods)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.44 - 25.00 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h /12 h

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Remarks on MMAD:

MMAD / GSD: The count median aerodynamic diameters (CMAD) and the geometric standard deviations (GSD) were recorded from the APS results and the mass median aerodynamic diameters (MMAD) were calculated from the CMAD and GSD values using the Hatch-Choate equation. The overall mean values (all chambers combined, except air control) for the CMAD, MMAD, and GSD during the pre-animal exposure period were 0.72, 1.06, and 1.42, respectively. During animal study, CMAD ranged between 0.56 gm and 0.68 gm, and GSD values were between 1.22 and 1.41. The corresponding calculated MMAD ranged between 0.64 gm 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 for the test article and air control exposures.
- Method of holding animals in test chamber: The cages with the animales were located in a Hazleton 1000, whole-body, inhalation exposure chamber. There were 24 compartments (12 per side) in each cage unit. One cage unit was used to house all animals assigned to a particular exposure group for the study.
- System of generating particulates/aerosols:
• The test article generation system was designed to simulate a possible manufacturing process failure which could result in release of a pressurized stream of liquified structural analogue into the atmosphere of the work place, where likely exposure would be inhalation of vaporized structural analogue and/or condensation aerosols of the test article.
• This generation system was designed to produce the highest reasonably or likely achievable concentration of structural analogue generated from its liquid state and allowed to achieve room temperature equilibrium conditions.
• The primary component of the generation system was a Sonimist® spray nozzle (Model HS600-2, Heat Systems Ultrasonics, Inc., Farmingdale, New York). A fine stream of heated liquid test article was discharged at a variable rate at the outlet of this nozzle where it was dispersed by a jet of compressed air producing a 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 chemical reservoir that was heated and pressurized for the delivery of the structural analogue to the Sonimist ® Nozzle. A 3 gallon galvanized canister was used as the reservoir and was modified to accept an internal core thermocouple and a 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 article 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 (reservoir core, reservoir skin, nozzle skin and carrier air line
skin) of the generation was controlled and measured.
• 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.
- Exposure System
• The test article atmosphere within the plenum chamber was transported into a single manifold system that supplied the three animal exposure chambers.
• The total concentration of structural analogue 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.
• Control rats were exposed in a chamber that received HEPA and activated charcoal-filtered room air only. The air handling system for the control chamber was separate from the test article generation and delivery system.
- Temperature, humidity, pressure in air chamber: 22.22 ± 2.78 °C, 55 ± 15 % recorded three times per day during the 1st, 3rd and 6th hour of animal exposure.
- Air flow rate and air change rate: Chamber air flow rates were controlled by orifice plate tube flowmeters and valves. Flow rates through the chambers were controlled to 15 ± 2 air changes per hour.
- Method of particle size determination:
Particles were assumed to be normally distributed with respect to the logarithm of their diameters for calculation of aerosol mass median aerodynamic diameters (MMAD) and geometric standard deviations (GSD). The aerodynamic diameters were defined as the diameter of unit density spheres which have the same settling velocity as particles of the test aerosol. 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 exposures, particle size distributions were measured once per exposure chamber (including air control) per day.
- Treatment of exhaust air: The exhaust air from all the chambers passed through Cambridge Sidelock® HEPA and Prefilter Assemblies (Cambridge Filter Corp., Syracuse, NY) to remove particulate structural analogue before combining the outflow into a Scrubber. The 'scrubbed' air was then vented outside the building.

TEST ATMOSPHERE
- Aerosol Concentration
• Because generation of structural analogue atmospheres under the test conditions produce both particulate and vapor phase emissions, a method was devised to sample both phases simultaneously. A calibrated rotameter and flow controller was used to draw an air sample from each chamber or sample location. The sample was first drawn through a 25 mm glass fiber filter mounted in an openfaced 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 entrained
in the sampling air stream and was directed into a Infrared Analyzer where the total vapor concentration was measured.
• The sample flow rate was set at 5 liter per minute while sample duration varied according to chamber test article 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 of structural analogue and were also expressed as the percent of total mass.
The Miran 1A Infrared Analyzer was equipped with a 20 meter variable pathlength gas sample cell. A standard calibration loop consisting of an injection port and circulation pump was added for calibration purposes. In this way, by knowing the volume of the sample cell and the volume of the injected amount of test material, the concentration in the cell was calculated. The absorbance (or in this case the analog output in VDC) was observed for a given injection or calibration point. The data were plotted and regressed such that input at an absorbance valve was derived for each known mass concentration value.
- Calibration
• The Miran 1A was calibrated during the pre-study development. First, a suitable wavelength was selected by completing a full infrared spectrum scan of structural analogue. Because of the physical properties of structural analogue at room temperature (solid phase), preventing injection of the test compound in liquid form, the structural analogue was dissolved in reagent grade methanol. A methanol scan was also performed. By comparing the scans, a wavelength of 8.25 gm at a detection cell pathlength of 20.25 meters was selected to be the most specific for structural analogue without interference from methanol and water vapor.
• Using known amounts of structural analogue standard prepared in methanol the absorbance value were recorded. A standard was prepared in reagent grade ethanol at a concentration of 0.204502 g/mL. A series of injections into the Miran closed-loop calibration system were conducted in triplicate. The Miran detection cell was maintained at 70°C to prevent condensation of structural analogue. The calibration was conducted over a concentration range of 4 ppm to 167 ppm (18 mg/m³ to 724 mg/m³).
- Samples taken: During the animal studies, all locations (as described above) were sampled once per hour with the gravimetric aerosol/Miran vapor sampling system, except that gravimetric samples were not collected from the room air, the air control chamber, or the Mystaire ® scrubber exhaust.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography (GC) of impinger samples was the chosen technique. One hundred twenty-five mL gas washing bottles 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 backflushed with methanol to retrieve condensed material. The samples were reconstituted to 100 mL volume and analyzed directly using a gas chromatograph.
For the GC a 30 m x 0.75 mm Supelco SPB-1 column with a 1.5 micron film thickness was used. The oven was set at 70°C with the injector and detector at 90°C. The carrier flow was at 20 mL/min. Injection volumes were constant at 0.6 L. To establish a standard calibration curve, feference test material was dissolved in reagent grade methanol between the ranges of 25 gg/mL to 764 gg/mL. The correlation coefficient for the curve was 0.999.
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. The results show that there was good agreement between gravimetric/vapor determination and GC determination methods.

Duration of treatment / exposure:

14 days

Frequency of treatment:

6 hours/day, 5 days/week for a total of 10 exposures

Remarks:

Doses / Concentrations:
0, 67, 200, 670 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 84.4, 227, and 731 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

other: filtered air alone

Details on study design:

- Dose selection rationale: The exposure concentrations for the study were selected to simulate, at the highest level, the theoretical worst-case exposure condition; with lower concentration values chosen to evaluate the concentration response. The Inhalation exposure system was designed to simulate release of heated, liquified structural analogue into ambient atmosphere and to deliver a near-saturated atmosphere of respirable test article to the study animals. Preliminary studies showed that 670 mg/m³ represented the approximate-saturation concentration of the respirable fraction (comprised of both vapor and aerosol phases) of structural analogue in absence of air under dynamic air-flow conditions. The middle and low concentrations represented approximated half-log interval decreases from the maximum achievable concentration. The lowest concentration level examined, was at least 2 to 3 orders of magnitude greater than the average concentration of the test article measured in the workplace.
- Post-exposure recovery period in satellite groups: None, animals were killed one day after the last exposure.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes,
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes,
- Time schedule: twice daily (in the morning, at least 6 hours apart, before 10:00 a.m. and after 2:00 p.m., 7 days per week) before and after each daily exposure or at similar intervals on nonexposure days for clinical evidence of toxicity or other abnormalities.
The clinical evaluations included a thorough examination of the animals general condition, exterior appearance, external orifices and observable mucous membranes. The animals were handled and allowed to move to evaluate general behavior, coordination and general neuro-muscular function. All
observations were recorded on a Data Management System.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight values of all animals were recorded on Study Day -8 (within 48 hours of receipt) and again on Study Day -2 for randomization and assignment to study groups. The body weights of animals assigned to the study were determined on Study Day 1, prior to the first exposure and again on Study Days 8, 14, and prior to scheduled necropsy.

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: shortly before necropsy
- Anaesthetic used for blood collection: Yes (identity), propylene glycolfree sodium pentobarbital
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: all animals
- The following parameters were examined:
• red blood cell count (RBC, 10e6 cells per microliter)
• hematocrit (HCT, percent)
• hemoglobin (Hgb, g/dL)
• mean corpuscular volume (MCV, femtoliters)
• mean corpuscular hemoglobin (MCH, picograms)
• mean corpuscular hemoglobin concentration (MCHC, percent)
• white blood cell count (WBC, 10³ per microliter)
• platelet count (PLAT, 103 platelets per microliter)
Smears for differential cell counts were also made from the blood samples and were stained using a modified Wright-Giemsa stain.
• The relative number of nonsegmented neutrophils (BAN),
• segmented neutrophils (SEG),
• eosinophils (EOS),
• basophils (BAS),
• lymphocytes (LYM), and
• monocytes (MON) was determined for each animal.
• The absolute number of each cell type per microliter was also calculated.
• The number of nucleated red blood cells per 100 white blood cell (nRBC/100 WBC) was also determined.
• The total reticulocyte count (RETIC) was determined after red blood cells were pre-stained with new methylene blue and a smear prepared.
• The number of reticulocytes per 100 red blood cells was counted microscopically and the results reported as the percentage of red cells which were
reticulocytes.
CLINICAL CHEMISTRY: Yes, from the retro-orbital Sinus
- Time schedule for collection of blood: shortly before necropsy
- Animals fasted: yes, approximately 12 hours prior to necropsy, water was provided ad libitum until the time of necropsy
- How many animals: All animals
- The following parameters were examined:
• blond urea nitrogen (BUN, mg/dL)
• creatinine (CRET, mg/dL)
• fasting glucose (GLU, mg/dL)
• total protein (TP, g/dL)
• albumin (ALB, g/dL)
• globulin (GLO, A:G ratio)
• cholesterol (CHOL, mg/dL)
• lactate dehydrogenase (LDH, IU/L)
• serum alanine aminotransferase (ALT, IU/L)
• serum aspartate aminotransferase (AST, IU/L)
• alkaline phosphatase (ALP, IU/L)
• bilirubin (Bil, mg/dL)
• calcium (CA, meg/L)
• sodium (NA, meg/L)
• potassium (K, meg/L)
• chloride (CL, meg/L)
• phosphorus (IP, mg/dL)
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes,
- Each animal was killed by exsanguination
- 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.

HISTOPATHOLOGY: Yes, liver, kidney, heart, mainstem bronchi, lung trachea, nasal cavity
- The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (two cross-sections), and trachea (crossand longitudinal sections), and all gross lesions suspected to be exposure-related, from all animals of each exposure group were blocked for slides, sectioned, stained with hematoxylin and eosin (H&E), and submitted for light microscopic examination. The lungs were sectioned so as to present a maximal section of the mainstem bronchi.
- The nasal cavity was prepared in four sections using the landmarks described by Young (Fundam. Appl. Toxicol., 1: 309-312, 1981).
- All other tissues collected from all other animals were preserved and held in formalin for possible future histopathological evaluation.

Details on results:

CLINICAL SIGNS AND MORTALITY
- All animals survived to study termination.
- Clinical signs of toxicity were confined to male and female rats exposed to the high-concentration (670 mg/m³) of structural analogue. A red nasal discharge was observed in all animals in the high-concentration 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.
- Nasal discharge, red: Males: day 1: 10/10, day 2 : 2/10, day 4 - 8: 10/10 and day 12 - 14: 10/10; Females: day 1: 10/10, day 4 - 8: 10/10 and day 12 - 14: 10/10;
- There were no other clinical or behavioral changes observed in the high concentration group that were thought to be related to structural analogue exposure.
- In addition, all other rats at the middle and low exposure concentration group were similar to control animals in behavior and in general health and clinical appearance.

BODY WEIGHT AND WEIGHT GAIN
- At randomization and on Study Day 1, all exposure groups had group mean body weights that were statistically similar to that of their respective control group. The 200 and 670 mg/m³ male exposure groups has 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 670 mg/m³ female group 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.
- All other male and female groups had group mean body weight values that were similar (p > 0.05) to those of their respective control group.

- The terminal body weights of male rats exposed to 200 and 670 mg/m³ of structural analogue were significantly less (p < 0.05 and 0.01, respectively) than the control group (after 8 days: control: 178.8 g; 200 mg/m³: 168.7 (5.6 %) and 670 mg/m³: 160.8 g (10.1 %); after 14 days: control: 185.4 g; 200 mg/m³: 173.9 (6.2 %) and 670 mg/m³: 181.2 g (2.3 %)). The group mean terminal body weights of females (the 670 mg/m³ exposure group) were also significantly decreased (p < 0.01) compared to control animals (after 8 days: control: 131.7 g; 670 mg/m³: 120.1 g (8.8 %); after 14 days: control: 132.3 g; 670 mg/m³: 117.5 g (11.2 %)).
FOOD CONSUMPTION
- no data

FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no data

HAEMATOLOGY
- Platelet Count was significantly changed in males of the mid and high dose group: control: 749.70 x 10³/µL; 200 mg/m³: 646.90 x 10³/µL; 670 mg/m³: 704.90 x 10³/µL.
- Hemoglobin was significantly changed in males of the high dose group: control: 16.71 g/dL; 200 mg/m³: 16.78 g/dL; 670 mg/m³: 16.19 g/dL.
- WBC Differential Count / Monocyte was significantly changed in females of the mid dose group: control: 0.70 x 10³/µL; 200 mg/m³: 1.80 x 10³/µL; 670 mg/m³: 1.11 x 10³/µL.
- These changes were small in magnitude and not considered to be related to structural analogue exposure.

CLINICAL CHEMISTRY
While there were some statistically significant changes (Creatinine: 670 mg/m³ females (control: 0.63 mg/dL; 670 mg/m³: 0.556 mg/dL); Total Protein: 200 mg/m³ females (control: 6.48 g/dL; 67 mg/m³: 6.22 g/dL); Albumin 67 mg/m³ females (control: 4.43 g/dL; 67 mg/m³: 4.28 g/dL); Globulin 670 mg/m³ males (control: 2.10; 670 mg/m³: 1.94); A/G Ratio 670 mg/m³ males (control: 2.11; 670 mg/m³: 2.36); Alanine Aminotransferase 670 mg/m³ females (control: 41.90 IU/L; 670 mg/m³: 53.44 IU/L); Alkaline Phosphatase 670 mg/m³ males (control: 413.80 IU/L; 670 mg/m³: 363.30 IU/L)), these changes were small in magnitude, were not concentration-dependent and were within the normal physiological ranges for this strain of rats. Therefore, none of the variations in clinical pathology parameters were considered to be due to structural analogue exposure.

URINALYSIS
- no data

NEUROBEHAVIOUR
- no data

ORGAN WEIGHTS
• Kidney: Absolute kidney weight value of male (7.5 %) and female (12.4 %) rats in the 670 mg/m³ group were significantly greater (p < 0.05 and 0.01, respectively) than control animals. Kidney to body weight ratios were also significantly increased (p < 0.01) in the male (22.6 %) and female (25.4 %) 670 mg/m³ exposure groups.
• Heart: There were no significant differences between the exposure and control animals in the absolute mean heart weights. However, there was a significant increase (p < 0.01) in organ-to-body weight ratios for the male (11.8 %) and female (9.2 %) high exposure (670 mg/m³ ) groups
compared to control animals.
• Lung: There were no significant differences (p > 0.05) between the exposure and control groups in absolute mean lung weights.
However, the female high concentration (670 mg/m³) group, had significantly elevated (p < 0.05) lung-to-body weight ratios (9.5 %) relative to control rats.
• Liver: There were no significant differences in group mean absolute liver weights between any groups. However, the male (21.8%) and female (12.4 %) high-concentration (670 mg/m³) 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. These lesions consisted of an apparent cystic left ovary in one air control female, and 3 low-concentration group females, and a red, discolored mediastinal lymph node in one middle-concentration (200 mg/m³) male.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Lesions considered to be related to the inhalation exposure of structural analogue were confined to the nasal cavity of the high-concentration group animals of both sexes and were present in the same anatomical location and at essentially the same degree of severity from both sexes.
- These changes were identified 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 lesion was 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 exudate, 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 proteinaceous fluid within the Lumen. Six of the high-concentration group females
and three of the high- concentration group males (670 mg/m³) 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 exposure to the test article or secondarily as a result of the inflammation, could not be definitively ascertained.
- Exposure-related lesions involving the nasal cavity in the high-concentration group (670 mg/m³) male and female rats are summarized in the following
table:
• Moderate, necrosis/degeneration of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, suppurative, inflammation of the olfactory epithelium: Males: 10/10, Females: 10/10
• Moderate, squamous, metaplasia, of the olfactory epithelium. Males: 10/10, Females: 10/10
• Adhesions, of the turbinate-dorsal wall: Males: 1/10, Females: 1/10
• Adhesions, of the turbinate-septum: Males: 2/10, Females: 5/10
- After examining the respiratory tract tissues from the high concentration and air-control rats, these same tissue sections collected from the low- and mid-concentration rats of both sexes were trimmed, processed, stained with hematoxylin and eosin and examined microscopically in an attempt to establish a no effect level (NOEL) for this particular compound. All tissues required to be examined histopathologically were present from the low-and middle-concentration rats of both sexes with the exception of the larynx from one middle-concentration (200 mg/m³) female rat, which was reported to be missing at trim. Histopathologically, the lesions involving the nasal cavities seen in the high-concentration animals were not present in the low- and middle-concentration rats of both sexes.
- Therefore, the NOEL for this spectrum of lesion in rats of both sexes under the exposure conditions used in this study 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 the exposure to structural analogue. Other than these small pulmonary inflammatory foci, no other lesions involving the lungs or the rest of the lower respiratory tract (nasopharynx, larynx, trachea) were observed in control or exposed rats of either sex.
- In conclusion, the whole-body inhalation exposure of rats to structural analogue for 10 days at a concentration of 670 mg/m³ resulted in a moderate degree of necrosis/degeneration of the olfactory epithelium lining of the nasal cavity in areas covering the dorsal meatus, adjacent nasal septum, and nasal turbinates. Associated changes consisted of a serofibrinous/suppurative inflammatory response, squamous metaplasia of affected epithelium and adhesions between the ethmoid turbinates and the nasal septum or the wall of the dorsal meatus. The lesions were of approximately the same extent and severity in both sexes with the exception of the adhesions which were more prevalent in the high-concentration group female rats. Microscopic lesions were identified in levels II, III, and IV of the nasal cavity with those involving level IV being the most extensive.
- Histopathologic examination of the respiratory tract tissues collected from the lower concentration group rats of both sexes did not reveal the nasal lesions seen in the high-concentration group rats. No changes in the lower respiratory tract (nasopharynx, larynx, trachea, and lungs) considered to be related to the inhalation of structural analogue were found. These irritation-related effects, seen only at the highest exposure level, were not unexpected for a respiratory irritant material, and could be explored further through additional evaluations, if warranted. A NOEC for this effect was determined to be 200 mg/m³.

Dose descriptor:

NOEC

Remarks:

local

Effect level:

>= 200 mg/m³ air

Sex:

male/female

Basis for effect level:

other: At highest dose, 670 mg/m3, microscopic findings included necrosis/degeneration, inflammation, and squamous cell metaplasia of the olfactory epithelium and nasal septum adhesions.

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

>= 200 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Decreased group mean body weight values (18 %).

Dose descriptor:

LOEC

Remarks:

local and systemic

Effect level:

670 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Decreased group mean body weight values (18%); red nasal discharges; necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions.

Critical effects observed:

not specified

The test atmosphere generation and delivery system used in this study functioned well and as designed. Heated, pressurized liquid structural analogue was delivered into a plenum of air held at room temperature, where the material vaporized and condensed, achieving equilibrium (under dynamic air flow conditions). The atmosphere within the plenum was effectively saturated at the nominal air flow of 350 L/min used to entrain and deliver the test atmosphere. The liquid structural analogue delivered to the plenum stabilized into a biphasic atmosphere, consisting of vapor and a fine condensation solid aerosol. The percentage of the total mass concentration present in aerosol phase within the animal exposure chambers was directly proportional to the total structural analogue concentration. Therefore, as portions of the saturated manifold atmosphere were diluted in the middle- and low-concentration chambers, a lower percentage of the chamber atmosphere was in particle phase.

Observation of toxic effects during this repeated exposure inhalation study was generally limited to the highest exposure level of structural analogue. Male and female rats exposed to 670 mg/m³ structural analogue had decreased group mean body weight values relative to controls of approximately 10 percent. The reduced body weights were apparent on Study Day 8 and continued until study termination. Rats exposed to 670 mg/m³ also showed red nasal discharges that were apparent after the end of each daily exposure, which was indicative of upper respiratory tract irritation and ulceration. Microscopic examinations showed that rats exposed to 670 mg/m³ of structural analogue had necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions. Squamous cell metaplasia is a dedifferentiation change that may become progressive and more proliferative in nature with continued exposure and is often considered to be a pre-neoplastic lesion.

While male and female rats exposed at the 670 mg/m³ level showed significant increases in absolute kidney weight relative to control and increases in several organ-to-body weight ratios, the toxicological significance of these findings is unclear in the absence of clinical chemistry and microscopic evaluations. Male rats exposed to 200 mg/m³ structural analogue had smaller body weight decreases of about 6 percent on Study Day 8.

No clinical signs of toxicity or treatment-related microscopic findings were seen in rats exposed to 67 or 200 ppm structural analogue. There were no concentration-related changes in clinical pathology studies that were considered to be biologically significant or exposure-related.

The observed lesions are considered to be typical of a respiratory irritant and are not unexpected following exposure to such a compound. The changes were noted only after repeated exposure to the highest concentration (670 mg/m³) used in this study, which would classify this test material as a mild to moderate acute respiratory irritant.

In conclusion, based on the results of this study the no-observable adverse effect concentration for structural analogue following repeated, 6 -hour exposures is 200 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

200 mg/m³

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 a subacute toxicity study (Ohara et al, 1999) 2,3,6-Trimethylphenol (CAS No. 2416-94-6, purity 99.67%) was administered by oral gavage to Sprague-Dawley rats (5-10 animals/sex/dose) at dose levels of 0, 100, 300,1000 mg/kg bw/day for 28 days. At the end of exposure, satellite animals of the control and high dose groups were observed without treatment for 14 days.

In the 1000 mg/kg group of both sexes, body weight gain and food consumption were suppressed. Similar changes were sporadically found in the 300 mg/kg treated animals. Yellowish urine was observed in both sexes of the 1000 mg/kg group. In female animals given 1000 mg/kg, proteinuria with increased excretion of bilirubin and urobilinogen was observed. The RBC count, hemoglobin content and hematocrit were decreased in the 1000 mg/kg females. Plasma levels of inorganic phosphorus were increased and that of albumin decreased in males of the 300 mg/kg and 1000 mg/kg groups. Weight of the adrenal glands was decreased in the males. Weights of the spleen and the liver were increased in 1000 mg/kg females. Hypertrophy with ground glass appearance of centrilobular hepatocytes, and hemosiderin deposition, dilatation of sinus in the spleen, and diffuse squamous cell hyperplasia of the forestomach were observed in both sexes of the 1000 mg/kg group. Most of the changes disappeared after withdrawal. Changes in hematology and histopathology in the lung (slight bleeding) were not fully reversible within the 14-day recovery.

Therefore, the NOAEL for the 28-day repeated dose oral toxicity test of 2,3,6-trimethylphenol was considered to be 100 mg/kg/day for male and female rats and the LOAEL of 300 mg/kg bw/day.

  

In a further subacute toxicity study (BASF, 1996), 2,3,6-Trimethylphenol (CAS 2416-94-6, purity 99.7%) was administered to Wistar rats (5 animals/sex/dose) in the diet at dose levels of 0, 600, 3000, 15000 ppm (0, ca. 55, 273, 1346 mg/kg bw/day) for 28 days.

Food consumption and body weight were determined once a week. Evident signs of toxicity or mortality were recorded at least once a day. Clinicochemical and hematological examinations were carried out during the last week of the administration period. All animals were subjected to gross-pathological assessment, followed by histopathological examinations. Substance-related findings in the high dose group (15,000 ppm, about 1,346 mg/kg body weight), were decreased food consumption, body weight and impairment of body weight change in males and females. Additionally, hematological parameters such as a decrease in red blood cells, hemoglobin and hematocrit in both sexes, decreases in mean corpuscular hemoglobin concentration in females, increases in polychromasia and cholesterol in both sexes, in mean corpuscular volume and mean corpuscular hemoglobin in females were observed. Increases of absolute spleen weight in females and of relative spleen weights in males and females as well as of extramedullary erythropoiesis and hemosiderosis of the spleen in males and females and a slight increase of erythropoiesis in the bone marrow of two males and two females were also determined.

In the mid dose group (3,000 ppm, about 273 mg/kg body weight) a decrease in hematocrit in females was observed and in the low dose group (600 ppm, about 55 mg/kg body weight) no substance-related changes were detected.

Thus, substance-related effects were seen at 15,000 ppm in both sexes and at 3,000 ppm in females. Target organs were spleen, bone marrow and erythron.

The only toxic effects observed were signs of marked anemia at the high dose (> 1,000 mg/kg body weight) with subsequent enhancement of hematopoiesis in bone marrow and spleen. Only a marginal sign of this effect was seen at the mid dose in females.

Therefore, the no observed adverse effect level (NOAEL) under the conditions of this study was 3,000 ppm (about 273 mg/kg body weight) for males and 600 ppm (about 55 mg/kg body weight) for females.

 

 

 A further study (RTI 2005) used a structurally similar analogue. This substance shares the functional phenolic hydroxyl group and the analogue approach was supported by evaluation of the compounds using several computational tools (Smithers REACH Services. 2,3,6-Trimethylphenol & Two Structural Anologues for REACH - Biomet Review. Report. 2010 – see Section 13).

Male and female CD® (Sprague-Dawley [SD]) F0 rats were administered TMP orally by gavage at 0, 10, 100, and 200 mg/kg/day at a dose volume of 5 ml/kg/day in Mazola® corn oil, 10 animals/sex/dose, for 2 weeks of prebreed exposure (males and females), 2 weeks of mating (males and females), and 3 weeks of gestation and lactation each (F0 females) for F0 parents, and direct dosing of selected F1 offspring from weaning through scheduled sacrifice, at least 7 weeks postweaning. Five additional F0 males per group from the control and 200 mg/kg/day groups were designated as recovery animals and held without dosing for 2 weeks after the F0 male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high-dose group. Five additional females each from the 0 and 200 mg/kg/day groups (designated "28-day females") were not mated and were terminated after 28 days of dosing. Similarly, 5 females each from the 0 and 200 mg/kg/day groups (designated "28-day recovery females") were dosed for 28 days and held without dosing for an additional 2 weeks as for the recovery group of males. The F0 males and the 28-day females were, therefore, tested according OECD 407. Doses were selected for this study based on the results of a 10-day dose range-finding (RF) study. In this study, TMP 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.

Body weights and feed consumption for the F0 males and females were recorded weekly during the prebreed period, for F0 females during gestation and lactation, and for selected F1 offspring from weaning through scheduled sacrifice. Clinical signs were recorded at least once daily for F0 males and females and F1 offspring. Functional Observational Battery (FOB), including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations, was performed on all initial animals and at least once per week for F0 animals and on 5 F1 females and 5 F1 males once midway during the postwean exposure period. 5 F0 males and females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy. Grip strength was also assessed for the 5 F1 males and F1 females per group selected for FOB during the last week of the postweaning exposure period. All F0 parental animals, nonselected F1 weanlings, and retained F1 adults were necropsied with complete histologic evaluation of the 28-day females and for 5 selected F0 and F1 males and females in the 0 and 200 mg/kg/day groups. F1 litters were culled on pnd 4 to yield, as nearly as possible, 5 males and 5 females per litter. At weaning, at least 1 female and 1 male (whenever possible) from each F1 litter 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. Hematology, clinical biochemistry, and urinalysis (28-day females and males only) assays were performed at necropsy for all 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group.

There was no adult F0 parental toxicity in either sex. There were no indications of toxicity in the recovery males, 28-day females, or recovery females. F1 postweanlings exhibited no systemic toxicity in either sex at any dose.

In conclusion, in the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg/day.

Conclusion

In the study of Ohara et al. (1999), 2,3,6-trimethylphenol (CAS No. 2416-94-6, purity 99.67%) was administered by oral gavage to the animals, whereas, in the second study (BASF 1996) the test compound was given in the diet. Since, bolus gavage dosing is expected as worst case compared to feeding, the NOAEL for the 28-day repeated dose oral toxicity test of 2,3,6-trimethylphenol of 100 mg/kg/day for male and female rats was used for exposure assessment and DNEL calculation. 

Dermal

There are no data for repeated dermal exposure.

Inhalation

An inhalation study used the structurally similar analogue. This substance shares the functional phenolic hydroxyl group and the analogue approach was supported by evaluation of the compounds using several computational tools (Smithers REACH Services. 2,3,6-Trimethylphenol & Two Structural Anologues for REACH - Biomet Review. Report. 2010 – see Section 13).

In the inhalation study, the test substance (structural analogue to 2,3,6-trimethylphenol) was administered to rats (10 animals/sex/dose) by repeated exposures, conducted on 10 consecutive working days over a 14-day period at concentrations of 67, 200 and 670 mg/m³), or an air control (0 mg/m³).

 

Animals were evaluated based on clinical condition, body weight change, clinical pathology profiles and anatomical pathology assessments. Exposure concentrations were used to simulate theoretical worst-case exposure conditions. Thereby, heated, pressurized and liquefied test substance was delivered into a plenum of air held at room temperature, where the material vaporized and condensed, achieving equilibrium (under dynamic air flow conditions). The atmosphere within the plenum was effectively saturated at the nominal air flow of 350 L/min used to entrain and deliver the test atmosphere. The liquid test substnce delivered to the plenum stabilized into a biphasic atmosphere, consisting of vapor and a fine condensation solid aerosol. The percentage of the total mass concentration present in aerosol phase within the animal exposure chambers was directly proportional to the total test substance concentration.

Male and female rats exposed to 670 mg/m³ test substance had decreased group mean body weight values relative to controls of approximately 10 percent and showed red nasal discharges that were apparent after the end of each daily exposure, which was indicative of upper respiratory tract irritation and ulceration. At the highest dose administered, 670 mg/m³, microscopic examination revealed necrosis/degeneration, inflammation, and squamous metaplasia of the olfactory epithelium and nasal septum adhesions, findings not unexpected following exposure to a respiratory irritant.

Squamous cell metaplasia is a dedifferentiation change that may become progressive and more proliferative in nature with continued exposure and is often considered to be a pre-neoplastic lesion.

No clinical signs of toxicity or treatment-related microscopic findings were seen in rats of the low and mid dose group. There were no concentration-related changes in clinical pathology studies that were considered to be biologically significant or exposure-related.

The observed lesions are considered to be typical of a respiratory irritant and are not unexpected following exposure to such a compound. The changes were noted only after repeated exposure to the highest concentration, which would classify this test material as a mild to moderate acute respiratory irritant.

In conclusion, based an the results of this study the no-observable effect level (NOEC) for the structural analogue following repeated, 6-hour exposures is 200 mg/m³.

Due to the corrosive (skin corrosion Cat. 1B, H314) properties of the structural analogue, this value is rather conservative compared to the irritating (skin irritation Cat. 2, H315) properties of 2,3,6-trimethylphenol and was estimated as overpredictive for risk assessment of 2,3,6-trimethylphenol. 

Justification for classification or non-classification

Based on reliable studies performed with the test compound 2,3,6 -trimethylphenol and structurally similar analogues, classification regarding STOT RE according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 is not warranted.