2,6-xylenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed per OECD 422 and following GLP. A subset of the animals tested were evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. A justification for the read-across is provided.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

structural analogue

IUPAC Name:

structural analogue

Constituent 2

Reference substance name:

2,4,6-trimethylphenol

EC Number:

208-419-2

EC Name:

2,4,6-trimethylphenol

Cas Number:

527-60-6

Molecular formula:

C9H12O

IUPAC Name:

2,4,6-trimethylphenol

Details on test material:

- Stability under test conditions: Stable in vehicle (corn oil) for at least 35 days
- Storage condition of test material: Ambient or refrigerated conditions.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: P = 63 days old
- Housing: singly in polycarbonate cages with bedding except during mating.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77 degrees F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light cycle
IN-LIFE DATES: From: 2003-09-2 To: 2004-01-06

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All dosing formulations were analyzed for concentration verification. Standards for acceptable accuracy of mixing were that the means of the analyzed samples was within +/- 10% of the nominal concentration and the relative standard deviation for triplicate samples did not exceed 10%.

Duration of treatment / exposure:

F0 females were dosed premating through the day prior to necropsy.
Recovery males, females and 28 days females were dosed for 28 days.
F1 animals were dosed post-weaning until the day before scheduled necropsy, at least 7 weeks duration.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100, 200 mg/kg/day
Basis:
other: nominal concentration at a dose volume of 5 mL/kg/day

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a dose range finding study, the test substance was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore, 300 mg/kg/day was considered too toxic for the OECD 422 study design.
- Rationale for animal assignment (if not random): Randomization stratified by body weight

Examinations

Observations and examinations performed and frequency:

PARENTAL OBSERVATIONS:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and included changes to skin and fur, eyes, mucous membranes, respiratory and circulatory sytems, autonomic and central nervous systems, somatomotor activity, and behavior patterns.
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreeding, for F0 females during gestation (gd 0,7,14, and 21) and lactation (pnd 0, 4, 7, 14, and 21). Body weights of the 28 day females and and recovery males and females were recorded on a weekly basis until termination. Body weight gains were computed.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

LITTER OBSERVATIONS:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, a maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, euthanized,
necropsied and completed external and visceral examinations performed. Survival indices were calculated at least weekly through weaning (pnd21).
GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or
found dead.
OTHER:
At weaning at least one male and one female from each litter (when possible) were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for retained F1 females included examination of vaginal patency and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last three weeks of the postwean exposure period prior to scheduled sacrifice. For each retained male offspring, observations or cleavage of the balanopreputial gland began at day 35 and continued until preputial separation. Androgenic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected F1 adult males and females per dose group. Body weights for selected F1 offspring from weaning through scheduled sacrifice were taken. Functional Observation Battery (FOB), including cage side observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations was performed on 5 F1 females and 5 F1 males once midway during the postwean exposure period. Grip strength was also assessed for the 5 F1 males and 5 F1 females per group selected for FOB during the last week of the post weaning exposure.

Sacrifice and pathology:

-All F0 parental animals, 28-day females, recovery males and females, adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of FO females were examined for the number of nidation (implantation) scars.

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

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

Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females in the control and high-dose groups. Since no treatment-related findings were identified, no histopathology on lower dose groups was performed.

Other examinations:

OTHER:
Urinalysis: Prior to necropsy, 5 F0 males per group, randomly selected at the end of the mating period, and the 28-day females were singly housed overnight in metabolism cages. The total amount of time the animal was in the chamber and the amount of urine collected were recorded. The urine was evaluated for appearance and by dipstick analysis for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes.

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

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

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



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

In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected parental F0 males and females per dose group.

Statistics:

Appropriate statistical analyses were used throughout the assay.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

post dose rooting - consistent with taste aversion

Mortality:

mortality observed, treatment-related

Description (incidence):

post dose rooting - consistent with taste aversion

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Treatment-related clinical observations of F0 males included rooting post-dosing in 5 males at 200 mg/kg/day. No other findings exhibited a treatment- or dose-related pattern of incidence or severity.
All 10 F0 females/group survived to scheduled sacrifice

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

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

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

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

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

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

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


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

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

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

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

OTHER FINDINGS (OFFSPRING):
FOB which was performed midway through the postweaning period for 5 of the F1 males and females/group showed no significant difference for home cage observations, handling observations, sensory and neuromuscular observations, or open field observations.Grip strength was also evaluated. Forelimb grip strength was unaffected across groups. Hindlimb grip strength was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no indications of toxicity (systemic, reproductive, developmental, or neurological) in either the paraental or F1 generation, the recovery males or females or the 28 day female dose groups evaluated in this study.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

RECOVERY MALES:

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

RECOVERY FEMALES:

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

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

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

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

Applicant's summary and conclusion

Conclusions:

In conclusion, the test substance, administered by gavage once daily at 0, 10, 100, and 200 mg/kg/day to parental rats, from prebreed through mating, gestation, and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice, resulted in no adult F0 parental toxicity and no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals.
Therefore, systemic NOAEL was at least 200 mg/kg/day.
A justification for the use of read-across has been provided.