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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available short-term toxicity data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 September 1991 - 4 October 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
No detailed clinical observation and no neurobehavioural observations (compared to current OECD 407 guideline version, adopted 3 October 2008)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No further details.
Species:
rat
Strain:
other: Charles River Crl: SD BR VAF Plus rats
Details on species / strain selection:
The rat was chosen since it has been shown to be a suitable model for this type of study and is the animal recommended in the test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: At delivery, the rats were 28 ± 1 days old. Thus, taken into account the acclimation period, the animals were 36 ± 1 days old at study initiation
- Weight at study initiation: mean (males): 158.25 g; mean (females): 138.5 g
- Fasting period before study: no
- Housing: 5 rats per cage according to sex
- Diet: Biosure LAD 1 Diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: Analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substances and micro-organisms likely to be present in feed components and which, if in excess of specified amounts, might have had an undesirable effectron the test system. In addition, routine physical and chemical examination of water at source were conducted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 49 - 66
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Details on route of administration:
No further details.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a suspension in corn oil. The test material was heated in a water bath to ca. 64°C in order to obtain a liquid for weighing and formulation. The weighed test material was the mixed with warm (64°C) corn oil and the mixture warmed (if necessary) to re-melt any solidified test material. The mixture was stirred using a spatula and the further mixed using a homogeniser. Lower concentrations were made by serial dilution. A series of formulation were prepared freshly each day at concentrations of 0.6, 2 and 6% w/v. Prior to commencement of the study (Day 1), the chemical stability and homogeneity of formulations in corn oil were assessed and were found to be satisfactory. Prior to dosing, the test substance formulation were mixed by inversion (x 20). Subsequent mixing using a magnetic occurred for a period of at least 10 minutes before dosing commenced. Dosing was completed within one hour of the commencement of stirring,The dosage volume was 5 mL/kg bw/day

VEHICLE
- Concentration in vehicle: 0.6, 2 and 6%
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of concentrations of 3,5-dimethylphenol in dose formulations prepared for Days 1 and 28 of dosing was conducted by high performance liquid chromatography using ultra-violet detection.
Mean concentration of 3,5-dimethylphenol in dose formulation:
- Day 1:
Control / Nominal inclusion: 0 mg/mL / mean analysed concentration (mg/mL) ND (< 0.075 mg/mL) / Relative mean eror (%): -
30 mg/kg bw/day / Nominal inclusion: 6 mg/mL / mean analysed concentration (mg/mL) 5.99 / Relative mean eror (%): -0.2
100 mg/kg bw/day / Nominal inclusion: 20 mg/mL / mean analysed concentration (mg/mL) 20.5 / Relative mean eror (%): +2.5
300 mg/kg bw/day / Nominal inclusion: 60 mg/mL / mean analysed concentration (mg/mL) 62.1 / Relative mean eror (%): +3.5

- Day 28:
Control / Nominal inclusion: 0 mg/mL / mean analysed concentration (mg/mL) ND (< 0.075 mg/mL) / Relative mean eror (%): -
30 mg/kg bw/day / Nominal inclusion: 6 mg/mL / mean analysed concentration (mg/mL) 5.88 / Relative mean eror (%): -2.0
100 mg/kg bw/day / Nominal inclusion: 20 mg/mL / mean analysed concentration (mg/mL) 19.4 / Relative mean eror (%): -3.0
300 mg/kg bw/day / Nominal inclusion: 60 mg/mL / mean analysed concentration (mg/mL) 58.1 / Relative mean eror (%): -3.2
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 times / week
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A prelimiary oral toxicity study was conducted with three groups of six rats (three males and three females) treated with 3,5-dimethylphenol, formulated as 5, 10 or 20% w/v suspensions in corn oil. The fomrulationwas administered orally, using a syringe and rubber catheter, at a dosage volume of 5 mL/kg bw/day to achieve dosage levels of 250, 500 and 1000 mg/kg bw/day. An additional group of three male and three female animals received the vehicle alone as a concurrent control. The animals were treated once daily, for seven consecutive days. All rats were killed on Day 8. There were no mortalities. Clinical signs observed at 1000 mg/kg bw/day included a post-dose increase in salivation on Day 1 and from Day 4 onwards for the majority of rats (salivation was steined red on Day 7 for two males and one female). This sign was accompanied by less frequent cases of pilo-erection and lethargy; noisy respiration was also recorded on most days for one male and less commonly for one further male and one female. Signs were slight to moderate. Clinical signs for rats treated at 500 mg/kg bw/day were limited to a post-dose increase in salivation from Day 4 for all animals with cases of pilo-erection and lethargy (Days 1 and 2) or noisy respiration (Day 4) among females. These signs were slight to moderate. There were no clincial signs observed for rats treated at 250 mg/kg bw/day or any of the control group animals. A macroscopic post mortem observationwas undertaken following seven days of treatment and liver, spleen and kidney weights were recorded for all animals. Organ weights for control and treated animals showed no notable variation and individual values were within the expected ranges for rats of this age and strain. There were no macroscopic changes noted during necropsy. The results of this investigation with 3,5-dimethylphenol indicated that 1000 mg/kg bw/day would be tolerated as a high dosage for the twenty-eight day study in the rat. However, following consultation with the Sponsor, it was concluded that dosages should be chosen taking into account the toxicity data from a 10-week study with the 2,6-analogue of dimethylphenol. Results from this study indicated that 300 mg/kg bw/day would be a suitable high dosage, with 100 and 30 mg/kg bw/day chosen as the lower levels to provide a suitable dose relationship.
- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
- Rationale for selecting satellite groups: no satellite group
Positive control:
No positive control.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily (signs of ill health, behavioural changes and toxicosis) or twice daily (dead or moribund animals)
- Cage side observations checked : signs of ill health, behavioural changes and toxicosis, look for dead or moribund animals

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period. Gravimetric measurement of water consumption was initiated during Week 2 following a suspected treatment-related effect.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken on Day 27
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: Yes
- How many animals: all rats prior to termination (Week 4)
- Parameters checked: Packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet count, absolute indices: mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and total white blood cell count (WBC), differential white blood cell count (Diff): neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)Monocytes (M), cell morphology, thrombotest (TT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken on Day 27
- Animals fasted: Yes
- How many animals: all rats prior to termination (Week 4)
- Parameters checked: Glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (AP), total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, albumin (Alb), globulin (Glob; by substraction: Total protein - Albumin), albumin / globulin ratio (A/G; by calculation from total protein and albumin concentrations), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphorus (P) , creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Day 29, after 28 days of treatment)

Organ weights:
adrenals, brain, kidneys, liver, ovaries, testes (with epididymides)

Macroscopic pathology:
Adrenals, aorta, brain (medullary, cerebellar and cortical sections), caecum, colon, duodenum, eyes, head, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen, sternum (for bone and marrow section), stomach (glandular and forestomach), testes (including epididymis), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any other macroscopically abnormal tissue

HISTOPATHOLOGY: Yes (for all rats of Group 1 (control group) and Group 4 (high dosage group) killed on Day 29)

Tissues examined:
adrenals, heart, kidneys, liver, spleen, stomach (glandular and forestomach), testes and epididymides from male rats of Groups 1 (control group) and 4 (high dosage group)
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. Body weight data were analysed using weight gains. The following sequence of statistical tests was used for body weight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:
(ii) Bartlett’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
(iv) Analyses of variance were followed by Student's ’t’ test and Williams’ test for a dose-related response, although only the one thought more appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't'-test and Williams' test (Shirley's test).

Organ weight analysis was initally carried out using analysis of variance as outlined above on absolute organ weights. Covariate analysis of organ weight data (with final body weight as covariate) was also perfomed using adjusted weights for organs where a correlation between organ weight and body weight was established at the 10% level of significance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 300 mg/kg bw/day: persistent increase in post-dosing salivation in both sexes (all animals) from Day 4 to termination, commonly accompanied by wet fur; the increased salivation varied from slight to severe in nature.
- 100 mg/kg bw/day: sporadic incidences of increased post-dosing salivation, slight or moderate in degree, throughout the study in all animals although the signs were less frequent and less marked in the females than in the males; wet fur was also noted for male rats from Day 16 to 18;
Post-dose salivation is a common finding in orally dosed rat studies and was considered to have arisen as a result of unpalatability of the test substance, therefore, this finding was considered to be of no toxicological importance.
- 30 mg/kg bw/day: indreased post-dosing salivation, slight in nature, in two rats of each sex on one occasion (Day 14) only, which was considered to be of no consequences as this finding at this incidence is occasionally seen in control rats.
In addition: greasy fur seen in all rats particularly during the latter half of the study, and scabs on the shoulder region of three rats; these signs were considered to be coincidental; greasy fur is commonly observed following administration of com oil and the scabs are probably a result of injury sustained during fighting with cage-mates.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 300 / 100 mg/kg bw/day:
Slightly lower than control body weight gains (-12% and -14%) for male rats receiving 100 or 300 mg/kg bw/day (although the difference was not statistically significant in comparison with controls); these decreases were reflected in reduced group mean body weights throughout the treatment period; these differences reflected the slightly lower food consumption values noted for these groups; intergroup variation among females revealed no such trend.
- 30 mg/kg bw/day:
No effects observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 300 / 100 mg/kg bw/day:
Slightly lower than control food consumption values (-7% and -9%) throughout the study for male rats receiving 100 or 300 mg/kg bw/day; this was in line with the lower body weight gains noted for these groups; for females the weekly consumption among treated animals was slightly higher than for the controls at all time points, but in view of the lack of body weight changes this was considered to be coincidental.
- 30 mg/kg bw/day:
No effects observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 300 / 100 mg/kg bw/day:
Monitoring of water consumption by visual assessment maintained throughout Weeks 1, 3 and 4 indicated increased consumption by male rats treated at 300 mg/kg bw/day and by female rats treated at 100 and 300 mg/kg bw/day; measurements performed during Week 2 showed higher than control water consumption values for males receiving 300 mg/kg bw/day (+60%) and females receiving 100 or 300 mg/kg bw/day (+88 and 90%); although slightly higher values were also obtained for the remaining treated groups (between 8 and 28%), these were considered unlikely to be a result of treatment in view of the slight degree of the increases and the fact that there was only one cage/sex/group.
- 30 mg/kg bw/day:
No effects observed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
- Clinical signs (Please refer also to Table 1 & 2 for further details):
There were no mortalities.
- 300 mg/kg bw/day: A persistent increase in post-dosing salivation was recorded for both sexes (all animals) from Day 4 to termination, commonly accompanied by wet fur. The increased salivation varied from slight to severe in nature and was seen in .
- 100 mg/kg bw/day: Sporadic incidences of increased post-dosing salivation, slight or moderate in degree in all animals, were seen throughout the study although the signs were less frequent and less marked in the females than in the males. Wet fur was also noted for male rats from Day 16 to 18. Post-dose salivation is a common finding in orally dosed rat studies and was considered to have arisen as a result of unpalatability of the test substance. Therefore, this finding was considered to be of no toxicological importance.
- 30 mg/kg bw/day: Indreased post-dosing salivation, slight in nature, was noted for two rats of each sex on one occasion (Day 14) only and was considered to be of no consequences as this finding at this incidence is occasionally seen in cotrol rats.
Other findings were limited to greasy fur seen in all rats particularly during the latter half of the study, and scabs on the shoulder region of three rats. These signs were considered to be coincidental, greasy fur is commonly observed following administration of com oil and the scabs are probably a result of injury sustained during fighting with cage-mates.

- Body weight / body weight gain (Please refer also to Table 3 & 4 for further details):
- 300 / 100 mg/kg bw/day:
Slightly lower than control body weight gains (-12% and -14%) were noted for male rats receiving 100 or 300 mg/kg bw/day although the difference was not statistically significant in comparison with controls; these decreases were reflected in reduced group mean body weights throughout the treatment period. These differences reflected the slightly lower food consumption values noted for these groups. Intergroup variation among females revealed no such trend.
- 30 mg/kg bw/day:
No effects observed.

- Food consumption (Please refer also to Table 5 for further details):
- 300 / 100 mg/kg bw/day:
Slightly lower than control food consumption values (-7% and -9%) were seen throughout the study for male rats receiving 100 or 300 mg/kg bw/day and this was in line with the lower body weight gains noted for these groups. For females the weekly consumption among treated animals was slightly higher than for the controls at all time points, but in view of the lack of body weight changes this was considered to be coincidental.
- 30 mg/kg bw/day:
No effects observed.

- Water consumption (Please refer also to Table 6 for further details):
- 300 / 100 mg/kg bw/day:
Monitoring of water consumption by visual assessment was maintained throughout Weeks 1, 3 and 4. This indicated increased consumption by male rats treated at 300 mg/kg bw/day and by female rats treated at 100 and 300 mg/kg bw/day. Measurements performed during Week 2 showed higher than control water consumption values for males receiving 300 mg/kg bw/day (+60%) and females receiving 100 or 300 mg/kg bw/day (+88% and +90%). Although slightly higher values were also obtained for the remaining treated groups (between 8 and 28%), these were considered unlikely to be a result of treatment in view of the slight degree of the increases and the fact that there was only one cage/sex/group.
- 30 mg/kg bw/day:
No effects observed.

- Haematology:
Intergroup variation among male rats revealed no significant change in comparison with controls in all dose groups.
- 300 mg/kg bw/day, females:
Slightly lower packed cell volume, haemoglobin and red cell count recorded for females treated at 300 mg/kg bw/day all achieved significance (P≤0.05) in comparison with controls. Individual values for treated rats all fell within the laboratory background data ranges (PCV, %, females: 5th percentile - 44; median - 49; 95th percentile - 55, n = 91; Hb, g/dL, females: 5th percentile - 14.0; median - 15.2; 95th percentile - 16.1, n = 92; RBC, x 10^6/mm3, females: 5th percentile - 5.5; median - 6.4; 95th percentile - 7.1, n = 91) and these changes were considered to have arisen by chance.
There were no other statistically significant changes recorded for female rats both in this dose group or in the other two dose groups.
The occurrence of slight polychromasia and anisocytosis is not uncommon among young laboratory rats and at the frequency seen in this study (5/40) they were not considered to be treatment-related.

- Biochemistry:
- 300 mg/kg bw/day, males:
A slightly elevated chloride ion level was recorded for male rats treated at 300 mg/kg bw/day achieving significance (P<0.05) in comparison with controls. However, individual values, with one exception (male high dose animal), fell within the laboratory background data range (Cl, mEq/L, males: 5th percentile - 93; median - 96; 95th percentile - 99, n = 91) and the change was considered to be coincidental.
There were no statistically significant differences noted for male or female rats in the other parameters measured both in this dose group or in the other two dose groups.

- Organ weights (Please refer also to Table 7 & 8 for further details):
- 300 / 100 mg/kg bw/day:
A significantly (P<0.05) decreased combined testis and epididymides weight was recorded for rats treated at 300 mg/kg bw/day in comparison with controls (-14%). Individual unadjusted values generally fell within the expected range (testis and epididymides, g, males: 5th percentile - 3.29, median - 3.90; 95th percentile - 4.56, n = 92) and the statistical analyses may have been affected by one slightly high value among the control animals and a low weight recorded for one rat of Group 4 (high dose group).
Unadjusted adrenal weights for females receiving 100 and 300 mg/kg bw/day were slightly but not significantly higher than controls (+2.7% and + 9.9%) although individual values, except for one animal, all fell within the laboratory background range (adrenals, mg, females: 5th percentile - 55.3; median - 69.1; 95th percentile - 86.3, n = 92).
For females treated at 100 and 300 mg/kg bw/day the unadjusted kidney weights were higher than controls (+13% and + 14.4%), achieving significance (P<0.05) at the high dosage. Adjusted weights, taking final bodyweight into consideration, indicated a small, significant (P<0.05) decrease in weights for the high and intermediate dosage groups. Overall, the difference from controls for adjusted weights was small and considered to have arisen by chance. Adjusted and unadjusted weights for male kidneys revealed no significant differences between treated and control group.

Female liver weights were higher than controls for rats receiving 300 mg/kg bw/day with unadjusted (P<0.05) and adjusted, taking final body weight into consideration, (P<0.01) values achieving significance in comparison with controls. However, individual unadjusted values generally fell within the laboratory background range (liver, g, females: 5th percentile - 10.4; median - 13.0; 95th percentile - 16.5, n = 92) and the statistical analysis may have been affected by a low value recorded for one control rat. The difference was considered to be coincidental.

Following review of the microscopic pathology results and the lack of macroscopic changes, organ weight analysis did not reveal any differences from control that were considered to be of toxicological importance.

- 30 mg/kg bw/day:
No effects observed.

- Macroscopic pathology:
No changes attributable to treatment.

- Microscopic pathology:
- 300 mg/kg bw/day:
Liver - There was an increase in centrilobular hepatocyte vacuolation in male rats receiving 300 mg/kg/day when compared with the control rats. However, as the degree of vacuolation in the high dose rats is within the range seen in untreated rats at this laboratory and there were no histopathological findings which correlate with the increased liver weights, this is considered unlikely to be related to treatment.
Incidental findings:
Testis - Moderate, unilateral tubular atrophy in a single male rat receiving 300 mg/kg bw/day may have contributed to the decrease in organ weight for the group.
Adrenals - Moderate cortical hypertrophy in a single female rat /receiving 300 mg/kg bw/day may have contributed to the increase in organ weight recorded for the group,

All other findings in both this dose group and the other two dose groups were considered to be spontaneous in origin and of no toxicological importance.
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed until the highest dose tested.
Key result
Critical effects observed:
no

Table 1: Clinical signs ovserved in male animals following oral administration of 3,5-dimethylphenol

Group/dosage mg/kg bw/day

Clinical signs

Earliest appearance (Day)

Last appearance (Day)

Maximum consecutive duration (days)

(Animal number)

Maximum severity of observed signs

1 ♂ / Control

 

 

 

 

(1)

(2)

(3)

(4)

(5)

Greasy fur

20

29

10

A

A

A

A

A

Scabs on right shoulder region

21

29

9

/

B

/

/

/

2 ♂ / 30

 

 

 

 

(6)

(7)

(8)

(9)

(10)

Increased salivation

14

14

1

A

A

/

/

/

Greasy fur

20

29

10

A

A

A

A

A

Scabs on left shoulder region

21

29

9

/

/

/

/

B

3 ♂ / 100

 

 

 

 

(11)

(12)

(13)

(14)

(15)

Increased salivation

6

28

3

B

B

B

B

B

Wet fur

16

18

3

A

A

A

A

A

Greasy fur

19

29

11

B

B

B

B

B

4 ♂ / 300

 

 

 

 

(16)

(17)

(18)

(19)

(20)

Increased salivation

4

28

25

C

B

B

B

B

Wet fur

6

19

4

B

B

B

B

B

Scabs on left shoulder region

21

29

9

/

/

/

A

/

A Slight response

B Moderate response

C Severe response

Table 2: Clinical signs ovserved in female animals following oral administration of 3,5-dimethylphenol

Group/dosage mg/kg bw/day

Clinical signs

Earliest appearance (Day)

Last appearance (Day)

Maximum consecutive duration (days)

(Animal number)

Maximum severity of observed signs

1 ♀ / Control

 

 

 

 

(21)

(22)

(23)

(24)

(25)

Greasy fur

25

29

5

A

A

A

A

A

2 ♀ / 30

 

 

 

 

(26)

(27)

(28)

(29)

(30)

Increased salivation

14

14

1

A

/

/

A

/

Greasy fur

23

29

7

A

A

A

A

A

3 ♀ / 100

 

 

 

 

(31)

(32)

(33)

(34)

(35)

Increased salivation

5

19

4

B

B

B

B

B

Greasy fur

23

29

7

A

A

A

A

A

4 ♀ / 300

 

 

 

 

(36)

(37)

(38)

(39)

(40)

Increased salivation

4

28

25

B

B

B

B

B

Wet fur

6

25

9

B

B

B

B

B

Greasy fur

16

29

14

B

B

B

B

B

A Slight response

B Moderate response

C Severe response

 

Table 3: Body weights - group mean values (g)

Week

Group and dosage (mg/kg bw/day)

1 ♂

2 ♂

3 ♂

4 ♂

1 ♀

2 ♀

3 ♀

4 ♀

Control

30

100

300

Control

30

100

300

Dosing

 

 

 

 

 

 

 

 

0

160

158

157

158

139

136

137

142

1

234

231

224

229

173

174

170

175

2

309

304

291

295

199

202

199

203

3

378

370

351

352

217

224

219

224

4

432

418

395

391

236

243

238

248

 

Table 4: Body weight gains - group mean values (g)

Week

Group and dosage (mg/kg bw/day)

1 ♂

2 ♂

3 ♂

4 ♂

1 ♀

2 ♀

3 ♀

4 ♀

Control

30

100

300

Control

30

100

300

Dosing

 

 

 

 

 

 

 

 

1

75

73

67

70

34

38

32

32

2

74

73

67

67

26

28

29

28

3

70

66

60

57

18

22

21

22

4

54

49

43

39

19

20

19

23

Group mean cumulative total

272

260

238

233

97

107

101

105

% of control

/

96

88

86

/

110

104

108

 

Table 5: Food consumption - group mean values (g)

Week

Group and dosage (mg/kg bw/day)

1 ♂

2 ♂

3 ♂

4 ♂

1 ♀

2 ♀

3 ♀

4 ♀

Control

30

100

300

Control

30

100

300

Dosing

 

 

 

 

 

 

 

 

1

176

173

165

168

117

124

122

123

2

199

199

187

183

113

123

125

124

3

215

213

201

190

113

130

130

129

4

207

209

188

181

116

126

126

129

Group mean cumulative total

797

794

741

722

459

503

503

505

% of control

/

100

93

91

/

110

110

110

 

 

Table 6: Water consumption – group mean values (g)

Week

Group and dosage (mg/kg bw/day)

1 ♂

2 ♂

3 ♂

4 ♂

1 ♀

2 ♀

3 ♀

4 ♀

Control

30

100

300

Control

30

100

300

Dosing

 

 

 

 

 

 

 

 

1.1

29.4

32.2

34.0

49.0

25.4

28.8

42.6

45.0

1.2

32.2

32.4

33.0

49.0

25.8

26.8

44.2

44.8

1.3

33.0

33.8

33.4

50.8

21.8

32.6

49.2

47.0

1.4

30.2

35.0

36.0

51.0

21.0

32.0

48.8

51.2

1.5

32.6

34.0

36.0

51.4

24.6

30.2

47.8

46.2

1.6

32.4

37.2

35.0

51.8

30.8

34.6

48.8

53.6

2

34.4

37.2

37.8

56.2

27.4

39.8

50.4

46.6

Group mean cumulative total

255

276

280

410

202

258

379

383

% of control

/

108

110

160

/

128

188

190

 

Table 7: Organ weights (males) – group mean values (Week 5)

Group /

Body wt.

Brain

Liver

Kidneys

Adrenals

 Testis and epididymides

dosage mg/kg bw/day

g

g

g

g

mg

g

1 ♂

 

A

K

A

A

 

Control

427

2.04

25.6

4.13

56.4

4.18

 

 

(2.01)

(3.98)

(54.2)

 

2 ♂

413

2.02

27.5

3.73

56.5

3.72

30

 

(2.01)

(3.67)

(55.6)

 

3 ♂

390

1.97

22.5

3.42

59.2

3.93

100

 

(1.99)

(3.51)

(60.6)

 

4 ♂

386

2.01

24.3

3.92

53.3

3.60*

300

 

(2.03)

 

(4.04)

(55.1)

 

Blank: Non-significant

* P<0.05 in comparison with controls using Williams' test

K: Kruskal- Wallis analysis

A: Data adjusted for final body weight; adjusted means are given in parentheses

 

Table 8: Organ weights (females) – group mean values (Week 5)

Group /

Body wt.

Brain

Liver

Kidneys

Adrenals

Ovaries

dosage mg/kg bw/day

g

g

g

g

mg

mg

1 ♀

 

 

A

A

 

A

Control

236

1.80

11.3

2.22

67.7

100.6

 

 

(11.6)

(2.56)

(102.0)

2 ♀

242

1.86

12.4

2.32

62.3

98.0

30

 

(12.3)

(2.31)

(97.3)

3 ♀

236

1.83

12.2

2.51

69.5

102.3

100

 

(12.6)

(2.55)*

(103.9)

4 ♀

247

1.88

13.5*

2.54*

74.4

111.2

300

 

 

(13.0)**

(2.48)*

 

(108.9)

Blank: Non-significant

* P<0.05 in comparison with controls using Williams' test

** P<0.01 in comparison with controls using Williams' test

A: Data adjusted for final body weight; adjusted means are given in parentheses

 

 

Conclusions:
In an oral gavage study conducted according to OECD 407 and under GLP the NOAEL for 3,5-dimethyl phenol (CAS 108-68-9) was set at the highest dose tested, 300 mg/kg bw/day for male and female rats due to the lack of adverse effects.
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 July 2004 - 17 Sept 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR VAF/Plus
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory, Raleigh, North Carolina
- Age at study initiation: 9 weeks (males and females)
- Weight at study initiation: 333 - 365 g (males); 205 -237 g females
- Fasting period before study:
- Housing: F0 generation rats were individually housed except during the cohabitation and postpartum periods. During cohabitation, each pair of rats were housed in the male rat's cage. Each dam and delivered litter was housed in a common nesting box during the postpartum period.
- Diet: Chow (#5002),ad libitum
- Water: Tap water,ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12






 
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test substance was prepared as a solution in the vehicle and administered orally by gavage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC/FID analysis of dosing preparation concentration, stability and homogeneity.
Duration of treatment / exposure:
28 days for males and 54 days for females.
Frequency of treatment:
Daily administration beginning 14 days prior to cohabitation and continuing until the day before sacrifice.
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
245 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 rats per sex per group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least 2/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: daily
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

Observations were performed for viability, clinical signs of toxicity, food consumption, body weight gain, functional observational battery and motor activity, hematology, clinical chemistry, developmental toxicity and reproductive performance, gross and microscopic post-mortem examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 male and 5 female F0 generation animals
- Parameters examined: total leukocyte count, differential leukocyte count (neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell); RBC indices (mean corpuscular volume, mean corpuscular haemogloblin, mean corpuscular haemoglobin concentration), platelet count, prothombin time and activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Not specified
- How many animals: 5 male and 5 female F0 generation animals
- Parametersexamined: blood urea nitrogen, alanine aminotransferase activity, aspartate transaminase activity, alkaline phosphatase, glucose, cholesterol, calcium, chloride, phosphorous, potassium, sodium, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, creatinine, triglycerides

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once
- Dose groups that were examined: Functional observational battery evaluations were performed once on 5 male and 5 female F0 generation animals.

 
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

F0 generation:
Gross pathological examination was performed on 5 male and 5 females/group of the F0 generation. An initial examination of external surfaces and all orifices, as well as the cranial, thoracic and abdominal cavities, including contents. The epidodymides, seminal vesicles with coagulating gland and prostate were retained.

Dams with no suriving pups were sacrificed after the last pup was found dead, missing or presumed cannibalised. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

Organ weights:
The following organs were weighed from 5 male and 5 females/group of the F0 generation at necropsy:
liver, spleen, kidneys, brain, adrenals, heart, thymus. ovaries, testes, uterus (with cervix) and epidiymides. F1 generation pups that died before the initial examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were considered stillborn; pups with lungs that floated were considered liveborn and to have died shortly after birth. Pups with gross lesions found on days 2 to 4 postpartum were preserved for possible future evaluation. On day 5 postpartum , pups were sacrificed abd examined for gross lesions. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross sectioned brain.
Statistics:
Body weight, weight gains and reproductive endpoints analysed by ANOVA and Dunnett's. Reproductive data analysed by Fisher's exact test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
urine stained fur in males and females of high dose group
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative weights of kidney, liver and ovaries were increased in the high dose group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Due to clinical observations (urine-stained fur, increased kidney, liver and ovarian relative weight).
Critical effects observed:
no

All rats survived the treatment. In males, urine stained fur was observed at the 245 mg/kg bw/day level. Body weight gain and food consumption were unaffected by treatment. Mating frequency was reduced at the 245 mg/kg bw/day level. Neurotoxicity was not observed during the study and there were no treatment related effects observed at gross necropsy or histopathologically. Urine staining of the fur was observed in females at the 245 mg/kg bw/day level. F1 animals showed no treatment related clinical or necropsy signs. Haematology and clinical pathology parameters were unaffected.

Reproductive indices are discussed under section 7.8.1.

Conclusions:
The test substance mixed xylenols was administered to rats by oral gavage at 0, 30, 100 and 245 mg/kg/day. The general toxicological No Observable Adverse Effect Level was shown to be 100 mg/kg bw/day.

The reproductive NOAEL is discussed under section 7.8.1.

Data source

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-xylenol
EC Number:
202-461-5
EC Name:
2,5-xylenol
Cas Number:
95-87-4
Molecular formula:
C8H10O
IUPAC Name:
2,5-dimethylphenol

Results and discussion

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Due to clinical observations (urine-stained fur, increased kidney, liver and ovarian relative weight).
Remarks on result:
other: Source, mixed xylenols, Merisol, 2005, OECD 422

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

In the result table above the most critical value of the two source substances of the read-across approach is given. The NOAEL of the second source substance 3,5-Xylenol (CAS 108-68-9) was set at 300 mg/kg bw/day (the highest dose tested) in an oral repeated dose toxicity study in rats according to OECD 407 and under GLP due to the lack of adverse effects (BG Chemie, 1993).

Applicant's summary and conclusion

Conclusions:
The available subacute repeated dose oral toxicity studies conducted on 3,5-Xylenol (CAS 108-68-9) and mixed xylenols resulted in NOAELs for general systemic toxicity of 300 and 100 mg/kg bw/day, respectively. As detailed in the analogue justification, it is considered that the target and the source substance are unlikely to lead to differences in repeated dose toxicity potential. Therefore, the results can be taken for the hazard assessment of the target substance 2,5-Xylenol (CAS 95-87-4). As a worst-case approach the lower NOAEL of 100 mg/kg bw/day of the mixed xylenols will be taken for risk assessment of the target substance 2,5-Xylenol (CAS 95-87-4).
Executive summary:

In an oral gavage study conducted according to OECD 407 and under GLP the NOAEL for the source substance 3,5-Xylenol (CAS 108-68-9) was set at the highest dose tested, 300 mg/kg bw/day, for male and female rats due to the lack of adverse effects. The sub-acute repeated dose oral toxicity study conducted on mixed xylenols according to OECD 422 and GLP result in a NOAEL of 100 mg/kg bw/day. Increases in liver and kidney weights were reported. No histopathological changes accompanied these increases in organ weights.