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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th April - 16th July 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
There are no significant deviations which affected the quality or the integrity of the study or the interpretation of the results in the report.
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
There are no significant deviations which affected the quality or the integrity of the study or the interpretation of the results in the report.
Principles of method if other than guideline:
The aim of this study was to evaluate the subchronic toxicity of m-xylene when administered to rats daily by oral gavage for at least 90 days.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
≥ 99.5 % pure

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material name: m-xylene
- CAS number: 108-38-3
- State: clear colourless liquid
- Source: Fluka Chemical Corporation, Hauppauge, New York
- Sponsor: Dynamac Corporation
- Lot No.of test material: 259 211
- Expiration date of the lot/batch: not specified
- Purity: 99.1% (pure by GC-analysis)

- The test substance was analysed for identity and purity prior to study initiation and for purity after study termination.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- During transit: stored frozen and protected from light
- Storage condition of formulated test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dosing preparations prepared using corn oil (vehicle) and were adjusted to 100% activity, based on a purity of 99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No further details specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Total number of animals: 168 males and 168 females
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 46 days
- Weight at study initiation: males = 203.7-246.4g and females = 142.1 - 174.8g
- Housing: rats were uniquely identified by ear tags and cage cards and were housed individually in elevated wire-mesh cages
- Diet (e.g. ad libitum): commercial rodent diet (Purina Certified Rodent Chow® #5002)
- Water (e.g. ad libitum): acidified tap water
- Acclimation period: acclimated to laboratory conditions for approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY: Contaminant analysis for the rodent chow 5002 diet and water can be found in the 'attached background material'

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-77
- Humidity (%): 31-69
> temperature and relative humidity in the animal were recorded twice daily (with few exceptions)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle was maintained daily via artificial illumination

IN-LIFE DATES: From: 11th April To: 14-16th July 1986

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The formulated test material was given to the animals by oral gavage. The dosing volume was 2.5 mL/kg
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions were prepared weekly.
- For each dose level, the amount of test material required was calculated and then weighed. It was transferred to a volumetric flask and then the vehicle, corn oil, was added to reach the final volume.
- The flask was capped and inverted by hand until thoroughly mixed.
- Groups and doses can be seen in Table 1 below.
Analytical verification of doses or concentrations:
yes
Remarks:
Formulated test material was analysed for purity, stability and dose concentration.
Details on analytical verification of doses or concentrations:
Stability results reveal that there was a small amount of test material lost after 21 days of storage at 5°C.
All samples analysed were within the specific limits ± 10% of target concentration
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control) and Group 5 (baseline)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
20 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment : Sprague-Dawley rats were employed since they have historically been used in safety evaluation studies and were requested by the EPA
- Animal selection: following physical and ophthalmologic examinations, a total of 85 clinically acceptable rats of each sex were randomly allocated to 4 treatment groups using a computer weight randomisation program, each composed of 20 animals/sex/group and 5 animals/sex were assigned to the baseline group employed for pre-treatment bloodwork.
- Animals not selected were assigned to another study or discarded without necropsy.
- Baseline groups were used for pre-treatment bloodwork and later discarded without necropsy.
Positive control:
No positive control was employed.

Examinations

Observations and examinations performed and frequency:
- All rats were observed daily for clear signs of toxicity, beginning about one hour after dosing
- A cursory examination was performed during dosing

PHYSICAL EXAMINATIONS: Yes
- Time schedule: performed at initiation and weekly thereafter

MORIBUNDITY/MORTALITY CHECKS: Yes
- Time schedule: twice daily

INDIVIDUAL BODY WEIGHTS: Yes
- Time schedule for examinations: recorded during the randomisation procedure, at initiation and weekly thereafter

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

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Performed by a board-certified veterinary opthalmologist
- Time schedule for examinations: prior to treatment, Week 13
- Dose groups that were examined: all animals prior to treatment and the last 10 survivors per sex per group during Week 13
- Pupils of both eyes were dilated prior to examination using 1% Atropine Sulphate

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation, weeks 5-13
- Anaesthetic used for blood collection: Yes - samples were collected via the orbital sinus while under carbon dioxide anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 animals/sex (Baseline group) prior to initiation and on the first 10 survivors/sex/group during weeks 5-13
- Parameters examined: please see Table 2 below

CLINICAL PATHOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation, weeks 5-13
- Animals fasted: Yes - samples were collected via the orbital sinus while under carbon dioxide anaesthesia
- How many animals: 5 animals/sex (Baseline group) prior to initiation and on the first 10 survivors/sex/group during weeks 5-13
- Parameters examined: please see Table 3 below

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- For all animals that died during the study and all survivors that were sacrificed after 13 weeks of treatment and were given a complete gross necropsy. Please see Table 4 below.
- Euthanasia was exsanguination whilst under sodium pentoarbital anaesthesia.
- At the terminal sacrifice, five animals per sex from the control group and 10 animals per sex from each treated group were selected for electron microscopy, the results of which will be available in a separate report.

HISTOPATHOLOGY: Yes
- All preserved tissues from all animals were embedded in paraffin, sectioned, and stained with haemoxylin and eosin.
- The following tissues were examined microscopically:
(1) All tissues from control and high-dose animals and from all animals not surviving to termination.
(2) Gross lesions, lungs, liver and kidneys from all remaining animals.
Other examinations:
ORGAN WEIGHTS
-The following organs were weighed for all terminally killed animals, except those selected for electron microscopy:
liver, kidneys, spleen, adrenal glands, brain with stem, heart, tested with epididymides, ovaries

TISSUE PRESERVATION
- Please see Table 5 below for all tissues that were preserved from each animal (when present) in 10% neutral buffered formalin.
Statistics:
- Cumulative survival data were analysed using the National Centre Institute Package (Life Table Analysis).
- Trend analysis of survival was evaluated at the 5.0% one-tailed probability level.
- Body weights, food consumption, appropriate clinical pathology data and organ weight data were compared between the control and treated groups of the same sex
- Tests for homogeneity of variances and ANOVA were evaluated at the 5.0% two-tailed probability level.
- Control vs. treated group means were evaluated using Dunnett's t-test at the 5.0% two-tailed probability level.
- Data transformations were performed where variances were heterogenous, which are indicated in the tables with an appropriate superscript next to the control group mean. The list of these superscripts can be seen in Table 6 below.
- When an effect is referred to as 'significant', it means that there is a statistically significant difference between the control group and the specified treated group of the same sex

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Urine stains were observed weekly in both sexes of Group 4 and thought to be possibly treatment-related.
Other common weekly signs observed were chromodacryorrhea and malocclusion, but these are not thought to be treatment-related.
Salivation was frequently observed in both sexes of Group 4 and only rarely in the other treated groups. It occurred just prior to dosing and the animals returned to normal by the one-hour post-dosing observations.
Other daily observations included hyperactivity, convulsions and epistaxis - they were only observed in the first three weeks of the study at a low frequency
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males: there was a significantly higher mortality in Group 3 (5 males died), but there was not a significant trend.

Females: there was significant trend and a significantly higher mortality in Group 3 (5 females died) and Group 4 (4 females died).

Males and females: In the 19 unscheduled deaths, all but one of the animals had foreign material in the alveoli and were determined to be related to gavage dosing accidents rather than being related to the test material (please refer to the histopathological findings section). This may also relate to the lung lesions, mottled lungs and failure of the lungs to collapse seen in males and females of Groups 3 and 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight gain over the course of the experiment was significantly decreased in Groups 3 and 4 for males and in Group 4 for females.
There was a clear relationship between dose and body weight reduction in both sexes.
The overall total mean body weights (Groups 1-4) were 15% and 8% lower than in the controls for males and females respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistical analysis was employed at three intervals - Weeks 1-5, 6-9 and 10-13.
Food consumption was significantly reduced during Weeks 1-5 for Group 4 males and during Weeks 6-9 for Group 3 and 4 males.
Food consumption was comparable for all female groups at all three intervals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only two animals (one Group 1 male and one Group 3 male) showed abnormalities at the terminal examination and thus were judged to be unrelated to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haemotocrit was significantly decreased in Group 4 females.
At Week 13, there were no significant changes to the haematology parameters in either sex.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significant changes were observed at the Week 5 interval:
- Group 4 males - potassium was increased and total bilirubin was decreased.
- Group 4 females - cholesterol and calcium increased
- Group 3 and 4 females - potassium was increased.

There were fewer significant changes at Week 13:
- Group 4 males - alanine aminotransferase was increased
- Group 4 females - calcium and cholesterol was increased
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group 4:
- the terminal body weights were significantly decreased in both sexes
- males - there were few significant changes in organ weights, however heart weight was decreased and relative brain and kidney weights were increased vs. control
- females - no significant changes in absolute or relative organ weights vs. control
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were few gross lesions observed.
The most commonly seen gross lesions were mottled lungs and failure of the lungs to collapse.
These observations were most frequently observed in both sexes of Group 3 and 4, but were only found in the animals found dead during the experiment.
The other lesions observed were sporadic and showed no apparent dose relationship
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Several spontaneously-occurring incidental lesions were observed in animals at the terminal sacrifice, but were concluded to be not related to treatment.

In the 19 unscheduled deaths (males Group 2-4; females Group 3-4), all but one of the animals had foreign material in the alveoli and were determined to be related to gavage dosing accidents rather than related to the test material.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
m-xylene
Effect level:
ca. 200 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: It has been reported as NOEL in the report
Key result
Dose descriptor:
NOAEL
Remarks:
m-xylene
Effect level:
ca. 100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: It was reported as NOEL in the report.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

Table 7: Summary incidence of clinical signsa

 

Males

Females

Observation

Group 1

Group 2

Group 3

Group 4

Group 1

Group 2

Group 3

Group 4

Malocclusion

2

2

2

0

1

0

0

0

Salivation

0

0

0

0

0

0

0

1

Urine stains

0

0

0

3

0

0

0

4

Alopecia

0

0

0

1

0

0

2

1

Sores

0

1

0

2

2

0

0

0

Bloody crust

1

0

0

2

1

0

0

0

Chromodacryorrhea

3

1

2

2

2

0

0

0

Small moveable tissue mass

0

0

0

1

0

0

0

0

aTotal incidence for entire study

Table 8: Overall mean body weight change (Week 0-13)

Sex

Group

Dose level (mg/kg)

Body weight

Mean

S.D.

N

Male

1

0

293.3

43.46

20

2

100

289.5

30.61

17

3

200

265.2S

30.37

15

4

800

219.5S

26.16

18

Female

1

0

115.9

18.00

20

2

100

113.2

14.06

20

3

200

107.2

14.86

16

4

800

98.1S

16.21

16

Sstatistically significant result

Table 9: Mean total food consumption

Sex

Group

Dose level (mg/kg)

 

Week

1-5

6-9

10-13

Male

1

0

Mean

860.0A

667.8

644.3

S.D.

66.03

48.31

52.29

N

10

20

9

2

100

Mean

831.4

640.6

611.6

S.D.

46.07

42.88

39.33

N

6

14

6

3

200

Mean

849.5

661.7S

614.6

S.D.

25.81

30.73

23.15

N

6

14

4

4

800

Mean

773.6S

612.0S

586.8

S.D.

59.64

55.08

28.92

N

8

14

5

Female

1

0

Mean

634.9

511.4

472.4

S.D.

56.91

46.89

28.32

N

8

13

9

2

100

Mean

650.1

505.4

478.0

S.D.

56.50

48.52

52.80

N

9

17

8

3

200

Mean

678.9

493.4

465.6

S.D.

38.24

50.78

40.62

N

5

15

6

4

800

Mean

656.8

479.7

488.8

S.D.

46.35

33.23

43.20

N

5

10

6

Adata analysed following log10transformation

SStatistically significant result

Table 10: Mean body weight changes and the percentage difference from controls.

Doses (mg/kg bw/day)

0

100

200

800

Males (g)

293.2

289.5

265.2 (s)

219.5 (s)

% difference from controls

--

1.26

9.5

25.1

Females (g)

115.9

113.2

107.2

98.1 (s)

% difference from controls

--

2.33

7.5

15.4

S = significant result

Applicant's summary and conclusion

Conclusions:
Based on the results of this study and following the assumption that the vehicle and/or test material aspiration were responsible for the deaths in the treated animals, the no-observed-adverse-effect level (NOAEL) of m-xylene was 100 mg/kg/day for males and 200 mg/kg/day for females.
Executive summary:

The sub-chronic toxicity of m-xylene was investigated in this study on male and female Sprague-Dawley rats. The rats were acclimated for approximately 2 weeks and were 46 days of age at the start of the study. The rats underwent physical and ophthalmologic examinations prior to the test and then 85 clinically acceptable rats of each sex were assigned to treatment groups, each composed of 20 rats/sex plus 5 rats/sex used in the baseline haematology group. The rats were gavaged daily at each sex were tested at each dose daily for at least 90 days.

 

Dosing solutions were prepared weekly. For each dose level, the calculated amount of test substance, m-xylene, was weighed and transferred to a volumetric flask. The vehicle, corn oil, was added to achieve the final volume and then the flask was capped and inverted by hand until thoroughly mixed. M-xylene was administered to the rats daily via oral gavage at the following dose levels; 0, 100, 200 and 800 mg/kg/day (Groups 1 -4 respectively). The control group (Group 1) received the vehicle only and the Baseline group (Group 5 0 mg/m^3) were used for pre-treatment bloodwork and later discarded without necropsy. Several observations were recorded, including clinical signs, body weights, food consumption, opthalmologic examinations, organ weights and organ-to-body weight ratios, mortality, gross pathology, clinical chemistry and histopathology was recorded.

 

Mortality was found to be significantly increased in male Group 3 and female Group 3 and 4, however based on histopathology findings, it is thought that the cause of these deaths was most likely to be due to vehicle and/or compound aspiration. In the 19 unscheduled deaths seen in males (Group 2 -4) and females (Groups 3 -4), all but one of the animals had foreign material in the alveoli and were determined to be related to gavage dosing accidents rather than being related to the test material, which may also relate to the lung lesions, mottled lungs and failure of the lungs to collapse seen in males and females of Groups 3 and 4, which were only observed in the animals found dead. Salivation was frequently observed in both sexes of Group 4 and only rarely seen in the other treatment groups. Urine stains were also observed weekly in both sexes of Group 4 and thought to be possibly-treatment related. Other commonly observed clinical signs were chromodacryorrhea and malocclusion, but were not thought to be treatment-related. Food consumption was significantly reduced for males during Weeks 1-5 for Group 4 males and during Weeks 6-9 for Group 3 and 4 males. The results remained comparable throughout the study for female rats. Only two animals showed abnormalities in the ophthalmological examination and thus it was concluded to be unrelated to the treatment. Few clinical pathology parameters were significantly changed and all were observed in Group 4, except for one seen in female Group 3. Few significant changes were seen in absolute or relative organ weights, all of which were seen in Group 4 males, where heart weight was decreased and relative brain and kidney weights were increased.

The mean body weight change was significantly lower than controls for males in Group 3 (200 mg/kg bw/day) and Group 4 (800 mg/kg bw/day) and for females in Group 4 (800 mg/kg bw/day) (Table 10). The percentage difference in mean body weight change between the control and each dose group for males and females were calculated. The percentage difference for males was 9.5% and 25.1% for Group 3 and 4 males respectively and 15.4% for Group 4 females. Reduced absolute body weight of approximately >10% is generally considered to be a sign of general toxicity, therefore the dose-related reductions in Group 3 and 4 males and Group 4 females are considered to be adverse and are therefore have been listed as NOAELs, whilst they have been reported as NOELs in the report.

Based on the results of this study and following the assumption that the vehicle and/or test material aspiration was responsible for the deaths in the treated animals in groups 3 and 4, the no-observed-effect level (NOAEL) of m-xylene was 100 mg/kg/day for males and 200 mg/kg/day for females.