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Toxicological information

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:
17 April - 23 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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Protocol required bloodwork to be taken in Week 4 but was actually performed during Week 5. We consider that 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
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Protocol required bloodwork to be taken in Week 4 but was actually performed during Week 5. We consider that 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 purpose of the study was to evaluate the subchronic toxicity of p-xylene when administered to Sprague-Dawley rats daily by oral gavage for at least 90 days.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
p-xylene
EC Number:
203-396-5
EC Name:
p-xylene
Cas Number:
106-42-3
Molecular formula:
C8H10
IUPAC Name:
1,4-xylene
Details on test material:
IUCLID4 Test substance: as prescribed by 1.1 - 1.4
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material: p-xylene
- CAS number: 106-42-3
-State: clear colorless liquid
- Source: Fluka Chemical Corporation, Hauppauge, New York (in three shipments - March 6, April 17, April 30 1986)
- Lot No.of test material: 235 855
- Expiration date of the lot/batch: not specified
- Purity: 99% (by GC-analysis)
> the test material was analysed for purity and identity prior to study initation and repeated at termination for purity only.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- During transit: 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 formulations were prepared using corn oil (vehicle) and were adjusted to 100% activity based on the purity

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
No further details specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, North Carolina
-Total number of animals: 105 males and 105 females
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 45 days
- Weight at study initiation: 199.1 - 257.8g (males) and 144.9-192.1g (females)
- Fasting period before study: not specified
- 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: approximately two weeks

DETAILS OF FOOD AND WATER QUALITY: Contaminant analysis for the rodent chow® 5002 diet and water are attached to the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 71-78
- Humidity (%): 31-69
- Temperature and relative humidity in the animal room were recorded twice daily (with few exceptions)
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: 17th April To: 21st- 23rd July 1986

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The formulated test material was administered to the animals by oral gavage daily for 13 weeks; control animals recieved the vehicle only. 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 was calculated, weighed and then 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.
- Groups and doses can be seen in Table 1 below.

Analytical verification of doses or concentrations:
yes
Remarks:
The formulated test material was analysed for purity, dose concentration and stability
Details on analytical verification of doses or concentrations:
Stability results revealed 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 (at least 90 days)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (group 1) and Baseline (Group 5)
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/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment : Sprague-Dawley rats were used since they have historically been employed in safety evaluation studies and were requested by the US EPA.
- Animal selection:Following physical and opthalmologic examinations, 85 clinically acceptable rats of each sex were assigned to treatment groups using a computer weight randomisation programme, 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.
- The baseline groups were used for pretreatment bloodwork and later discarded without necropsy.

Positive control:
No positive control was employed.

Examinations

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

CAGE SIDE OBSERVATIONS: Yes
- Looking for clear signs of toxicity
- Time schedule: Daily starting one hour after completion of dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Complete physical examiniations
- Time schedule: at initiation and weekly thereafter

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

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

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined Yes
- Time schedule: weekly

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Performed by a board-certified veterinary opthalmologist. 1% Atropine Sulphate was used to dilate pupils of both eyes prior to examination.
- Time schedule for examinations: Prior to treatment and on the last 10 survivors per sex per group during Week 13
- Dose groups that were examined: all groups

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
- Samples were collected via the orbital sinus while under carbon dioxide anaesthesia
- Time schedule for collection of blood: prior to initiation and during weeks 5 and 13. (Protocol required bloodwork to be taken in Week 4 but was actually performed during Week 5).
- Animals fasted: yes overnight
- How many animals: Five animals per sex (baseline group) and the first 10 survivors per sex per group during week 5 and 13
- Parameters tested: please see Table 3 below

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals that died during the course of the study and all survivors were sacrificed after 13 weeks of treatment and were given a complete gross necropsy. Please see Table 4 below.
- Euthanasia was exsanguination while 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 leesions, lungs, liver and kidneys from all remaining animals.
Other examinations:
ORGAN WEIGHTS
-The following organs were weighed for all terminally killed animalls, 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):
The most commonly observed weekly clinical signs were malocclusion, chromodacryorrhea, sores and salivation.
Only salivation showed a compound-related effect and was seen in both sexes in Group 4. Observed every week except the first week, generally just prior to dosing and then the animals returned to normal by the one-hour post-dosing observations.
Sores were mostly seen on the ears, most likely due to the ear tags (method of identification)
Daily observations included sensitivity to touch and salivation. The former had such a low incidence (observed twice during the study) that it was not considered important. Salivation, however, was observed frequently during every study week except the first, and it occurred only in Group 4 (both sexes). Generally, it was observed just prior to dosing and the animals were back to normal by the one-hour post-dosing observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males: a significant trend for increased mortality and in Group 4 there was a significantly higher mortality
Females: no significant differences in mortality and no significant trend

Intra-alveolar foreign material was found in the lungs of nearly all of the unscheduled deaths in males and females, and were determined to be associated with congestion and appeared secondary to vehicle and/or test material aspiration rather than being related to the test material (please refer to the histopathological findings section).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of both sexes in Group 4 were lower than the other groups.
There was no significant difference in total body weight gain between the control and treated groups of either sex or a significant difference in mean body weights at Week 13.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was generally highest in Group 4 of both sexes. Total food consumption was significantly increased in the Group 4 females during Weeks 10-13.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were seen in any of the animals at the terminal examination
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant differences observed in any haematology parameters in the treated groups of either sex at Week 5.
The only significant difference at Week 13 was in segmented neutrophils, which were decreased in the Group 3 males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Few significant differences were observed in any clinical chemistry parameters at week 5: all the Group 4 females showed increased levels of phosphorus, cholesterol and alanine aminotransferase.
At week 13, there were no significant changes seen in either sex.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were observed between the control and treated groups of either sex in any of the absolute or relative organ weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were few gross lesions observed at necropsy, which were infrequent and concluded to show no dose relationship.
In unscheduled deaths, mottled lungs and failure of lungs to collapse were seen more frequently than other types of lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- In both treated and control animals, lesions were found in the kidneys and liver and included:
(1) kidneys - e.g. tubular regeneration, focal mononuclear cell infiltrates, interstitial fibrosis
(2) liver - e.g. mononuclear cell infiltrates, subacute inflammation, fatty change
-1 male in Group 3 and 3 males in Group 4 (all unscheduled deaths) - cytoplasmic hyaline inclusions were observed.
- The nature of these inclusions was unclear and they were not considered to be biologically significant, since they were not present in terminally sacrificed rats, they were observed with a low overall incidence and only seen in male rats.
- In nearly all of the unscheduled deaths, intra-alveolar foreign material was present in the lungs that was often accompanied by congestion and thus thought to be secondary to vehicle and/or test aspiration.
- Various incidental lesions were observed in all groups, which were not treatment-related.
- No specific compound-related histopathologic alterations were present in the tissues examined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
Although the report lists 200 mg/kg bw/day as a NOEL, the LOA XWG considers 200 mg/kg bw/day as a NOAEL based on significantly reduced M and F body weight at 800 m/kg bw/day (Table 1).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Please see the 'Attached background material' section for all tables

Table 1: Mean body weight changes and the percentage difference from controls.

Doses (mg/kg bw/day)

0

100

200

800

Males (g)

305.9

320.7

327.8

272.2

% difference from controls

--

-4.8

-7.16

11.02 (s)

Females (g)

135.1

134.9

148.2

120

% difference from controls

--

0.15

-9.7

11.2 (s)

S = significant result

Applicant's summary and conclusion

Conclusions:
Based on the results of this study and the conclusion that vehicle and/or test material aspiration was the cause of all unscheduled deaths in the treated animals, the no-observed--adverse-effect-level (NOAEL) of p-xylene was determined to be 200 mg/kg/day.
Executive summary:

This 90-day study was undertaken in order to evaluate the sub-chronic toxicity of the test substance, p-xylene (99% purity) in rats. Four doses were tested in this study: 0, 100, 200 and 800 mg/kg/day (Groups 1 -4 respectively) and Group 5 (0 mg/kg/day) was a baseline group that was used solely for pre-treatment bloodwork (animals assigned to this group were later discarded without necropsy). Physical and opthalmologic examinations were performed on male and female Sprague-Dawley rats used in this study. Twenty animals/sex were assigned to each dose group and were dosed daily for at least 90 days.

Dosing solutions were prepared weekly. For each dose level, the calculated amount of test substance, p-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. . 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.

Results show that mortality was present in all treatment groups, but significantly increased with a significant trend in Group 4 males, whilst there were significant differences seen in the female groups. 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 and are not treatment-related, since intra-alveolar foreign material was found in all of the unscheduled deaths in males and females. The most commonly observed clinical signs were malocclusion, chromodacryorrhea, sores and salivation. Salivation was increased in both sexes of Group 4. It was a sign observed weekly, generally just prior to dosing and then the animals returned to normal one-hour post-dosing. Sores were seen mostly on the ears and were most likely due to the ear tags (method of identification). There were no ocular abnormalities or significant differences seen in the haematology parameters, but there were a few significant differences identified in the clinical chemistry parameters in Group 4 females at Week 5, however these returned to normal levels at Week 13. Overall mean body weight across all treatment groups were slightly decreased, whilst food consumption was slightly increased in Group 4 males and females. In terms of histopathology, lesions were discovered in the liver and kidneys of control and treated animals and foreign material was found in the lungs of nearly all unscheduled deaths, often accompanied with congestion thus indicating it was secondary to vehicle and/or test material aspiration. Among the animals that died on test, mottled lungs and failure of the lungs to collapse were detected in males and females from groups 2 -4, however these lesions were considered to be unrelated to p-xylene exposure. No compound -related histopathologic alterations in the tissues examined.

The mean body weight change was significantly lower than controls for males and females in Group 4 (800 mg/kg bw/day) (Table 1). The percentage difference in mean body weight change between the control and each dose group for males and females were calculated. The percentage differences for males was 11.02% and 11.2% for females in Group 4. Reduced absolute body weight of approximately >10% is generally considered to be a sign of general toxicity. Although the report lists 200 mg/kg bw/day as a NOEL, LOA XMG considers the 200 mg/kg bw/day as a NOAEL based on significantly reduced male and female body weight at 800 mg/kg/day.

Based on the results of this study and the conclusion that vehicle and/or test substance aspiration was the cause of all unscheduled deaths in treated animals. The NOAEL of p-xylene was determined to be 200 mg/kg/day.