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Administrative data

short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-05-23 to 1990-04-27
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP study according to OECD technical guideline.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Presentation: Clear, pale yellow liquid
- Receipt date: 30 March 1988, Preliminary study; 23 May 1988, Main study
- Batch-no: Probe no 633274, Prod. no 048828-04
- Purity: 98.2%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Ltd., Manston Road, Margate, Kent, England
- Age at study initiation: 6 weeks old on delivery
- Weight at study initiation: male: 159-160g female: 145-146g
- Housing: 5 of the same sex in suspended polypropylene cages fitted with stainless steel mesh tops and floors; each group was kept in a in seperate ventilated cabinet to prevent cross-contamination; dimensions (56cm x 38cm x 18cm)
- Diet: access to a weighed quantity of standard quality controlled laboratory rat food (LAD1)
- Water: ad libitum

- Temperature (°C): 16.5-21.5°C
- Humidity (%): 50-76%

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
clean air
Details on inhalation exposure:
- The test substance atmosphere was produced by metering 2,4-XYLIDINE to stainless steel concentric jet atomisers.
- These were mounted in the base of glass dispersion columns into which diluent air was fed.
- Each column was connected to the inlet duct of an exposure chamber.
- In order to facilitate the production of a vapour atmosphere and to offset the cooling effect of the atomiser tip, air fed to the atomisers was passed through a copper coil immersed in a water bath maintained at 80°C.
- Different exposure concentrations were achieved by metering the test substance to the atomisers at different rates, appropriate to achieve the desired concentrations.
- Due to slight variation between atomisers and elutriation characteristics of the exposure chamber systems, adjustments were made to individual feed rates in order to achieve and maintain the desired target concentrations.
- The necessary adjustments were made initially during preliminary work, but also as the study progressed.
- Using this system, a proportion of the test substance would be expected to vaporise, therefore the test atmosphere, particularly at the high dose level, would be expected to contain a mixture of droplets and vapour of the test substance.
- The exposure chambers were constructed of stainless steel and glass.
- Each of the 4 chambers used was approximately 0.5 m^3 in volume.
- The chambers were of square plan section fitted with a pyramidal base and top.
- A square-shaped 3-inch tubular perforated exhaust plenum was fitted in the base of each chamber, below exposure cage level.
- A perforated dispersion plate was fitted at the point of air/aerosol entry. A 1.5-inch drainage duct fitted with a ball valve was present in the centre base of the chamber and connected with a drainage system.
- Exposure cages, constructed of stainless steel mesh were supported on a frame work, mid-way inside the chamber.
- Each cage held 5 rats of the same sex and 2 such cages were used per group.
- The chambers were each fitted with six ports for withdrawal of chamber air samples for analysis.
- Routinely a mid-centre port at the same level as the rats' breathing zone was used for sample collection.
- Diluent inlet air (oil-free dried compressed air) at a rate sufficient, together with the atomiser air flow, to result in a total chamber air flow of 100 litres per minute, entered at the base of the glass dispersion column, mixed with the atomised solution of the test substance and entered the chamber via the inlet duct and chamber dispersion plate.
- Air flows were monitored by tapered tube flow-meters, mounted at the front of a purpose-built stainless steel trolley. The generation apparatus was also mounted on the trolley.
- A magnehelic pressure gauge (0 - 100 mm water gauge) was connected with each chamber by a nylon tube. This was used to monitor chamber internal pressure.
- Extraction of the chamber was accomplished by means of individual air handling units containing coarse and fine filtration media, together with activated charcoal.
- Extract flow was adjusted using gate valves, mounted in the extract ducting between the chamber and filters. The internal pressure within each chamber was set to 10 mm water gauge below ambient when operational.
- A separate exposure chamber was used for each group of rats. The air control animals were exposed using a similar system to that used for the test groups receiving the test substance.
- The animals were removed from their holding cages and placed within the wire mesh exposure cages in the chamber appropriate to each group.
- The chamber doors were sealed after a wet and dry bulb thermohygrometer was placed within the chamber and diluent air turned on.
- Syringes were filled with the test substance and mounted on Precidor infusion pumps.
- The initial volume of each syringe was recorded.
- The infusion pump mechanism was advanced until a drop of test solution was seen at the tip of the atomiser.
- The air flow to the atomisers was turned on and the internal pressure of the chambers adjusted to 10 mm water gauge using the gate valves.
- Exposure commenced when the infusion pumps were switched on and operating at the required rate.
- After six hours exposure the infusion pumps were switched off and the volume of test substance remaining in the syringes recorded.
- The diluent and atomiser air supply were each turned off, a bung removed from a sample port and the gate valves opened to facilitate rapid clearance from the chamber.
- The rats were unloaded from the chamber into their respective holding cages following a period of chamber clearance, generally 20 - 30 minutes for test group animals.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
- The concentrations of 2,4-XYLIDINE present in the exposure chambers used were determined on 3 occasions during each exposure.
- Samples of test atmosphere were withdrawn at 2 litres per minute through fritted bubblers containing acetone as the trapping medium.
- The air volume was recorded using an in-line wet-type gas meter.
- The samples were made up to a standard volume of 25 ml.
- The samples were analysed by flame ionisation chromatography using external standards.
- Samples of chamber atmosphere were removed once during each exposure.
- Chamber air was withdrawn through a May Multistage Liquid Impinger* at a rate of 10 litres per minute
- Diluent air flow was monitored continuously using tapered tube rotometers and recorded at 30-minute intervals throughout each exposure.
- The chamber internal pressure relative to ambient was monitored continously by magnetic pressure gauges and recorded at 30-minute intervals throughout exposure.
- The wet and dry bulb temperatures of the thermohygrometer in each chamber were recorded at hourly intervals throughout each exposure.
- The chamber relative humidity was calculated from these data.

Duration of treatment / exposure:
6hrs per day, 5 days per week, 4 weeks in total
Frequency of treatment:
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.033 mg/L
analytical conc.
Doses / Concentrations:
analytical conc.
Doses / Concentrations:
analytical conc.
No. of animals per sex per dose:
5 per sex and dose
Control animals:
Positive control:


Observations and examinations performed and frequency:
- Clinical signs during exposure were recorded as a group response where all visible animals appeared to be responding similarly or a proportion were affected.
- Group responses seen during exposure were not transferred to the individual clinical signs sheets, and are reported in the text of this report.
- At other times animals were examined twice each day, usually prior to loading and immediately following unloading from the chambers on exposure days, and in the morning and afternoon on non-exposure days.
- An entry was made in the individual clinical signs sheets twice daily, even if abnormalities were not seen.

- Each rat was weighed for allocation to groups (all rats), prior to the start of exposure, weekly during the study and at autopsy.

- The quantity of food consumed by each cage of rats was recorded weekly, commencing one week prior to the start of exposures, until the end of the study.

- The amount of water consumed by each cage of rats was recorded daily commencing on the day of the first exposure until the end of the study.

Sacrifice and pathology:
After 20 exposures all rats were sacrificed. The rats were anaesthetised by intraperitoneal injection of Pentobarbitone sodium and killed by exsanguination.

Macroscopic pathology and organ weight analysis:
At autopsy a detailed macroscopic examination of all rats was performed. The following organs were dissected from each rat and weighed:
Adrenals, brain, heart, kidneys. liver, lungs, ovaries, pituitary, prostate, testes (with epididymides) and uterus

Samples or the whole of various tissues/organs, together with any macroscopically abnormal entities were preserved in buffered 10% Formalin. Microscopic examination of the liver, spleen, lungs, trachea, heart, adrenals. kidneys, stomach and any gross abnormalities was performed on all rats in group 1 (air control) and 4 (high dose).
In addition the liver was examined on all rats in groups 2 and 3 and the spleen was examined on all male rats in groups 2 and 3.
Other examinations:
Haematology/Clinical chemistry:
- Samples of blood were withdrawn from the orbital sinus during Week 4 of the study.
- The samples were removed while the rats were lightly anaesthetised with ether.
- All rats were deprived of food overnight prior to removal of blood. Water was available during this overnight period.

All statistical analyses were carried out separately for males and females.
Analyses were carried out using the individual animal as the basic experimental unit. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, 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 animals with 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) Except for pre-exposure data, analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only Williams' test was reported. The Kruskal-Wallis analyses were followed by Shirley's test, the non-parametric equivalent of the 't' and Williams' tests.
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might affect the organ weights.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
eye irritation
mortality observed, treatment-related
Description (incidence):
eye irritation
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
slightly reduced
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased liver and kidney weight
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
- No unscheduled deaths.

- Signs indicative of an irritant effect of the test material were seen during exposure.
- At the higher dose levels (0.1 and 0.3 mg/L) the signs included partial or total closure of the eyes; rubbing of the chins on the mesh floor of the exposure cage; reduced response to a knock on the chamber wall; and slow breathing with head movements.
- At the low dose level partial closing of the eyes and a reduced responsiveness were seen.
- Lethargy noted in high dose rats following each exposure.
- Complete recovery from this effect overnight between exposures.

- Dose-related reduction in bodyweight gain by all male 2,4-XYLIDINE groups compared with control group.
- Minimal effect on low dose rats.
- There was also a reduction in rate of bodyweight by female rats exposed to 2,4-XYLIDINE but the effect was not strictly dose-related.

- Slightly reduced food consumption for all groups exposed to 2,4-XYLIDINE.
- The reduction was greatest in male rats in Group 4 (High dose) and female rats in Group 3 (Intermediate dose).
- The reduction in Group 2 (Low dose) was minimal.

- Increased water consumption in groups exposed to 2,4-XYLIDINE.
- The effect was most marked in Group 4 (High dose) in which male and female rats drank 80% or 40% respectively more than control rats.
- In Groups 2 (Low dose) and 3 (Intermediate dose) the effect was very small and not consistently dose-related.

- Increase in platelet count for 2,4-XYLIDINE exposed groups. This was dose-related for male rats.
- Myeloid/erythroid ratios, calculated from terminal marrow smear, indicated no effect on bone marrow.

- Increased GPT, /GT and bilirubin levels in high dose rats. Increased calcium and cholesterol in all groups exposed to 2,4-XYLIDINE.

- Liver enlargement noted in intermediate and high dose rats. Crater-like depressions in the forestomach of high dose rats.

- Dose-related increase in liver weight, especially when values adjusted using bodyweight as covariate.
- Slight increase in kidney weight suspected.

- Minimal diffuse hepatocyte enlargement in 3/5 males and 3/5 females from the high dose (0.3 mg/L) group and minimal centrilobular hepatocyte enlargement in 2/5 males and 2/5 females in the intermediate dose (0.1 mg/L) group.
- The hepatocyte enlargement in these animals was considered to be related to the increased liver weights recorded.
- Additional liver changes observed in the high dose (0.3 mg/L) rats only were:
- foci of single cell necrosis in the majority of both males and females;
- foci of extramedullary haemopoiesis in 2/5 males; minimal bile duct hyperplasia in 1/5 males.

- A minimal increase in extramedullary haemopoiesis was present in 4/5 high dose (0.3 mg/L) male rats compared to control male rats.

- Minimal foci of acanthosis in the non-glandular stomachs of 2/5 males and 1/5 females from the high dose (0.3 mg/L) group were considered to represent the crater-like depressions reported.
- Since focal acanthosis was not detected in the stomachs of any control rats the possibility that this was a treatment-related change cannot be excluded.
- There were no treatment-related changes detected in the low dose (0.03 mg/L) rats.

Effect levels

Dose descriptor:
Effect level:
0.033 mg/L air (analytical)
Basis for effect level:
other: based on effects on body weight gain and changes in plasma GPT

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

From the results presented in this report a NOAEC for 2,4-Xylidine of 0.03 mg/L is established.
Executive summary:

The present study indicates that inhalation of 2,4-Xylidine by rats at concentrations of 0.1 or 0.3 mg/L over a 4-week period causes changes indicative predominantly of effects on the liver. At the higher dose level there were also changes in the stomach and an elevation of water consumption. At the lowest dose level used in this study (0.03 mg/L) there were no microscopic pathological changes. However there were slight effects on weight gain and food consumption, signs of eye irritation and statistically significant changes in plasma GPT and calcium. It is concluded that the NOEL for inhalation of 2,4-Xylidine by rats over a 4-week period is 0.03 mg/L.