4-chloro-o-xylene

Endpoint summary

Description of key information

ORAL

NOAEL <50 mg/kg/day, Sprague-Dawley rat, OECD 407 (Marr, 2005)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 407 guideline; under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

See overall remarks field below

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD[SD] IGS BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: 7 weeks at start of treatment
- Weight at study initiation: males 198.6-201.4 g; females 177.6-181.4 g
- Housing: Housed individually in solid-bottomed polycarbonate cages with stainless-steel wire lids.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 4 degrees C
- Humidity (%): 49 - 62
- Photoperiod: 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was chosed as a suitable vehicle based on previous studies with an analog to this compound.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the concentration of the test substances in the dosing suspensions were performed on all doses prior to dosing using GC. The following conditions were used:
- Instrument: HP Model 5890
- Injector: Autosampler HP Model #7673
- Detector: FID
- Column: Capillary DB-5MS (30m x 0.32 mm ID, 0.5 um film)
- Data system: Atlas 2000 Version R2 (Lab systems)
- Mode: Thermal gradient
- Carrier gas: Helium @ 2.36 ml/min (measured at 80 degrees C)
- Temperature: Detector: 270 degrees C; Injector: 250 degrees C
- Column program: 80 degrees C to 250 degrees C at 15 degrees C/min, hold at 250 degrees C (4 min) [total time = ~ 15 min)
- Injection: Split, with split ratio at 50:1
- Injection volume: 1.0 ul
- Retention time: Analyte: ~4.1 min; internal standard: ~7.7 min

Duration of treatment / exposure:

Animals were exposed to the test material daily for 28 days. Recovery animals were held for an additional 2 weeks without dosing.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 50, 250 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

8 animals of each sex per treatment group. An additional 5 animals of each sex were used in the 0 mg/kg/day and 1000 mg/kg/day treatment groups as recovery animals.

Control animals:

yes

Details on study design:

- Dose selection rationale: Doses were selected based on a 10d range-finding study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observation for mortality were made twice daily and general condition of all animals checked daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes (except for recovery animals)
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Parameters checked: red blood cell (RBC, nucleated RBC), white blood cell (WBC; total/differential and corrected), platelet (PLT), haemoglobin concentration (HGB), haemocrit (HCT), red cell distribution width (RDW), mean platelet volume (MPV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular concentration (MCHC) and prothrombin time.

CLINICAL CHEMISTRY: Yes (except for recovery animals)
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- Parameters checked: albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), total cholestrol, creatinine, glucose, total protein, sodium, potassium and chloride.

URINALYSIS: Yes (excluding recovery animals)
- Time schedule for collection of urine: Prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocytes.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all (including recovery animals)
- Battery of functions tested: home-cage observations, handling observations, open-field observations, physiological observations, sensory and neuromuscular observations (including grip strength). In the 4th week of exposure, all animals were assessed for auditory function and motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

- For body weights, organ weights, feed consumption, grip strength, auditory startle response and motor activity, treatment groups were compared to the concurrent control group using either parametric ANOVA or robust linear regression methods.
- Body weights and organ weights from the recovery animals were analysed using the Student's t-test.
- Binary outcomes (FOB) were analysed using Fisher's Exact Test for overall heterogeneity among dose groups followed by pairwise comparisons of exposed groups to the control if the overall test was statistically significant.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment related mortalities were observed in all male and female dose groups (including recovery animals).
- Males: clinical observations included rooting postdosing (at 250 and 1000 mg/kg/day groups) and salivation prior to dosing (in 1000 mg/kg/day group). Although these are considered to be behavioural responses and not a toxic sign.
- Recovery males: no significant findings.
- Females (including recovery females): Rootingpost dosing (at 250 and 1000 mg/kg/day groups) and salivation prior to dosing (in 1000 mg/kg/day group). These were considered to be behavioural responses rather than a toxic sign. In the 1000 mg/kg/day group, 4/13 females displayed dorsal recumbancy postdosing only on day 3 - this is an equivocal finding due to occurrence on a single day of the study.
- Recovery females: no significant findings.

BODY WEIGHT AND WEIGHT GAIN
- Males: A significant reduction in male body weight in the 1000 mg/kg/day group was observed on days 7 and 21 (6-7% decrease), but the weight difference on day 14 and 27 were not statistically significant. A significant reduction in body weight gain was observed in the 1000 mg/kg/day group when compared to the control group from day 0 to 7 (24% reduction) and day 0 to 27 (13% reduction). No significant reductions in body weight or body weight gain were observed in the 50 and 250 mg/kg/day treatment groups.
- Recovery males: Body weight change was significantly increased in the 1000 mg/kg/day group when compared to the control group between days 35 to 42.
- Females: A significant reduction in female body weight in the 1000 mg/kg/day group was observed on days 7, 14 and 21. For body weight change, a significant decrease from day 0 to 7 was observed in the 1000 mg/kg/day group (46% decrease) and in the 250 mg/kg/day group (34% decrease); a significant decrease was also observed in the 1000 mg/kg/day group from day 0 to 27.
- Recovery females: A significant reduction in body weight was observed on day 42 in the 1000 mg/kg/day group. A statistically significant difference in terminal body weight between the control and 1000 mg/kg/day group was observed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Males: A significant treatment-related reduction on food consumption was observed from day 0 to 7. A significant increase was also observed in the 250 and 1000 mg/kg/day groups during days 21-27.
- Recovery males: no significant findings.
- Females: A significant reduction in food consumption was observed in the 250 and 1000 mg/kg/da from days 0 to 7 and days 14-21. A significant reduction in food consumption was also observed in the 50 mg/kg/day group but only for days 14-21.
- Recovery females: no significant differences.

HAEMATOLOGY
- No significant findings in both males and females.

CLINICAL CHEMISTRY
- Males: A significant increase in BUN and albumin was observed in the 1000 mg/kg/day group.
- Females: Significant increase in total protein, albumin, total cholestrol and alanine aminotransferase was observed in the 1000 mg/kg/day group.

URINALYSIS
- Males: Urine pH was significantly reduced in the 1000 mg/kg/day group.
- Females: No significant findings.

NEUROBEHAVIOUR
- Males (including recovery males): Functional observation battery data for all males were not significantly different during treatment, although a decrease in average pupil size was observed in the 250 mg/kg/day group during week 2. Percent abnormal arousal was significantly increased at 250 and 1000 mg/kg/day groups during week 3. Abnormal gait was observed at 50 and 250 mg/kg/day groups during week 3. Motor activity was significantly reduced in the 1000mg/kg/day group on day 22. These observations were considered not to be treatment-related due to the lack of dose response. No treatment-related effects were observed on auditory startle.
- Recovery males: no significant findings.
- Females (including recovery females): In week 3 abnormal arousal was significantly increased across all dose groups with 1,4,4 and 7 displaying depressed arousal at 0, 50, 250 and 1000 mg/kg/day groups respectively. In the 250 mg/kg/day dose group an increase in the average number of defecations was observed during weeks 2 and 3. No significant treatment-related effects were observed on auditory startle or motor activity.
- Recovery females: no significant findings.

ORGAN WEIGHTS
- Males: Liver weight was significantly increased at 1000 mg/kg/day. Paired kidney weights, relative to body weight was significantly increased in the 1000 mg/kg/day group due to the slightly reduced body weight. No significant changes were observed in the absolute and relative organ weights of the brain, thymus, heart, spleen, paired adrenal glands, paired testes and paired epididymides.
- Recovery males: no significant findings.
- Females: Significant decreases in brain and heart weights were observed in the 1000 mg/kg/day group and a significant decrease in spleen weight was observed in the 250 mg/kg/day group. (these changes were deemed equivocal due to the magnitude of the change and lack of consistency with males).
- Recovery females: Liver and paired kidney weights were significantly increased in the 1000 mg/kg/day group relative to terminal body weight.

GROSS PATHOLOGY
- No significant findings in both males and females (including recovery animals).

HISTOPATHOLOGY: NON-NEOPLASTIC
- Males: In the 1000 mg/kg/day group, minimal (5/8 males) and mild (1/8 males) showed centrilobular hepatocyte hypertrophy. In the 250 mg/kg/day group, minimal hypertrophy was seen in 4/8. No males of the 50 mg/kg/day group displayed signs of centrilobular hepatocyte hypertrophy. In the 1000 mg/kg/day group, minimal (3/8 males) and mild (5/8 males) hyperplasia of the epithelium in the urinary bladder was observed; hyperplasia of the epithelium of the renal pelvis (1/8 males) was also observed in this dose group. In the 50 and 250 mg/kg/day dose groups, minimal hyperplasia of the epithelium of the urinary bladder was observed in 2/8 and 3/8 males respectively.
- Recovery males: Hypertrophy of the liver was absent, but hyperplasia of the urinary bladder epithelium was observed in 5/5 males.
- Females: In the 1000 mg/kg/day group, minimal (4/8 females) and mild (4/8 females) showed centrilobular hepatocyte hypertrophy; no lesions were observed in the 250 mg/kg/day group.
In the 1000 mg/kg/day group, minimal (2/8 females) and mild (6/8 females) hyperplasia of the epithelium in the urinary bladder was observed; hyperplasia of the epithelium of the renal pelvis (2/8 females) was also observed in this dose group. In the 50 and 250 mg/kg/day dose groups, minimal hyperplasia of the epithelium of the urinary bladder was observed in 3/8 and 7/8 females respectively. No hyperplasia of the epithelium of the renal pelvis was observed.
- Recovery females: Hypertrophy of the liver was observed in 3/5 females in the 1000 mg/kg/day group. Minimal hyperplasia of the transitional epithelium of the urinary bladder was observed in 4/5 females of the 1000 mg/kg/day group. Hyperplasia of the epithelium of the renal pelvis was absent.

Dose descriptor:

NOAEL

Effect level:

< 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No NOAEL could be determined based on the findings of hyperplasia in the urinary bladder at 50, 250 and 1000 mg/kg treatment groups.

Critical effects observed:

not specified

The results of the homogeneity study indicates that the procedure for mixing the test material with vehicle (corn oil) produced uniform formulations suitable for use in this toxicity study.

The results of the stability study indicated that dose formulations of the test material in vehicle at 2 and 200 mg/l were stable for at least 35 days when stored in a refridgerator and were stable for at least 4 hours at conditions that simulate dosing. Analyses of the dose formulations also showed that they were 95.1% to 101% of the nominal concentrations.

Conclusions:

Urinary bladder lesions were observed in all treatment groups and complete recovery of these lesions did not occur in the high-dose recovery animals. Therefore under the conditions of this study, the author could not determine a NOAEL based on the dose-related findings of hyperplasia in the urinary bladder at 50, 250 and 1000 mg/kg/day groups. A NOAEL of <50 mg/kg/day seems appropriate.

Executive summary:

The study was performed to evaluate the potential toxicity of the test material to male and female Crl:CD[SD] IGS BR) rats when administered orally by gavage for 28 consecutive days. The study was performed according to GLP and was designed to meet the requirements of OECD Guidlelines for Testing of Chemicals No. 407. Four groups of 8 males and 8 female rats were doses with the test material in corn oil at 0, 50, 250 and 1000 mg/kg bw/day, once daily for 28 days. An additional 5 female and 5 male rats were used in the 0 and 1000 mg/kg bw/day and these acted as the recovery groups (a 2-week recovery postdosing period without administration of test material to assess if recovery occurred from any possible treatment-related effects identified in the high-dose group).Clinical observations were recorded daily. Body weight and food consumption were recorded weekly. In addition, a functional observational battery was conducted weekly which included home-cage observations, handling observations, open-field observations, sensory and neuromuscular observations (including grip strength) and physiological observations. In the fourth week of exposure all animals were assessed for auditory function and motor activity. Animals were killed on day 28 (recovery animals were killed 2 weeks later) and examined post mortem. At termination, cardiac blood samples were taken for haematology and blood chemistry investigations. Selected organs were weighed and specified tissue/organs were processed for histopathological examination. Tissue/organs from control and high dose animals were examined together with all macroscopic abnormalities in any group.

Analytical data confirmed the suitability of the dosing preparation used in the study. There were no adverse treatment-related effects on clinical observations, functional observational battery, or motor activity. There were no toxicologically significant effects on haematology. Body weight was reduced in males and females at 1000 mg/kg bw/day. Effects of treatment at 250 mg/kg bw/day were seen in females for body weight change early in the dosing period and for feed consumption (g/day). No treatment-related effects during the in-life portion of the study were observed in males or females at 50 mg/kg bw/day. After the recovery period, body weights of males were equivalent in the control and high-dose group, whilst female high-dose recovery group was lower than control. Absolute and relative liver weights were affected in males and females dosed at 1000 mg/kg bw/day which did not completely recover during the 2-week period. Microscopic lesions (centrilobular hepatocellular hypertrophy) of the liver were seen in males at 250 and 1000 mg/kg bw/day and only at 1000 mg/kg bw/day in females. Urinary bladder lesions (epithelial hyperplasia) were seen in all doses in both males and females. Complete recovery of the urinary bladder lesions did not occur in the high-recovery animals. Under the conditions of this study, a NOAEL could not be determined based on the dose-related findings of hyperplasia in the urinary bladder at 50, 250 and 1000 mg/kg/day groups, but a NOAEL of <50 mg/kg/day seems appropriate.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be high.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the key study, the repeated dose oral toxicity of the test material was evaluated in male and female Crl:CD[SD] (IGS BR) rats in study conducted in accordance with OECD 407 under GLP conditions.

The test material was administered in corn oil by gavage to four groups each consisting of 8 male and 8 female rats at doses of 0, 50, 250 or 1000 mg/kg bw/day, once daily for 28 days. An additional 5 female and 5 male rats were used in the 0 and 1000 mg/kg bw/day and these acted as the recovery groups (a 2-week recovery post-dosing period without administration of test material to assess if recovery occurred from any possible treatment-related effects identified in the high-dose group).

There were no adverse treatment-related effects on clinical observations, functional observational battery, or motor activity. There were no toxicologically significant effects on haematology. Body weight was reduced in males and females at 1000 mg/kg bw/day. Effects of treatment at 250 mg/kg bw/day were seen in females for body weight change early in the dosing period and for feed consumption (g/day). No treatment-related effects during the in-life portion of the study were observed in males or females at 50 mg/kg bw/day.

After the recovery period, body weights of males were equivalent in the control and high-dose group, whilst female high-dose recovery group was lower than control. Absolute and relative liver weights were affected in males and females dosed at 1000 mg/kg bw/day which did not completely recover during the 2-week period. Microscopic lesions (centrilobular hepatocellular hypertrophy) of the liver were seen in males at 250 and 1000 mg/kg bw/day and only at 1000 mg/kg bw/day in females. Urinary bladder lesions (epithelial hyperplasia) were seen in all doses in both males and females. Complete recovery of the urinary bladder lesions did not occur in the high-recovery animals. Under the conditions of this study, a NOAEL could not be determined based on the dose-related findings of hyperplasia in the urinary bladder at 50, 250 and 1000 mg/kg/day groups, but a NOAEL of <50 mg/kg/day seems appropriate.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Only one study available.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: urinary bladder

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to repeated dose toxicity as STOT RE Category 2 (H373: May cause damage to organs).

 

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance requires classification with respect to repeated dose toxicity as Xn; R48/22 Harmful: danger of serious damage to health by prolonged exposure if swallowed.