Benzene, 1-chloro-2,3-dimethyl-

Endpoint summary

Description of key information

At a dose of 50 mg/kg bw/day (lowest dose tested) no effects were observed in a reliable study performed according to OECD 407. Little effects were also observed in the high dose group (1000 mg/kg bw/d, males: dose-related increase in incidence of minimal (5 of 8 animals) or mild (1 of 8 animals) eosinophilic droplets in the renal cortex) and the mid dose group (250 mg/kg bw/d, males: minimal eosinophilic droplets were observed in 5 of 8 animals.). However incidences were high, the severity of the effect is low. Therefore a factor of 3 was selected to extrapolate from the LOAEL of 250 mg/kg bw/d to a NOAEL of 83.3 mg/kg bw/d (250 mg/kg bw/d /3 = 83.3 mg/kg bw/d).

A sub-chronic toxicity study (90 days) does not need to be conducted because a reliable short-term toxicity study (28 days) is available showing toxicity effects according to the relevant criteria for

classifying the substance, for which the observed NOAEL-28 days, with the application of an appropriate uncertainty factor, allows the extrapolation towards the NOAEL-90 days for the same route of

exposure

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:

From 21 Aug 2003 to 14 Apr 2004

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)

Version / remarks:

27 July 1995

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 42 days
- Weight at study initiation: 207.3-211.7 (males), 176.5-184.1 (females)
- Fasting period before study: no
- Housing: individually
- Diet: Purina Certified Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Prepared prior to start of dosing (storage stability study indicated that formulations of 2 and 200 mg/ml were stable for 35 days of storage in amber bottles in the refrigerator or ambient temperature)
Dosing volume: 5 ml/kg bw

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days, recovery groups (control, high dose) 14 days post exposure without dosing

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

8, in recovery groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 10 days range finding study with slightly reduced body weight gain at the highest dose of 1000 mg/kg bw/day
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behavior pattem

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during quarantine and weekly thereafter until necropsy, including once during the recovery period
Parameters: FOB, including home-cage observations, handling observations, open-field observations, sensory and neuromuscular observations (including grip strength), and physiological observations

BODY WEIGHT: Yes
- Time schedule for examinations: recorded initially and then weekly throughout the study and recovery period

FOOD CONSUMPTION:
- Time schedule for examinations: weekly throughout the study and during the recovery period

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 (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 5/8 animals from all groups
- Parameters checked : red blood cell (RBC, nucleated RBC), white blood cell (WBC; total/differential and corrected), and platelet (PLT) counts; hemoglobin concentration (HGB); hematocrit (HCT); red cell distribution width (RDW); mean platelet volume (MPV); the RBC indices including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC); and prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: 5/8 animals from all groups
- Parameters checked : albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), total cholesterol, creatinine, glucose, total protein and sodium, potassium, and chloride

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: not specified
- Parameters checked: glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at day 20 of study
- Dose groups that were examined: all groups
- Battery of functions tested: auditory function, motor activity, and grip strength

IMMUNOLOGY: No

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (all animals from control and high dose groups, and other groups, if effects were evident)

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. The homogeneity of variance assumption was examined via Levene's Test. If Levene's Test indicated lack of homogeneity of variance (p<0.05), robust regression methods (available in the REGRESS procedure of SUDAAN® Release 8; RTI, 2001) were used. Body weights and organ weights from the recovery animals were analyzed using the Student's t-test. Binary outcomes (FOB) were analyzed using Fisher's Exact Test for overall heterogeneity among dose groups, followed by individual pairwise comparisons of exposed groups to control if the overall test was statistically significant. All tests were two-tailed. Exact two-sided p-values for standard nonparametric tests were provided for ordinal outcomes (FOB). This included exact p-values for the Kruskal-Wallis Test for overall heterogeneity among dose groups, followed by individual pairwise comparisons of exposed groups to control if the overall test was statistically significant. Grip strength and hindlimb foot splay were analyzed using either a parametric ANOVA or robust linear regression methods.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clear dose-response pattern of incidence or severity included rooting (in the mid and high dose groups) and salivation (in the high dose group) postdosing, which were considered to be due to taste aversion to the dosing formulation.
Animals of both sexes of the high dose group were lethargic postdosing, which was considered to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male was euthanized moribund on day 23. Clinical signs included diarrhea and dehydration, but the exact cause of death could not be determined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Significant decreases in male body weight in the 1000 mg/kg/day group compared to controls on sd 7, 14, 21, and 27 (-9% at each time point).
Significant reduction in male body weight change in the 1000 mg/kg/day group compared to controls from sd 0 to 7 (42% decrease) and for sd 0 to 27 (23% decrease).
No significant reductions in body weight on any study day in the 50 and 250 mg/kg,/day groups.
Statistically significant (14%) decrease in terminal body weight between the control and high-dose recovery males.

Females: No significant changes in body weight, but significant decrease in female body weight change at 1000 mg/kg/day. At 250 mg/kg bw/day: only transient reduction on days 0-7 of study

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in feed consumption of high dose males and females on days 0-7 of study

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

Description (incidence and severity):

Males: There were significant decreases in total protein in the 250 and 1000 mg/kg bw/day groups compared to the control group, and a significant decrease in sodium concentration in the 1000 mg/kg/day group.

Females: There was a significant increase in total cholesterol at 1000 mg/kg bw/day compared to controls.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine pH was significantly reduced in both sexes at 1000 mg/kg bw/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Males: FOB data for all males were not significantly different across groups during the 4 weeks of the dosing period except for an increase in the percent with abnormal arousal in an open field at 1000 mg/kg/day (62% compared to 15% reported in controls) in Week 1, and a significant but slight increase in the average muscle tone score during handling observations at 250 mg/kg bw/day in Week 2. Neither of these effects was consistent across weeks and therefore, neither were considered related to treatment. On study day 20, there were no significant treatment-related effects on motor activity or auditory startle. There were significant decreases in forelimb grip strength at 250 and 1000 mg/kg bw/day compared to controls when the measurement was conducted in conjunction with the motor activity and auditory startle measurements on day 20, but not when conducted as part of the FOB.

Females: There were no significant differences among groups in FOB data for all females up to week 3 of study. In week 4, there was a significant decrease in average approach response score in the 50 and 1000 mg/kg bw/day groups compared to controls. The difference in approach response was not dose related, did not occur in other weekly FOB determinations, and the value for the treated groups in week 4 was similar to the control group during the week 3 observations. Therefore, these differences were not considered to be treatment-related. Overall, there were no clear dose-response patterns in FOB.
There were significant treatment effects on auditory startle responses measured over 5 consecutive 10-trial blocks on day 20. In every 10-trial block, there was a significant decrease (up to a 61%) in the average force of jump at 1000 mg/kg bw/day compared to controls, and in blocks 3 and 4, there were significant decreases at 250 mg/kg bw/day compared to controls. In addition, the average force of jump in the other blocks for the 250 mg/kg bw/day group was reduced from the control values. There were statistically significant reductions in motor activity during 2 consecutive 10-minute blocks at 1000 mg/kg bw/day compared to controls, however total activity was not significantly different across treatment groups. Since these differences were not consistent across time blocks, and the total motor activity counts were not significantly different across groups, they were not considered treatment-related. There were no significant treatment¬related effects on grip strength performed on day 20.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: relative liver weight was significantly (12%) greater in the 1000 mg/kg bw/day group compared to the control group, and relative paired kidney weight that was significantly (16%) greater in the 1000 mg/kg bw/day group compared to controls. The significant increases in male liver and kidney weights (relative to body weight) were considered to be due to the reduced body weight. Relative to brain weight, organ weights were unaffected across all groups.
At 1000 mg/kg bw/day, in the recovery group there were significant decreases in absolute brain and thymus weights compared to control weights, and a significant 10% increase in paired kidney weight relative to sacrifice weight. There were no significant differences between groups for organ weights relative to brain weight.

Females: There were significant increases (28%) in liver weight and increases (12%) in paired kidney weights relative to terminal sacrifice weight in the 1000 mg/kg bw/day group compared to controls. There were no significant differences in organ weights relative to brain weight, with the exception of the liver that was significantly increased (17%) at 1000 mg/kg/day compared to controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males: dose-related increase in incidence of minimal (5 of 8 animals) or mild (1 of 8 animals) eosinophilic droplets in the renal cortex at 1000 mg/kg bw/day. Therefore, the kidneys from the mid dose and recovery animals were evaluated histopathologically. In the kidneys from the high dose recovery animals, mild eosinophilic droplets were observed in one animal, and in male kidneys at 250 mg/kg bw/day, minimal eosinophilic droplets were observed in 5 of 8 animals. Since lesions of the kidney were observed at the mid dose, low-dose kidneys were evaluated. No lesions were observed in the kidneys at 50 mg/kg bw/day.
Treatment-related microscopic findings were observed in one male in the 1000 mg/kg bw/day recovery group with eosinophilic droplets in the epithelial cells of the renal tubules.

Females: increase in incidence of minimal (4 of 8 animals) or mild (3 of 8 animals) centrilobular hypertrophy of the liver at 1000 mg/kg bw/day. Therefore, the livers from the mid dose and recovery animals were evaluated histopathologically. In the livers from the high dose recovery animals, minimal centrilobular hypertrophy was observed in one animal. No lesions were observed at 250 mg/kg/day.
One female in the 1000 mg/kg bw/day recovery group was diagnosed with centrilobular hypertrophy considered to be related to treatment during the dosing phase.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Key result

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

histopathology: non-neoplastic

Remarks on result:

other: effects not fully reversible in recovery groups

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

nervous system

Organ:

other: behavioural effects

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Dosing dilutions represented 103, 106 and 103 % of nominal values for low, mid and high dose groups.

Conclusions:

Under the conditions of this subacute toxicity study with recovery groups, the NOAEL was 50 mg/kg bw/day.

Executive summary:

Eight male and eight female Sprague-Dawley rats per group (study animals) and an additional five male and five female rats in the 0 (control) and 1000 mg/kg/day groups (recovery animals) were administered the test item, 1-chloro-2,3-dimethylbenzene, orally by gavage at 0, 50, 250, or 1000 mg/kg bw/day in corn oil for 28 days, including recovery groups without dosing for 2 weeks post exposure. There were treatment-related effects in males and females at 1000 mg/kg bw/day, as evidenced by reductions in body weight and observations of lethargy. Reductions in male forelimb grip strength occurred in measurements conducted on day 20 at 250 and 1000 mg/kg bw/day. An apparent dose-related decrease in female auditory startle response was also observed at 250 and 1000 mg/kg bw/day on day 20. A dose-related increase in the incidence of eosinophilic droplets in the epithelial cells of the renal tubules in males was observed at 250 and 1000 mg/kg bw/day. Liver hypertrophy, with associated increase in liver weight, in females was observed at 1000 mg/kg bw/day. No treatment-related effects were observed in males or females at 50 mg/kg bw/day. The effects on grip strength and auditory startle response were consistent with the observations of lethargy and were typical of exposure to organic solvents. The liver hypertrophy in the females was considered an adaptive response to the treatment and not a toxic response. After the recovery period, body weights of male rats remained reduced showing little indication of recovery. In addition, a single incidence of the kidney lesion in males and liver hypertrophy in females was observed in the recovery groups, suggesting partial recovery from effects in these organs. In conclusion, the treatment resulted in a no observable adverse effect level (NOAEL) of 50 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

83.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study reliable without restrictions

System:

urinary

Organ:

kidney

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

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species-specific effects of the substance. Therefore the results are regarded as relevant for humans.

Additional information

Sprague-Dawley rats were administered the test item, 1-Chloro-2,3-dimethylbenzene, orally by gavage at 0, 50, 250, or 1000 mg/kg bw/day in corn oil for 28 days, including recovery groups without dosing for 2 weeks post exposure. There were treatment-related effects in males and females at 1000 mg/kg bw/day, as evidenced by reductions in body weight and observations of lethargy. Reductions in male forelimb grip strength occurred in measurements conducted on day 20 at 250 and 1000 mg/kg bw/day. An apparent dose-related decrease in female auditory startle response was also observed at 250 and 1000 mg/kg bw/day on day 20. A dose-related increase in the incidence of eosinophilic droplets in the epithelial cells of the renal tubules in males was observed at 250 and 1000 mg/kg bw/day. Liver hypertrophy, with associated increase in liver weight, in females was observed at 1000 mg/kg bw/day. No treatment-related effects were observed in males or females at 50 mg/kg bw/day. The effects on grip strength and auditory startle response were consistent with the observations of lethargy and were typical of exposure to organic solvents. The liver hypertrophy in the females was considered an adaptive response to the treatment and not a toxic response. After the recovery period, body weights of male rats remained reduced showing little indication of recovery. In addition, a single incidence of the kidney lesion in males and liver hypertrophy in females was observed in the recovery groups, suggesting partial recovery from effects in these organs. Based on the LOAEL of 250 mg/kg bw/d a NOAEL of 83.3 mg/kg bw/d was calculated.

Justification for classification or non-classification

The observed effects require a classification specific target organ toxicity-repeated exposure, STOT RE Cat. 2, according to REGULATION (EC) No 1272/2008, 3.9.2.7.3, 3.9.2.9.5 and 3.9.2.9.7. Functional neurotoxic effects have been observed at 250 mg/kg bw/day. This is within the dose range requiring such a classification in subacute toxicity studies ( 10 < C ≤ 100 in 90 days studies, by a factor of 3 higher in 28 days studies).