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Administrative data

Description of key information

Repeated dose toxicity: Oral

Low Observed Adverse Effect Level (LOAEL) was considered to be in the range of 475-500 mg/kg body weight, when rats were treated with the given test chemical via oral route for 28 days.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
The effect of repeated (4 weeks) oral administration of test chemical at the dose levels of 400 -500 mg/kg/day on the morphology and microsomal drug metabolising enzyme activity of the liver was studied in rats.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CFY strain
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animal
TEST ANIMALS
- Source: Anglia Laboratory Animals, Alconbury, Huntingdon,
U.K.
- Age at study initiation: 8 weeks old
- Weight at study initiation:
Males: 200g; females: 170 g
- Housing: 5 rats/ polycarbonate cage
- Diet (e.g. ad libitum): The rats were allowed free access to food (Spratt's Laboratory Diet No. 1)
- Water (e.g. ad libitum): The rats were allowed free access to water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-27°C
- Humidity (%):45-55%
- Photoperiod (hrs dark / hrs light): 12 : 12 h.
Route of administration:
oral: gavage
Vehicle:
not specified
Details on oral exposure:
No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
4 weeks (28 days)
Frequency of treatment:
once daily for 4 weeks
Remarks:
Doses / Concentrations:
dose levels of 400–500 mg/kg/day (Rats were dosed with 400
mg/kg/day during the first week, and thereafter at 500 mg/kg/day.
)
Basis:
no data
No. of animals per sex per dose:
Total:40
10 :male ; 10 :female :400 mg/kg
10 male;10:female:500 mg/kg
Control animals:
yes
Details on study design:
No data available
Positive control:
No data available
Observations and examinations performed and frequency:
Observations and examinations performed & frequency

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week


OTHER:
BIOCHEMISTRY:
Microsomal suspensions of the liver were prepared for assay of cytochrome P-450 concentration and activity of aniline hydroxylase and p-nitrophenol glucuronyltransferase.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes ;At the termination of the study (24 h after the final dose of xylidine) the rats were sacrificed by cervical dislocation. Post-mortem examination was performed on all rats, including those dying during the study.

HISTOPATHOLOGY: Yes
Tissue samples of the liver were fixed in 10% neutral formalin solution,paraffin-embedded, sectioned and stained with haematoxylin and eosin and with Feulgen reaction. Fresh frozen sections were stained with haemalum and Oil Red O for detection of fat, with PAS for glycogen, and according to Wachstein and Meisel for glucose-6-phosphatase activity. For electron microscopical examination, small pieces of the livers were fixed in 4% glutaraldehyde in 0.05 M cacodylate buffer pH 7.3 for 2 h at +4°C and then post-fixed with 1% osmium tetroxide in 0.05 M cacodylate buffer pH 7.3 for 2 h at +4°C. The samples were dehydrated and embedded in epoxy resin. Survey sections were cut at 1 #m and stained with toluidine blue. Centrilobular areas in these sections were selected for ultrastructural examination.Silver-gold thin sections were then cut, picked up on uncoated grids,stained with lead citrate and examined electron microscopically.A Quantimet 720 Image analysing computer {Cambridge Instruments, Melbourn, Hertfordshire) was used to detect the Feulgen stained nuclei of the hepatocytes in the relevant centri- and perilobular regions. The nuclei count in a given area enabled the size of the hepatocytes to be calculated. The glucose-6-phosphatase activity was also measured on a Quantimet 720 using the densitometer module.
Other examinations:
Biochemistry:
The liver samples were placed in ice-cold 0.05 M Tris buffer (pH 7.4) containing 0.25 M sucrose and homogenised individually in 4 volumes of 0.05 M Tris buffer: 0.25 M sucrose solution (pH 7.4). The homogenates were centrifuged at 10 000 g for 20 min at 4°C, and the supernatant decanted. Microsomal fractions were prepared by centrifugation of this supernatant at 105 000 g for 1 h at 4°C. The microsomal pellet was suspended in 0.05 M Tris buffer: 0.25 M sucrose (pH 7.4) so that 1 ml of the suspension was approximately equivalent to 330 mg liver (wet wt). The microsomal suspension was kept at 4°C until used, within 2 h, for enzyme assays and cytochrome P-450 estimations. Concentrations of cytochrome P-450 in microsomal suspensions were assayed by the method of Omura and Sato and calculated from the molar extinction coefficient of 91 mM-1 cm-1.The activities of aniline hydroxylase and p-nitrophenol glucuronyltransferase were assayed in the microsomes at 37°C according to Wills and Pogell and Krisman .The protein concentration of the microsomal suspensions was determined by the method of Lowry et al. using bovine serum albumin as a standard.
Statistics:
Statistical significance between the values was calculated by Bartlett's t-test.
Clinical signs:
not specified
Description (incidence and severity):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study there were a few mortalities but none of them were due to the compounds administered.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A reduction in body weight gain was observed in all treated rats
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no essential difference in food consumption between the control and treated groups.
Food efficiency:
not specified
Description (incidence and severity):
not specified
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
not specified
Ophthalmological findings:
not specified
Description (incidence and severity):
not specified
Haematological findings:
not specified
Description (incidence and severity):
not specified
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose-6-phosphatase activity was evenly distributed in the control animals.

Biochemistry investigation:
The hepatic microsomal protein content in male and female rats treated with test chemical. A slight increase was evident in males dosed with test chemical, but not in female rats.

The hepatic microsomal cytochrome P-450 content was increased in males and females dosed with test chemical.

Total aniline hydroxylase activity was increased in all treated Rats.

Glucuronyltranferase activity (using p-nitrophenol as substrate) was increased in male and female rats dosed with test chemical and which was evident when results were expressed both in term of the total liver and per unit liver weight.
Urinalysis findings:
not specified
Description (incidence and severity):
not specified
Behaviour (functional findings):
not specified
Description (incidence and severity):
not specified
Immunological findings:
not specified
Description (incidence and severity):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Livers were enlarged, but had a normal gross appearance. Relative liver weight was significantly increased compared to controls in both male and female rats (p<0.05 and p<0.01, respectively).
Gross pathological findings:
not specified
Description (incidence and severity):
not specified
Neuropathological findings:
not specified
Description (incidence and severity):
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed a minimal fine droplet fatty change of the livers of comparable degree in both control and treated groups. Furthermore, there was an apparent enlargement of the hepatocytes, which was significant in the centrilobular region (p<0.01) for both males and females.

Staining with PAS demonstrated a centrilobular decrease in liver glycogen. This decrease was greater in males than in females.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
not specified
Other effects:
not specified
Description (incidence and severity):
not specified
Details on results:
not specified
Dose descriptor:
LOAEL
Effect level:
475 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
not specified
System:
other: not specified

Table:BODY AND LIVER WEIGHTS IN RATS TREATED WITH 400-500 mg/kg/day OF XYLIDINE ISOMERS FOR 4 WEEKS

GROUP

MALE

FEAMLE

No. of animals

Final body weight(g)a

Liver weight (g)

Liver wt. in % of final body wt

No. of animals

Final body weight(g)a

Liver weight (g)

Liver wt. in % of final body wt

Control

5

400 ± 5

14.88 ± 0.60

3.72 ± 0.09

3

270 ± 16

9.86 + 1.07

3.62 ± 0.17

Test chemical

4

373 ± 11

20.56 ± 0.60c

5.52 ± 0.13d

4

238±12

10.99±0.54

4.62±0.18b

aInitial mean body weights of males and females were 192 ± 3 g and 172 ± 2 g respectively.

Symbols for statistical significance (compared with the control group)bP < 0.05 ; cP < 0.01 ;dP< 0.001.

 

Table:QUANTIFICATION OF THE MEAN HEPATOCYTE SIZE IN RATS TREATED WITH 400-500 mg/kg/day OF XYLIDINE ISOMERS FOR 4 WEEKS

GROUP

MALE

FEAMLE

No. of animals

Mean hepatocyte area (μm2)

No. of animals

Mean hepatocyte area (μm2)

Periportal region

Centrilobular

region

Periportal region

Centrilobular

region

Control

5

524

531

3

428

416

Test chemical

4

623a

676b

4

487a

510b

Symbols for statistical significance (compared with the control group):aP <: 0.05;bP <: 0.01.

 

Table:DENSITOMETRIC ESTIMATION OF MEAN GLUCOSE:6-PHOSPHATASE ACTIVITY IN THE LIVERS OF RATS TREATED WITH 400-500 mg/kg/day OF XYLIDINE ISOMERS FOR 4 WEEKS

GROUP

MALES

FEMALES

Number of animals

Average density

Number of animals

Average density

CONTROL

5

51

3

45

TEST CHEMICAL

4

44a

4

45

Symbols for statistical significance (compared with the control group):aP < 0.05;bP < 0.01.

Table:HEPATIC DRUG-METABOLISING ENZYME ACTIVITY/rag MICROSOMAL PROTEIN IN RATS TREATED WITH 400-500 mg/kg/day OF XYLIDINE ISOMERS FOR 4 WEEKS

The values are expressed as meant S.E.M.

 

GROUP

MALES

FEMALES

Number of animals

Cytochrome P-450 (nmol/mg protein)

Aniline hydroxylase

(μg product/h/mg protein)

Glucuronyl-tranferase

(μg conjugate/h/mg protein)

Number of animals

Cytochrome P-450 (nmol/mg protein)

Aniline hydroxylase

(μg product/h/mg protein)

Glucuronyl-tranferase

(μg conjugate/h/mg protein)

CONTROL

5

0.252±0.011

1.78±0.18

3.83± 1.22

3

0.176±0.005

0.88±0.14

2.49 ±1.06

TEST CHEMICAL

4

0.342±0.038a

2.10±0.30

10.76±1.36b

4

0.246±0.008b

1.17±0.19

11.33±1.64c

Symbols for statistical significance (compared with control group):aP < 0.05;bP < 0.01;cP < 0.001.

 

Table:HEPATIC DRUG-METABOLISING ENZYME ACTIVITY/g LIVER TISSUE IN RATS TREATED WITH 400--500 mg/kg/day OF XYLIDINE ISOMERS FOR 4 WEEKS

The values are expressed as mean + S.E.M.

GROUP

MALES

Number of animals

Microsomal protein (mg/g liver)

Cytochrome P-450 (nmol/g liver)

Aniline hydroxylase

(μg product/h/g liver)

Glucuronyl-tranferase

(μg conjugate/h/g liver)

CONTROL

5

22.23 ± 1.82

5.53 ± 0.26

38.5 ± 2.4

78.2 ±18.6

TEST CHEMICAL

4

23.91 ± 1.41

8.23 ± 1.06b

50.3 ± 1.1

258.0 ± 38.6b

 

 

FEMALES

CONTROL

3

23.02 ± 1.76

4.03 ± 0.26

20.1 ± 3.1

58.7± 26.9

TEST CHEMICAL

4

23.42 ± 3.16

5.38 ± 0.97

26.0 ± 2.0

257.9 ± 33.8 b

 

  Symbols for statistical significance (compared with control group):aP < 0.05;bP < 0.01;

cP < 0.001.

Conclusions:
The low-observed-adverse-effect-level (LOAEL) was considered to be 475 mg/kg/day when Sprague-Dawley derived rats (CFY strain) male and female rats were treated with test chemical via gavage for 28 days.
Executive summary:

The effect of repeated (4 weeks) oral administration of test chemical at the dose levels of 400 - 500 mg/kg/day on the morphology and microsomal drug metabolising enzyme activity of the liver was studied in Sprague-Dawley derived rats (CFY strain). No mortalities due to test substance administration occurred during the study. A reduction in body weight gain was observed in all treated rats, especially the males, but this was not significant. There was no difference in food consumption between control and treated groups. Livers were enlarged, but had a normal gross appearance. Relative liver weight was significantly increased compared to controls in both male and female rats (p<0.05 and p<0.01, respectively). Microscopic examination revealed a minimal fine droplet fatty change of the livers of comparable degree in both control and treated groups. Furthermore, there was an apparent enlargement of the hepatocytes, which was significant in the centrilobular region (p<0.01) for both males and females. Staining with PAS demonstrated a centrilobular decrease in liver glycogen. This decrease was greater in males than in females. Glucose-6-phosphatase activity was evenly distributed in control animals but in treated animals there was a centrilobular decrease in enzyme activity. The hepatic microsomal protein content was increased in males, but not in females. The hepatic microsomal cytochrome P-450 content was increased in males and females. Total aniline hydroxylase activity was not increased.Glucuronyltransferase activity was increased, but not significantly, over controls.Therefore, The low-observed-adverse-effect-level (LOAEL) was considered to be 475 mg/kg/day when Sprague-Dawley derived rats (CFY strain) male and female rats were treated with test chemical via gavage for 28 days.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
475 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is Klimisch 2 and from Publication

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

Repeated dose toxicity: Oral

Data available from the various sources was reviewed to determine the toxic nature of the given test chemical. The studies are as mentioned below:

The effect of repeated (4 weeks) oral administration of test chemical at the dose levels of 400 - 500 mg/kg/day on the morphology and microsomal drug metabolising enzyme activity of the liver was studied in Sprague-Dawley derivedrats(CFY strain). No mortalities due to test substance administration occurred during the study. A reduction in body weight gain was observed in all treated rats, especially the males, but this was not significant. There was no difference in food consumption between control and treated groups. Livers were enlarged, but had a normal gross appearance. Relative liver weight was significantly increased compared to controls in both male and female rats (p<0.05 and p<0.01, respectively). Microscopic examination revealed a minimal fine droplet fatty change of the livers of comparable degree in both control and treated groups. Furthermore, there was an apparent enlargement of the hepatocytes, which was significant in the centrilobular region (p<0.01) for both males and females. Staining with PAS demonstrated a centrilobular decrease in liver glycogen. This decrease was greater in males than in females. Glucose-6-phosphatase activity was evenly distributed in control animals but in treated animals there was a centrilobular decrease in enzyme activity. The hepatic microsomal protein content was increased in males, but not in females. The hepatic microsomal cytochrome P-450 content was increased in males and females. Total aniline hydroxylase activity was not increased.Glucuronyltransferase activity was increased, but not significantly, over controls.Therefore, The low-observed-adverse-effect-level (LOAEL) was considered to be 475 mg/kg/day when Sprague-Dawley derived rats (CFY strain) male and female rats were treated with test chemical via gavage for 28 days.

In another experimental study,the daily oral administration by gavage of the test chemical in doses of 100 mg/kg bw for 1 week and then of 500 mg/kg bw for an additional 3 weeks to male SD-rats produced increased liver weight, proliferation of the reticular endothelial system, enlargement of hepatocytes, decreased glycogen and glucose-6-phosphatase in the liver, and an increase of p450 microsomal protein and glucuronyltransferase in the liver.

Thus based on the above studies, the Low Observed Adverse Effect Level (LOAEL) is considered to be in the range of 475 - 500 mg / kg body weight which is considered to be a toxic dose range and hence is likely to classify as per the criteria mentioned in CLP regulation.

Additional information

Justification for classification or non-classification

Based on the experimental data available, the given test chemical exhibits toxic nature upon repeated exposure by oral route of exposure. Hence, it is likely to classify as toxic as per the criteria mentioned in CLP regulation.