4-chloro-2,5-dimethoxyaniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-06-29 to 1993-10-28, report re-issued on 1994-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to previous OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

Constituent 2

Reference substance name:

2,5-dimathoxy-4-chloroaniline

IUPAC Name:

2,5-dimathoxy-4-chloroaniline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK), arrived on 08 July 1992
- Age: 28d +/- 1d
- Weight range of +/- 10% of the mean weight on arrival
- Acclimatisation: at least one week
- Caging in groups of five (as far as possible) according to sex
- Standard pellet laboratory rodent diet and drinking water: both ad libitum
- Animal room temperature: 19.5 - 23.5°C
- Relative humidity: 51 - 72%
- Air exchange: approximately 20 timer per hour
- Lighting: 12hrs artificial light / 12hrs darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: corn oil

Details on oral exposure:

- The test item was administered as a suspension in corn oil, via oral gavage using a syringe and rubber catheter at a dosage volume of 5mL/kg/day
- A series of formulations was prepared freshly each day at concentrations of 0.4, 2.0 and 10% w/v
- The physical and chemical stability and homogeneity of formulations in corn oil was assessed prior to the start of treatment and were found to be satisfactory
- The dosage levels of the test substance was selected on the basis of available toxicity data and a preliminary dietary
- Each animal received a constant dosage level based on its most recently recorded body weight
- Prior to dosing the test substance formulations were mixed by inversion (x10)
- Subsequent mixing using a magnetic stirrer occured for a period of at least 10 minutes before dosing commenced.
- Dosing was completed within one hour pf the commencement of stirring

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Once daily for twentynine consecutive days

Frequency of treatment:

Once daily for twentynine consecutive days

Doses / concentrations

Remarks:

Doses / Concentrations:
20 mg/kg/day, 100 mg/kg/day, 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs, body weight, water consumption, haematology; clinical chemistry, gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Clinical signs

There were no mortalities.

There were no signs of ill health or behavioural change observed in control animals.

In both male and female rats treated at 500 mg/kg/day, pilo-erection, hunched posture, a post-dose increase in salivation (all ranging from moderate to well-defined) and a reddish/brown staining on the cage tray paper were all frequently observed during the study. Lethargy and a "waddling gait" (both slight to moderate in nature) were seen intermittently. Ptosis (slight in nature) was evident in both sexes on Day 1 and in females only on Day 2.

At 100 mg/kg/day, a post-dose slight to moderate increase in salivation and a reddish/brown staining on the cage tray paper were routinely seen during the study. In addition, pilo-erection was noted infrequently in both males and females during the first half of the study, and a "waddling gait" was conspicuous in females on Day 5 only (both responses slight to moderate in degree).

For rats treated at 20 mg/kg/day, signs were confined to a post-dose slight increase in salivation noticed on a number of occasions during the study.

 

Bodyweight

In comparison with controls, male rats receiving 500 mg/kg/day showed slightly lower bodyweight gains for each week of the study, this variation reached statistical significance (P <0.05) at Weeks 1 and 2 and overall.Bodyweight gains were significantly low (P <0.01) at Week 4 for female rats of the intermediate and high dosages, but overall gain in these groups was not significantly affected.

 

Food consumption

Food consumption in male rats receiving 500 mg/kg/day tended to be slightly less than that of the concurrent controls during the first half of the study, but reached similar levels during the latter half of the study.Food consumption for low and intermediate dosage male rats and all female treatment groups showed no dosage-dependent variation.

 

Water consumption

When assessed by daily weighing of water bottles during the study, the volume of water consumed by male and female rats receiving 500 mg/kg/day was clearly greater than that of the controls, (overall for males by 117% and for females by 47%).Increases in consumption were also observed in both male and female rats receiving 100 mg/kg/day and to a lesser extent in male rats treated at 20 mg/kg/day in comparison with controls.

Water consumption in female rats receiving 20 mg/kg/day was similar to those of the controls.

Haematology

At 500 mg/kg/day significant disturbances (P<0.05 or P<0.01) were recorded in rats of both sexes for red blood cell parameters.These changes included lower packed cell volume (PCV), haemoglobin (Hb), red blood cell (RBC) count and for males only mean corpsular haemoglobin concentration (MCHC). For females only, an increase for mean cell volume (MCV) was also seen. In addition, increased numbers of reticulocyte were seen for both sexes.

Many of these disturbances were also seen in animals receiving 100 mg/kg/day but to a lesser extent. These achieved significance (P <0.05 or P <0.01) namely for lower PCV (males only), Hb, RBC (males only) and increased values for reticulocytes and thrombotest times (both for females only).

A slight variation in red cell parameters seen for animals treated at 20 mg/kg/day was not statistically significant.

Slight polychromasia was seen in all animals treated at 500 mg/kg/day, four male and four females at 100 mg/kg/day. Slight anisocytosis was evident for all animals treated at 500 mg/kg/day.

Thrombotest time was slightly but significantly reduced in comparison with controls for females receiving 500 or 100 mg/kg/day (P<0.05).

Intergroup variation in the remaining parameters measured, revealed no further significant differences between treated and control animals.

 

Biochemistry

Bilirubin concentrations were significantly increased for both males and females treated at 100 and 500 mg/kg/day (P <0.05 or P<0.01).

Cholesterol concentration was significantly increased for both males and females treated at 500 mg/kg/day and for females only there was an elevation in plasma protein and creatinine levels (P<0.05 or P<0.01).

Significant differences in enzyme level comprised a reduction in alkaline phosphatase activity for females at 500 mg/kg/day and a slight reduction of glutamic-oxaloacetic transaminase (GOT) activity for males at both 100 and 500 mg/kg/day.

Variation in electrolyte concentrations involved slight decreases in sodium and chloride levels at 500 mg/kg/day with the differences significant for females (P <0.05 or 0.01). Other statistically significant differences occurred for potassium and calcium ions among males, and phosphorus ions among females with significance extending down the groups; however, these differences in potassium and calcium ions were not dose dependent.

 

Bone myelograms

Statistically significant increased values (P <0.05 or 0.01) were achieved for early and late normoblasts in male rats receiving 500 mg/kg/day and total erythroid cells were increased although not significantly.In females total erythroid cell count was also higher than for controls, although again not significantly. In females at all three dosages there was an increase in neutrophil metamyelocytes, although this tendency was not dosage dependent.

Intergroup variation in the remaining parameters measured, revealed no further significant differences between treated control animals.

 

Organ weights

In both male and female rats treated at 500 mg/kg/day, significantly increased absolute and adjusted liver and spleen weights, and adjusted kidney weights were observed (P < 0.05 or P < 0.01).

Organ weight disturbances were also seen in animals receiving 100 mg/kg/day, and comprised,
increased absolute and adjusted liver weights (both sexes) and adjusted kidney weights (females only).

In comparison with controls reduced absolute testes weights were evident at all three dosages, but the tendency was not dose dependent.

Intergroup variation in the remaining parameters measured, revealed no further significant differences between treated and control animals.

 

Macroscopic pathology

The macroscopic examination performed at termination revealed no changes attributable to treatment with 2,5-dimethoxy-4-chloroaniline.

 

Microscopic pathology

 

Liver

Centrilobular hepatocyte enlargement in a single rat from each sex receiving 100 mg/kg/day, 5/5 male and 4/5 female rats receiving 500 mg/kg/day.

 

Kidneys

Degeneration of papillary tubules in 2/5 male rats receiving 100 mg/kg/day.

Necrosis of papillary tubules associated with tubular basophilia with/without dilated tubules with eosinophilic material, inflammatory cell infiltration into papilla and congestion of papilla in 4/5 male and 3/5 female rats receiving 500 mg/kg/day.

No histopathological change was observed to correlate to the increased spleen weights in rats receiving 500 mg/kg/day and reduced absolute testes weights in rats treated with the test compound.

All other findings observed are considered to be of no toxicological importance.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study 20 mg/kg/day represents a no toxic effect level for 2,5-dimethoxy-4- chloroaniline in the rat.

Executive summary:

This study was performed to assess the systemic toxicity to the rat of 2,5-dimethoxy-4-chloroaniline and to provide guidance in the choice of dosages for a subsequent 90 day study. The method followed was that outlined in Annex V, Part B, Method B7 to the EEC Directive 79/831/EEC and OECD Guideline for Testing of Chemicals No. 407, "Repeated dose oral toxicity -Rodent: 28-day or 14-day study".

 

2,5-Dimethoxy-4-chloroaniline was administered by gavage, once daily, to groups of five male and five female rats for twenty-nine consecutive days at fixed dosage levels of 20, 100 and 500 mg/kg/day.The test substance was prepared as suspensions in corn oil at concentrations of 0.4, 2.0 and 10% w/v. A further group of rats (five males and five females) was held as a concurrent control receiving the vehicle, corn oil, alone.

Bodyweights, food and water consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken prior to termination, Day 30. Animals were killed and examined macroscopically on Day 30. Histological examination of specified tissues in accordance with OECD Guideline No. 408 was then initiated.

 

The following comments in relation to real or possible treatment-related findings are made in summary:

 

Clinical signs. There were no mortalities.At 500 mg/kg/day, rats of both sexes frequently showed signs of pilo-erection, hunched posture, a post-dose increase in salivation and a reddish/brown staining on the cage tray paper. In addition, a "waddling gait", lethargy and ptosis were also seen on occasion. At 100 mg/kg/day a post-dose increase in salivation, reddish/brown staining on the cage tray paper, "waddling gait" and pilo-erection were also seen but with less frequency. In rats at the low dosage, signs were confined to infrequent incidences of increased salivation following dosing.

 

Bodyweight. Male rats receiving 500 mg/kg/day showed slightly lower bodyweight gains for each week of the study, this reached significance at Weeks 1 and 2 and overall. Bodyweight gains were significantly low at Week 4 only for female rats treated at 100 and 500 mg/kg/day.

 

Water consumption. The volume of water consumed by rats receiving 500 mg/kg/day was clearly greater than that of the controls. Increases in consumption were also observed in rats receiving 100 mg/kg/day and to a lesser extent in males only treated at 20 mg/kg/day.

 

Haematology. At 500 mg/kg/day significant disturbances were recorded for several red blood cell parameters. These changes included lower PCV, Hb, RBC count and for males only MCHC. For females only, an increase for MCV was also seen. In addition, increased numbers of reticulocytes were seen for both sexes. Many of these disturbances were also seen in animals receiving 100 mg/kg/day but to a lesser extent, with significance occurring for PCV (males only), Hb, RBC (males only) and increased values for reticulocytes (females only). Slight polychromasia was seen in all animals treated at 500 mg/kg/day and for the majority of rats at 100 mg/kg/day. Slight anisocytosis was evident for all animals treated at 500 mg/kg/day. Thrombotest time was slightly but significantly reduced in comparison with controls for females receiving 500 or 100 mg/kg/day.

 

Biochemistry. Significant increases were noted for bilirubin concentrations at 100 and

500 mg/kg/day. Cholesterol concentration was increased at 500 mg/kg/day and, for females only, there was an elevation in plasma protein and creatinine levels. Differences in enzyme level comprised a reduction in AP activity for females at 500 mg/kg/day and a slight reduction of GOT activity for males at both 100 and 500 mg/kg/day. Variation in electrolyte concentrations involved slight decreases in sodium and chloride levels at 500 mg/kg/day with the differences significant for females. Other statistically significant differences occurred for potassium and calcium among males, and phosphorus among females but differences were not dose dependent.

 

Bone myelograms. Significantly increased values were achieved for early and late normoblasts in male rats receiving 500 mg/kg/day.

 

Organ weights. In rats treated at 500 mg/kg/day, significantly increased absolute and adjusted liver and spleen weights, and adjusted kidney weights were observed. Also at 100 mg/kg/day, increased absolute and adjusted liver weights and adjusted kidney weights (females only) were observed. In addition, in comparison with controls, reduced absolute testes weights were evident at all three dosages, but the tendency was not dose dependent.

Microscopic pathology. In rats treated at 500 mg/kg/day the majority showed renal changes involving necrosis of papillary tubules, with associated changes, and in the livers centrilobular hepatocyte enlargement was observed. At 100 mg/kg/day a small proportion of the rats showed degeneration of papillary tubules and centrilobular hepatocyte enlargement.

 

At 20 mg/kg/day no treatment-related histopathological changes were observed.

 

Other findings. Changes seen in the other parameters measured, namely food consumption and macroscopic pathology were considered to be unrelated to treatment.