3,5-dimethylbenzoyl chloride

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:

25 November 1992 - 23 December 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylbenzoyl chloride

EC Number:

413-010-9

EC Name:

3,5-dimethylbenzoyl chloride

Cas Number:

6613-44-1

Molecular formula:

C9H9ClO

IUPAC Name:

3,5-dimethylbenzoyl chloride

Details on test material:

DMBC
Appearance: clear, colourless liquid
Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

other: CD rats of Sprague-Dawley origin (Crl:CD BR VAF PLUS)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, England.
- Age at study initiation: 35 ± 1 day old.
- Weight at study initiation: Weight range of 63 - 81 g on arrival.
- Housing: All rats were initially caged, as far as possible, in groups of five according to sex in metal cages with wire mesh floors.
- Diet (e.g. ad libitum): A standard pelleted laboratory rodent diet (Special Diet Services Rat and Mouse Maintenance Diet) was provided ad libitum, with the exception of the overnight period prior to the collection of samples. In this situation, animals wee fasted overnight.
The batches of diet used for the study had been analysed for nutrients, possible contaminants and micro-organisms.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 21.5 °C.
- Humidity (%): 42 - 54 % relative.
- Air changes (per hr): Approximately 19 per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light (0700 - 1900 hours) in each 24-hour period.

IN-LIFE DATES: From: To: 18 November 1992 - 23 December 1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

A 20 % w/v solution was prepared freshly each day by mixing the test substance with corn oil (high dose level). A series of formulations (3.0 and 0.3 % w/v) was prepared by further serial dilutions of the high dose level.

Formulations were prepared freshly each day.

Prior to dosing, the test substance formulations were mixed by inversion (x 20) and subsequently mixed using a magnetic stirrer for a period of at least 10 minutes before dosing commenced. Dosing was completed within one hour of the commencement of stirring.

The test substance was administered by oral gavage to rats using a syringe and rubber catheter at a dose volume of 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

APPARATUS AND INSTRUMENTATION
High performance liquid chromatograph (HPLC)
Pump: Spectra Physics SP8770.
Autosampler: Waters WISP 710B.
Detector: Waters 481.
Integrator: Spectra-Physics SP4270.

Sample extraction
A representative sample (approximately 1 mL) of test formulation was accurately weighed and extracted (mechanical shake, 15 minutes) with an accurately pipetted volume (50 mL) of acetonitrile. The extract was centrifuged (2000 rpm, 1 minute) and the supernatant was appropriately diluted using acetonitrile to provide a solution containing DMBC in the expected concentration range 2 - 4 µg/mL.
The final solution was filtered (Whatman PURADISC 25PP, 0.45µm) and the concentration of DMBC was quantified by high performance liquid chromatography using ultraviolet detection as detailed below.

Typical chromatographic conditions:
Analytical column: LiChrospher 100 RP-18e, 5 µm, 125 x 4 mm ID, Merck Ltd.
Guard column: LiChrospher 100 RP-18e, 5 µm, 4 x 4 mm ID, Merck Ltd.
Mobile phase: Acetonitrile/water (45/55 v/v).
Flow rate: 1.0 mL/minute.
Detector wavelength: UV, 230 nm.
Injection volume: 35 µL.
Integrator attenuation: 64.
Retention volume: 2 mL.

Calibration
A primary standard solution was prepared for each analytical occasion by dissolving an accurately weighed quantity (50 mg) of DMBC in acetonitrile. Solutions for instrument calibration, containing DMBC in the concentration range 1 - 5 µg/mL, were prepared by appropriate dilution of the primary standard using acetonitrile.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence, using the conditions detailed above.

Calculation
The peak response for DMBC in each calibration chromatogram was measured and calibration curves were constructed by linear regression of standard response versus standard concentration. The response of the peak observed at the characteristic retention volume in sample and procedural recovery chromatograms was measured and the concentration determined.
Results were corrected for the appropriate mean procedural recovery value at analysis.

Limit of detection
The limit of detection was determined as 0.075 mg/mL.

VALIDATION OF THE METHOD OF ANALYSIS
The analytical procedure was validated by fortifying a minimum of six samples (1 mL) of control vehicle with DMBC to concentrations of 1 mg/mL) and 200 mg/mL, which were analysed in accordance with the analytical procedure. The test substance was added either as a solution in acetonitrile (inclusion levels <20 mg/mL) or as neat test material (inclusion levels 320 mg/mL).

Procedural recoveries were determined for each inclusion level and analysed concurrently with test formulations.

DETERMINATION OF CONCENTRATIONS IN DOSE FORMULATIONS ANALYSED DURING THE STUDY
Representative samples (approximately 20 mL) of freshly-prepared dose formulations were thoroughly mixed by vigorous shaking and duplicate sub-samples (1 mL) were analysed in accordance with the analytical procedure.

DETERMINATION OF THE CHEMICAL STABILITY IN CORN OIL FORMULATIONS
Freshly-prepared specimen formulations (approximately 20 mL), containing DMBC at nominal concentrations of 1 mg/mL and 200 mg/mL, were each thoroughly mixed by repeated inversion. Samples (approximately 1 mL) were removed immediately for analysis, from points at approximately one-quarter, one-half and three-quarters the depth (representing the top, middle and bottom) of each formulation (0 hour). The remainder of each formulation was stored in the dark at ambient temperature. At a time-point representing storage for 4 hours and 24 hours after preparation, each formulation was re-mixed and sampled for analysis as above.

At each occasion, the three sub-samples from each formulation were analysed in accordance with the analytical procedure.

RESULTS
Mean results were within ± 7 % of nominal concentrations
The results indicate that DMBC produces homogeneous formulations in corn oil, which is chemically stable during storage for 24 hours at ambient temperature.

Procedural recovery data and the determination of stability obtained during method validation indicate that the analytical method is both precise and accurate: a mean procedural recovery value of 99.2 % ± 2.28 SD (n=8) was obtained for 1 mg/mL and 96.4 % ± 0.97 SD (n=8) for 200 mg/mL. Results for the analysis of test samples are corrected for the appropriate mean procedural recovery value at analysis.
The absence of a peak at the characteristic retention volume for DMBC in the control sample chromatogram demonstrates the specificity of the HPLC assay.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 150 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

TREATMENT PROCEDURE
The high dosage was selected on the basis of available toxicity data.
The test substance was administered by oral gavage to rats using a syringe and rubber catheter at a dose volume of 5 mL/kg/day. Control animals received a similar dose volume.
Each animal received a constant dosage based on its most recently recorded bodyweight.

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS
-Clinical signs
All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.

-Bodyweight
All rats were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.

-Food consumption
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

-Water consumption
Daily monitoring by visual appraisal was maintained throughout the dosing period. In addition, gravimetric measurements of water consumption was initiated on Day 15 and continued through Week 3 following observation of a suspected treatment-related effect during Week 2.

CLINICAL PATHOLOGY
-Removal of blood samples
Blood was withdrawn under light ether anaesthesia from the orbital sinus of all rats prior to termination (Week 4).
The collected blood samples were divided as follows:

EDTA anticoagulant tubes for haematological investigations; Citrate anticoagulant tubes for coagulation test and Heparin anticoagulant microtainer tubes for biochemical tests.
All tubes were then mechanically agitated for at least five minutes and the microtainer tubes were subsequently centrifuged for a minimum period of two minutes (3000 x gravity).

The estimations performed have been listed below, together with an abbreviated title, the methods and the units of measurement applicable:

HAEMATOLOGY
The following parameters were analysed with an Ortho ELT-1500 Analyser, using standard Ortho methodology:
Units
Packed cell volume (PCV) %
Haemoglobin (Hb) g/dL
Red blood cell count (RBC) x 10^6/mm3
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC) %
Calculated: (Hb (g/dL) x 100) / PCV (%)
Mean corpuscular volume (MCV) fL
Calculated: (PCV (%) x 10) / RBC (x 10^6/mm3)
Platelet count (Plts) x 10^3/mm3
Total white blood cell count (WBC) x 10^3/mm3


The following estimations were measured using the appropriate methodology:

Units

Thrombotest (TT) s

Differential white blood cell count (Diff) - namely:
Neutrophils (N) x 10^3/mm3
Lymphocytes (L) x 10^3/mm3
Eosinophils (E) x 10^3/mm3
Basophils (B) x 10^3/mm3
Monocytes (M) x 10^3/mm3


The percentage distribution of each cell type was determined by standard microscopy of a blood smear stained with modified Wright's stain counting 100 cells. Percentage values were then converted to absolute values by computer inevitably involving a `rounding off' in a proportion of the results. Hence, the measured total WBC may differ slightly from the calculated total for the differential count.

Additional blood film slides were prepared and examined for morphological abnormalities.
P Polychromasia
H Hypochromasia
A Anisocytosis
R Rouleaux formation
S Separate film report (generated for additional abnormalities)

NAD No abnormality detected
1 Slight
2 Moderate
3 Marked
4 Gross

BIOCHEMISTRY
The following parameters were analysed with a Hitachi 737 Clinical Chemistry Analyser:
Units
Glucose - using BCL Test Kit (hexokinase mediated) mg/dL
Total protein g/dL
Albumin (Alb) g/dL
Globulin (Glob) g/dL
Calculated: Total protein (g/dL) minus Alb (g/dL)
Albumin/Globulin ratio (A/G) -
Calculated from Total protein and Albumin concentrations
Urea nitrogen (Urea Nitr) mg/dL
Creatinine mg/dL
Alkaline phosphatase (AP) mU/mL
reaction temperature 30 °C
Glutamic-pyruvic transaminase (GPT) mU/mL
using BCL Test Kit, reaction temperature 30 °C
Glutamic-oxaloacetic transaminase (GOT) mU/mL
using BCL Test Kit, reaction temperature 30 °C
Total bilirubin (Bilirubin) mg/dL
Sodium (Na) mEq/L
Potassium (K) mEq/L
Calcium (Ca) mEq/L
Chloride (Cl) mEq/L
Inorganic phosphorus (P) mEq/L
Cholesterol (Chol) mEq/L

Sacrifice and pathology:

TERMINAL STUDIES
Termination
After twenty-eight days of treatment (Day 29) all animals were killed by carbon dioxide asphyxiation and a complete autopsy undertaken. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.

-Organ weight
The following organs from each animal were dissected free of fat and weighed:

adrenals ovaries
brain spleen
kidneys testes (with epididymides)
liver

Macroscopic pathology
The macroscopic appearance of the tissues of all rats tissues were preserved in 10% buffered formalin:

adrenals* aorta
brain (medullary, cerebellar and cortical sections) caecum
colon duodenum
eyes (Davidson's fluid as fixative) femur with joint head
heart* ileum
jejunum kidneys*
larynx liver*
lungs lymph nodes (cervical and mesenteric)
mammary glands oesophagus
ovaries pancreas
pharynx pituitary
prostate rectum
salivary gland sciatic nerve
seminal vesicles skeletal muscle
skin spleen*
sternum (for bone and marrow sections) stomach
testes (including epididymis)* thymus (where present)
thyroid (with parathyroid) tongue
trachea urinary bladder
uterus (with cervix) vagina
any macroscopically abnormal tissue*

* Tissues required for histopathological examination

Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax (m.p. 56 °C); sections were cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides was carried out for all rats of Group 1 (Control group) and Group 4 (High dosage group) killed on Day 29.
Examinations were extended to include livers for males and kidneys for males and females of the Intermediate and Low dose groups after changes were seen in these tissues at the High dosage.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following sequence of statistical tests was used for bodyweight gains, organ weight and clinical pathology data:

If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75 %), the proportion of values different from the mode was analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:

Bartlett's test (3) was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1 % level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.

If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose-related response.

If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).

Organ weight analysis was initially carried out using analysis of variance as outlined above on absolute organ weights. Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10 % level of significance.

Significant differences between control animals and those treated with the test substance have been expressed at the 5 % (* P<0.05) or 1 % (** P<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was no mortality but some clinical signs were seen. The effects are detailed below.

Mortality:

mortality observed, treatment-related

Description (incidence):

There was no mortality but some clinical signs were seen. The effects are detailed below.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a tendency for slightly higher food consumption in males and females treated at 1000 mg/kg/day in comparison with controls, particularly during the latter half of the study.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A dose-related trend in increased water consumption was seen. Details are given below.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The effects are detailed below.

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):

Significantly increased kidney and liver weights were seen for male and female rats. The effects are detailed below.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The effects are detailed below.

Histopathological findings: neoplastic:

not examined

Details on results:

OBSERVATIONS

-Clinical signs
Increased salivation after dosing was observed throughout the study for rats dosed at 1000 mg/kg/day and intermittently from Day 12 or 13 to termination in all rats dosed at 150 mg/kg/day. This was accompanied by wet fur throughout the study for rats dosed at 1000 mg/kg/day and on one occasion for all males dosed at 150 mg/kg/day (Day 19). Although, the incidence of these signs was greater at 1000 mg/kg/day, it was considered likely that this was a response to the palatability of the dose.
Red/brown staining on the fur was found in all rats dosed at 1000 mg/kg/day accompanied by red/brown staining around the urogenital region mostly in female rats dosed at 1000 mg/kg/day.
No clinical signs were observed for control animals.

-Food consumption (Table 1)
There was a tendency for slightly higher food consumption in males and females treated at 1000 mg/kg/day in comparison with controls, particularly during the latter half of the study.

-Water consumption (Table 2)
A dose-related trend in increased water consumption was seen in both male and female rats. Percentage increases of 53 and 76 % were recorded at 1000 mg/kg/day and 37 or 30 % at 150 mg/kg/day for males and females respectively in comparison with controls.
At 15 mg/kg/day, percentage increases over control values were 7 and 4 % for males and females respectively. These differences may have been coincidental.

CLINICAL PATHOLOGY

-Biochemistry (Table 3)
The AP levels for males and females and the GPT and GOT levels in females were significantly (P<0.01) elevated for rats treated at 1000 mg/kg/day in comparison with controls.
AP and GPT were also found to be significantly elevated (P<0.05) in female rats at 150 and 15 mg/kg/day in comparison with controls. Although these differences from controls at 150 and 15 mg/kg/day were not strictly dosage-related, it is possible that they were treatment-related.
Slightly lower cholesterol levels and increased glucose concentrations were observed for both males and females treated at 1000 mg/kg/day.
The phosphate concentration was significantly (P <0.05) depressed in all treated male rats in comparison with controls. All but one of the individual values for treated rats were within the laboratory background data range for this parameter in rats of the age and strain used (P, mEq/L, males: 5th percentile - 4.1; median - 4.7; 95th percentile - 5.5; n = 108) and this difference probably reflects the slightly higher than expected values recorded for the male controls (in particular animal No. 1).
There were no other significant differences between control and treated animals for the remaining parameters measured.

TERMINAL STUDIES

-Organ weights (Table 4)
Significantly (P<0.01) increased kidney and liver weights, absolute and adjusted for final bodyweight, were recorded for male and female rats respectively treated at 1000 mg/kg/day in comparison with controls.
There were no other significant differences between control and treated animals.

-Microscopic pathology (Table5)
The following treatment-related changes were observed:
Liver
Minimal centrilobular hepatocyte enlargement in two male rats dosed at 1000 mg/kg/day.
Kidneys
Dilated cortical tubules with basophilic flattened epithelium, graded as 'moderate" in one male rat and "minimal" in one female rat of the 1000 mg/kg/day dose group. This change was associated with casts in the lumen of dilated tubules in one male and one female rat of this group.
These changes were not observed in any of the control or treated male and female rats of other treatment groups.
All the other histopathological findings were considered of no toxicological importance.
No microscopic changes were detected which might explain the increased liver weights recorded for female rats receiving 1000 mg/kg/day, or the increased kidney weights recorded for male rats treated at 1000 mg/kg/day.

Effect levels

Dose descriptor:

other: NOTEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A clear NOEL could not be established for this study.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Food Consumption - Group Mean Values (g/rat/week)

Week	Group and Dosage (mg/kg/day)
	Males				Females
	1 Control	2 15	3 150	4 1000	1 Control	2 15	3 150	4 1000
Dosing 1 2 3 4	182 200 198 172	176 195 198 177	178 199 205 184	176 200 216 192	143 139 135 125	142 136 137 124	147 144 144 134	143 158 157 159
Group Mean Cumulative Value	752	746	766	784	542	539	569	617

 

Table 2 Water Consumption - Group Mean Values (g/rat/day)

Week	Group and Dosage (mg/kg/day)
	Males				Females
	1 Control	2 15	3 150	4 1000	1 Control	2 15	3 150	4 1000
Dosing 2.1 2.2 2.3 2.4 2.5 2.6 3.0	29.8 30.0 30.0 30.4 28.0 28.4 29.4	28.8 32.6 32.4 32.0 33.4 31.8 30.0	38.8 40.2 40.2 40.0 40.4 39.6 43.2	43.8 44.4 48.0 47.2 43.4 44.2 44.2	21.6 24.6 23.6 21.4 22.8 22.8 24.0	23.2 24.0 24.2 24.8 22.8 24.6 23.8	29.2 28.6 28.4 31.6 29.2 30.4 31.2	34.2 42.6 42.2 42.2 40.2 39.8 43.0
Group Mean Cumulative Value	206	221	282	315	161	167	209	284
% Difference From Control	-	7	37	53	-	4	30	76

 

Table 3 Biochemistry - Group Mean Values

Group/ Dosage (mg/kg/day)		Glucose mg/dL	Protein g/dL			A/G	Urea Nitr mg/dL	Creat-inine mg/dL	AP mU/mL	GPT mU/mL	GOT mU/mL	Bili-rubin mg/dL
			Total	Alb	Glob
Male	1/Control 2/15   3/150   4/1000	113   109   96   121	6.0   6.3   6.0   5.9	2.7   2.7   2.6   2.7	3.3   3.6   3.4   3.2	0.83   0.76   0.77   0.84	10   9   9   11	0.5   0.5   0.5   0.5	420   519   476   621**	29   29   30   34	63   62   66   67	0.1†   0.1   0.1   0.1
Female	1/Control 2/15   3/150   4/1000	107   90   100   120	6.2   6.1   6.2   6.2	2.8   2.7   2.7   2.9	3.4   3.3   3.5   3.3	0.82   0.82   0.78   0.87	12   13   13   10	0.5   0.5   0.5   0.5	244   339*   320*   437**	20   27*   24*   33**	51   56   57   63**	0.1†   <0.1   0.1   <0.2

 

Group/ Dosage (mg/kg/day)		Na mEq/L	K mEq/L	Ca mEq/L	P mEq/L	Cl mEq/L	Chol mg/dL
Male	1/Control   2/15   3/150   4/1000	142   141   141   140	3.9   3.9   3.8   3.9	5.5   5.4   5.5   5.4	5.7   4.9*   5.0*   5.1*	95   95   95   94	78   83   83   66
Female	1/Control   2/15   3/150   4/1000	141   141   140   140	3.8   3.7   3.5   3.8	5.4   5.4   5.5   5.4	3.9   4.0   3.7   4.4	97   97   96   95	87   76   86   72

†Frequency Analysis

*P<0.05

**P<0.01

 

Table 4 Organ Weights - Group Mean Values (Week 5)

Values adjusted for final bodyweight are given in parentheses.

Group/ Dosage (mg/kg/day)		Body Weight (g)	Brain (g)	Liver (g)	Spleen (g)	Kidneys (g)	Adrenals (mg)	Testes + Epidids (g)	Ovaries (mg)
Males	1/Control 2/15 3/150 4/1000	336 334 335 325	1.93 1.96 1.97 1.92	(19.9) (18.7) (19.4) (20.9)	(0.71) (0.78) (0.80) (0.67)	(2.77) (2.80) (2.94) (3.35)**	(53.4) (48.8) (48.3) (53.6)	3.69 3.71 3.73 3.78	- - - -
Females	1/Control 2/15 3/150 4/1000	226 228 233 232	1.81 1.86 1.87 1.79	(11.7) (11.4) (11.5) (15.9)**	0.59 0.69 0.57 0.58	(2.02) (2.25) (1.99) (2.10)	60.8 61.9 61.2 59.9	- - - -	87.4 98.6 99.3 93.6

**P<0.01

Table 5 Microscopic Pathology Incidence Summary

Animals on study Animals completed	Males
	Group 1	Group 2	Group 3	Group 4
	5 5	5 5	5 5	5 5
Heart Examined No abnormalities detected Myocardial fibrosis Degenerate muscle fibres	5 5 0 0	0 0 0 0	0 0 0 0	5 4 1 0
Liver Examined No abnormalities detected Centrilobular hepatocyte enlargement (Total)     Minimal Centrilobular hepatocyte vacuolation (Total)     Minimal Mononuclear cells Sinusoidal dilation/congestion	5 2 0 0 0 0 1 2	5 0 0 0 1 1 2 4	5 1 0 0 2 2 0 4	5 2 2 2 0 0 0 2
Spleen Examined No abnormalities detected	5 5	0 0	0 0	5 5
Kidneys Examined No abnormalities detected Focal subcapsular cortical scarring (Total)     Minimal Tubular basophilia Tubular dilation Congestion Dilated cortical tubules (basophilic flattened epithelium, Total)     Minimal Moderate Casts in lumen of dilated tubules Cortical scarring/cyst(s)	5 3 1 1 0 1 0 0 0 0 0 0	5 4 0 0 0 0 1 0 0 0 0 0	5 3 0 0 1 0 1 0 0 0 0 1	5 2 2 2 0 0 0 1 0 1 1 0
Testes Examined No abnormalities detected	5 5	0 0	0 0	5 5
Epididymides Examined No abnormalities detected	5 5	0 0	0 0	5 5
Adrenals Examined No abnormalities detected Vacuolated cortical tubules (Total)     Minimal	5 4 1 1	0 0 0 0	0 0 0 0	5 5 0 0
Lymph Nodes - Cervical Examined Lymphoid proliferation (Total)     Minimal     Moderate	4 4 2 2	0 0 0 0	0 0 0 0	1 1 0 1

 

Animals on study Animals completed	Females
	Group 1	Group 2	Group 3	Group 4
	5 5	5 5	5 5	5 5
Heart Examined No abnormalities detected Myocardial fibrosis Degenerate muscle fibres	5 4 0 1	0 0 0 0	0 0 0 0	5 5 0 0
Liver Examined No abnormalities detected Centrilobular hepatocyte enlargement (Total)     Minimal Centrilobular hepatocyte vacuolation (Total)     Minimal Mononuclear cells Sinusoidal dilation/congestion	5 3 0 0 0 0 2 0	0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0	5 4 0 0 0 0 1 1
Spleen Examined No abnormalities detected	5 5	0 0	0 0	5 5
Kidneys Examined No abnormalities detected Focal subcapsular cortical scarring (Total)     Minimal Tubular basophilia Tubular dilation Congestion Dilated cortical tubules (basophilic flattened epithelium, Total)     Minimal Moderate Casts in lumen of dilated tubules Cortical scarring/cyst(s)	5 4 0 0 0 0 1 0 0 0 0 0	5 5 0 0 0 0 0 0 0 0 0 0	5 5 0 0 0 0 0 0 0 0 0 0	5 3 1 1 0 0 0 1 1 0 1 0
Adrenals Examined No abnormalities detected Vacuolated cortical tubules (Total)     Minimal	5 5 0 0	0 0 0 0	0 0 0 0	5 5 0 0
Lymph Nodes - Cervical Examined Lymphoid proliferation (Total)     Minimal     Moderate	2 2 1 1	0 0 0 0	0 0 0 0	1 1 1 0

Applicant's summary and conclusion

Conclusions:

150 mg/kg/day was considered to be a NOTEL. The continuation of these changes to the low dosage mean that a clear no observed effect level (NOEL) could not be established for this study.

Executive summary:

This study was performed to assess the systemic toxicity of DMBC to the rat, in accordance with Annex V, Part B, Method B7 in the EEC Directive 84/449/EEC and OECD Guideline for Testing of Chemicals No. 407, "Repeated dose oral toxicity - Rodent: 28-day or 14-day study".

 

DMBC was administered by oral gavage, once daily, to groups of five male and five female rats for twenty-eight consecutive days, at fixed dosage levels of 15, 150 and 1000 mg/kg/day.

-Clinical signs

Increased salivation after dosing was observed at 1000 mg/kg/day and to a lesser extent at 150 mg/kg/day. Red/brown staining on the fur was found in all rats dosed at 1000 mg/kg/day accompanied by red/brown staining around the urogenital region mostly in female rats dosed at 1000 mg/kg/day.

-Water consumption

 A marked increase in water consumption was recorded for rats of both sexes receiving 1000 and 150 mg/kg/day.

-Biochemistry

 The AP concentration for males and females and the GPT and GOT concentrations in females were elevated for rats treated at 1000 mg/kg/day. Minor changes in cholesterol and glucose levels were also seen for these rats. The AP and GPT concentration in female rats was also found to be elevated at 150 and 15 mg/kg/day.

-Organ weight

Kidney and liver weights were increased for male and female rats respectively treated at 1000 mg/kg/day.

-Microscopic pathology

Minimal centrilobular hepatocyte enlargement in the liver was evident among males treated at 1000 mg/kg/day. Renal changes consisting of dilated cortical tubules with basophilic flattened epithelium, associated with casts in the lumen of the dilated tubules were seen in both sexes treated at 1000 mg/kg/day.

 

150 mg/kg/day was considered to be a NOTEL (no observed toxic effect level). The continuation of these changes to the low dosage mean that a clear no observed effect level (NOEL) could not be established for this study.