4-mesyl-2-nitrotoluene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December - 20 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-550-0

EC Name:

-

Cas Number:

1671-49-4

Molecular formula:

C8H9NO4S

IUPAC Name:

4-methanesulfonyl-1-methyl-2-nitrobenzene

Details on test material:

- Physical state: off-white powder
- Purity test date: 10 December 2004
- Expiration date of the lot/batch: December 2005
- Storage condition of test material: ambient temperature in the dark

Test animals

Species:

rat

Strain:

other: Alpk:APfSD (Wistar-derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 166-232 g (males); 166-232 g (females)
- Fasting period before study: none
- Housing: 4 per cage (sexes separately) in suitable for animals of this strain and the weight range expected during the course of this study
- Diet: CT1 diet ad libitum (except during urine collection)
- Water: mains water ad libitum (except during urine collection)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes: at least 15 changes/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 4 January 2005 To: 20 July 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulated as suspensions in 1% carboxymethyl cellulose (CMC). Stability data for the test substance in 1% aqueous CMC was not available at the start of dosing and, therefore, initially the dose preparations were prepared daily and administered to the animals as soon after preparation as possible. Once stability had been confirmed to be in excess of 7 days, the preparations were made weekly and divided into 7 aliquots which were stored, under appropriate conditions in the animal room. Dosing preparations were dosed orally at 1.0 mL/100 g bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentrations for all batches of dosing preparations analysed were within 9% of the nominal concentration. The homogeneity of the test substance in dosing preparations at concentrations of 0.5 mg/mL and 10 mg/mL, was determined and considered satisfactory, percentage deviations from the overall mean were within 4%. The reanalysis of the test substance in dosing preparations at concentrations of 0.5 mg/mL and 10 mg/mL when stored at room temperature was shown to be acceptable for 16 days, covering the period of storage and dosing in this study.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 5, 15, 100 mg/kg/day
Basis:
other: nominal in 1% CMC

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for this study were based on the results of a 28-day oral study in Alpk:ApfSD, Wistar derived, rats (Rattray N J, 1999. 4-mesyl-2 nitrotoluene (NMST): 28 day oral toxicity study in rats. CTL Report Number: CTL/P/6309).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to study start, daily immediately prior to dosing throughout the study and prior to scheduled termination

BODY WEIGHT: Yes
- Time schedule for examinations: daily immediately prior to dosing throughout the study and prior to scheduled termination

FOOD CONSUMPTION AND UTILISATION: Yes
- Time schedule for examinations: recorded continuously throughout the study for each cage of rats and food consumption was calculated, at weekly intervals, as a mean value (g food/rat/day) for each cage. The food utilisation value per cage was calculated as the bodyweight gained by the rats in the cage per 100g of food eaten.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-experimentally (all rats) and in week prior to termination
- Dose groups that were examined: control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: termination
- Anaesthetic used for blood collection: Yes (halothane Ph. Eur. vapour)
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters examined: haematocrit, haemoglobin, red blood cell count, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, total white cell count, differential white cell count, platelet count, prothrombin time, activated partial thromboplastin time, blood cell morphology, blood methaemaglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: termination
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters examined: alkaline phosphatase activity, alanine aminotransferase activity, aspartate aminotransferase activity, gamma-glutamyl activity, creatine kinase activity, total bilirubin, urea, creatinine, glucose, cholesterol, triglycerides, sodium, potassium, chloride, calcium, phosphorus (as phosphate), total protein, albumin, albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: week prior to termination over a 16-18 hour period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, appearance, colour, specific gravity, pH, glucose, ketones, bilirubin, protein, blood and (if appearance was abnormal) microscopic examination of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals)

ORGAN WEIGHTS: Yes (all animals surviving to termination)
- organs weighed: adrenal glands, brain, epididymes, testes / ovaries, heart, kidneys, liver, spleen, thymus, uterus (with cervix)

HISTOPATHOLOGY: Yes (all tissues listed from controls and 100 mg/kg/day groups; macroscopic abnormalities from all groups; testis and epididymis from all treated male groups)
- tissues examined: - adrenals, aorta, brain (cerebrum, cerebellum, pons), bone marrow (femur), caecum, colon, duodenum, epididymes, eyes (retina, optic nerve), femur (including stifle joint), Harderian gland, heart, ileum, jejunum, kidney, larynx, liver, lung, lymph nodes (cervical, mesenteric), mammary gland (females only), nerve (sciatic), nose, oesophagus, ovary, oviduct, Peyer's patches, pancreas, parathyroid gland, pharynx, pituitary gland, prostate gland, rectum, salivary gland, seminal vesicle, spinal cord (cervical, thoracic, lumbar), skin, spleen, sternum, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, uterus (with cervix), voluntary muscle.

Statistics:

All data were evaluated using analysis of variance and/or analysis of covariance for each specified parameter using the MIXED procedure in SAS (SAS Institute Inc. SAS/STAT 9.1 User's Guide, Cary, NC: SAS Institute Inc, 2004).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: no deaths and no adverse clinical observations

BODY WEIGHT AND WEIGHT GAIN: no compound-related effects

FOOD CONSUMPTION AND UTILISATION: no compound-related effects

OPHTHALMOSCOPIC EXAMINATION: no compound-related effects

HAEMATOLOGY: Haemoglobin was statistically significantly lower in top dose animals from both sexes as was the red blood cell count in females. In females, at 100 mg/kg/day methaemaglobin and activated partial thromboplastin time was statistically significantly higher than controls. White blood cell count (including lymphocyte count in females only) and platelet count in top dose animals was statistically significantly higher than controls. There were a number of other statistically significant differences from control values but these showed no evidence of a dose-response and were considered to be incidental to treatment.

CLINICAL CHEMISTRY: There were a number of statistically significant differences from control: plasma calcium levels higher than controls in both sexes at the top dose, phosphorus higher than controls at 15 and 100 mg/kg/day in males only and urea higher than controls at 100 mg/kg/day in males which were considered to be compound-related but were slight and unlikely to be of toxicological significance.

URINALYSIS: Urine volume was statistically significantly higher than controls in females given 5 or 100 mg/kg/day and urine specific gravity was statistically significantly lower than controls in females at all dose levels. These differences were inconsistent between the groups, showed no evidence of a dose response and were not consistent between the sexes and are therefore considered not to be compound-related. There were no compound-related differences from control values in any other urine parameters

NEUROBEHAVIOUR: There were no clinical changes except salivation seen in 1 male and 1 female at 100 mg/kg/day. This finding is considered to be compound-related but is likely to be an autonomic response to the unpalatability of the test substance and is considered to be of no toxicological significance.

ORGAN WEIGHTS: Testis and epididymis weight in animals given 100 mg/kg/day were statistically significantly lower than controls after adjustment for terminal bodyweight. Kidney weights in both sexes given 100 mg/kg/day were statistically significantly higher than controls after adjustment for
terminal bodyweight (approximately 5 and 10% in males and females respectively). Liver weights in both sexes given 100 mg/kg/day were statistically significantly higher than controls after adjustment for terminal bodyweight (approximately 23 and 20% in males and females respectively). Adrenal weight in females at 100 mg/kg/day was statistically significantly lower than controls after adjustment for terminal bodyweight. Thymus weight in females was statistically significantly higher than controls but there was no effect after adjustment for terminal bodyweight and therefore this difference from controls is considered not to be compound-related.

GROSS PATHOLOGY: At 100 mg/kg/day, macroscopic examination showed 9/12 males had reduced epididimydes and 11/12 males had
either reduced and/or flaccid testes compared to controls. There were no other compound-related macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC: At 100 mg/kg/day compound-related microscopic finding of bilateral tubular degeneration in the testis was seen in all males and bilateral reduction of spermatozoa in the epididymis was seen in 11/12 males. At dose levels of 15 or 5 mg NMST/kg/day bilateral (1 at 5 mg/kg/day) or unilateral (2 at 5 mg/kg/day and 1 at 15 mg/kg/day) tubular degeneration in the testis was noted. All other microscopic findings are considered to be incidental to treatment.

HISTOPATHOLOGY: NEOPLASTIC: no compound-related effects

Effect levels

1

Dose descriptor:

other: No NOEL identified

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Marginal degenerative changes in the testes at 15 and 5 mg/kg . Based on clear effects at 100mg/kg a relationship to treatment could not be excluded in this study.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

2

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: slight perturbations in some haematological parameters and in blood clotting function, an increase in liver weight and some perturbation of blood clinical chemistry parameters at 100 mg/kg/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Intergroup comparison of selected haematology parameters

	Dose level of the test substance (mg/kg/day)
	Males				Females
Parameter	0	5	15	100	0	5	15	100
haemoglobin	14.7	14.6	14.6	14.0**	14.8	14.6	14.5	14.1**
red blood cell count	8.44	8.62	8.58	8.18	8.00	8.01	7.87	7.63*
methaemaglobin	0.96	1.00	0.97	1.13	0.98	0.98	1.09	1.28*
activated partial thromboplastin time	25.9	25.0	25.2	26.8	24.4	25.7*	25.6	27.7**
white blood cell count	5.89	6.06	6.32	7.04*	4.30	4.61	5.21	6.01**
lymphocyte count	4.06	4.26	4.13	4.70	3.10	3.39	3.78	4.70**
platelet count	868	890	883	1010**	917	883	902	1038**
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 2: Intergroup comparison of selected clinical chemistry parameters

	Dose level of the test substance (mg/kg/day)
	Males				Females
Parameter	0	5	15	100	0	5	15	100
plasma calcium	2.77	2.79	2.79	2.85*	2.81	2.81	2.79	2.93**
phosphorus	1.95	1.92	2.21*	2.29**	2.40	2.14*	2.21	2.40
urea	7.73	7.82	7.40	8.83**	6.87	7.40	7.27	7.36
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 3: Intergroup comparison of selected organ weights (g)

		Dose level of NMST (mg/kg/day)
		Males				Females
organ		0	5	15	100	0	5	15	100
testis	actual	3.79	3.68	3.76	1.94**	-	-	-	-
	adjusted	3.80	3.69	3.76	1.93**	-	-	-	-
epididymis	actual	1.439	1.391	1.432	0.964**	-	-	-	-
	adjusted	1.440	1.391	1.432	0.963**	-	-	-	-
kidney	actual	3.26	3.29	3.31	3.37	2.02	2.06	2.04	2.28**
	adjusted	3.24	3.26	3.33	3.41*	2.03	2.06	2.08	2.24**
liver	actual	18.3	18.8	18.5	21.6**	9.5	9.6	9.7	12.6**
	adjusted	18.2	18.6	18.6	21.9**	9.5	9.5	10.0	12.3**
adrenal	actual	0.063	0.063	0.068	0.064	0.091	0.085	0.082	0.079
	adjusted	0.063	0.062	0.068	0.064	0.091	0.085	0.084	0.078*
thymus	actual	0.499	0.539	0.501	0.523	0.422	0.418	0.395	0.481*
	adjusted	0.495	0.532	0.504	0.531	0.422	0.418	0.396	0.479
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

 

 

Applicant's summary and conclusion

Conclusions:

Oral administration of 100 mg/kg/day for 91 consecutive days resulted in degenerative changes in the testes and reduction of spermatozoa in the epididymides. There were slight perturbations in some haematological parameters and in blood clotting function, an increase in liver weight and some perturbation of blood clinical chemistry parameters. At dose levels of 15 or 5 mg/kg/day some animals showed the degenerative change in the testis. The no observed effect level (NOEL) for females in this study was 15 mg/kg/day. An NOAEL was not established for males.

Executive summary:

Groups of twelve male and twelve female Alpk:APfSD (Wistar-derived) rats were dosed orally, by gavage, with 0 (control), 5, 15 or 100 mg/kg/day in 1.0% aqueous carboxymethyl cellulose (CMC) for 91 consecutive days.   Clinical observations, bodyweights and food consumption were measured throughout the study; a functional observation battery of tests and locomotor activity monitoring were performed during week 13. An ophthalmoscopic examination was performed on all animals pre-study and control and high dose group in week 12. Urine samples collected and analysed during week 13 of the study. At the end of the scheduled period, the animals were killed and examined post mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed and specified tissues were taken for subsequent histopathological examination. Oral administration of 100 mg/kg/day for 91 consecutive days resulted in degenerative changes in the testes and reduction of spermatozoa in the epididymides. There were slight perturbations in some haematological parameters and in blood clotting function, an increase in liver weight and some perturbation of blood clinical chemistry parameters. At dose levels of 15 or 5 mg/kg/day some animals showed the degenerative change in the testis. These were minor and showed no clear dose response but given the findings at the higher dose level a relationship to treatment could not be discounted in this study. The no observed adverse effect level (NOAEL) for females in this study was 15 mg/kg/day. An NOAEL was not established for males.