4-mesyl-2-nitrotoluene

Endpoint summary

Description of key information

NOEL (male/female) =15 mg/kg bw, rat, 28 day, gavage, OECD 407, Rattray 1999
NOEL (female) =15 mg/kg bw, no NOEL (male), rat, 90 day, gavage, OECD 408, Rattray 2005

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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

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).

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).

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

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)

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

Critical effects observed:

not specified

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)

 

 

 

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February - 7 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 170-200 g (males), 139-164 g (females)
- Fasting period before study: none
- Housing: 5 per cage, sexes separately in cages suitable for rats of this strain and weight range expected during the course of the study
- Diet: CT1 ad libitum
- Water: mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes: minimum of 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 March 1999 To: 7 June 1999

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). Each preparation was thoroughly mixed and subdivided into aliquots and fresh aliquots were used for each day of the study. Dosing preparations were shaken immediately prior to dosing. They were dosed orally at 1.0 mL/100 g bodyweight according to their daily bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean achieved concentration of NMST in 1% CMC were typically within 4 or 9% of nominal (except on sample prepared on 29 March 1999 for 2 days when the 25 mg/mL preparation was 28% from nominal). Homogeneity at 1.5 and 25 mg/mL was determined on samples prepared on 29 March 1999. At 1.5% concentrations were within 2% of the overall mean but at 25 mg/mL homogeneity was poor. Homogeneity was repeated on a new preparation (31 March 1999) and was within 1% of overall mean. The chemical stability of the test substance in the dosing preparations at concentrations of 0.1, 0.5 and 100 mg/mL was satisfactory for at least 21 days but the results obtained for achieved concentrations at 0.1 and 0.5 mg/mL after 9 and 21 days were slightly elevated from those obtained at days 1 and 3. This was considered not to have adversely affected the interpretation of the data.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 (control), 15, 150, 250 mg/kg/day
Basis:
other: nominal in 1% CMC

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results obtained in a preliminary oral dosing study in rats with doses of 0, 15, 150, 250, 500 and 1000 mg/kg/day in 1% CMC.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: soon after dosing and towards end of working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to start of study, immediately prior to dosing and immediately prior to termination on day 29.

BODY WEIGHT: Yes
- Time schedule for examinations: immediately prior to dosing and immediately prior to termination on day 29

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded continuously for each cage and calculated as a weekly mean (g food/rat/day) for each cage

OPHTHALMOSCOPIC EXAMINATION: No

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.

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.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 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: day 28 following dosing and day 29 prior to termination
- 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

HISTOPATHOLOGY: Yes (all tissues listed from controls and 250 mg/kg/day groups; macroscopic abnormalities from all groups; liver and lung from all treated groups, brain from all treated female groups and testis and epididymis from all treated male groups)
- tissues examined: - adrenals, brain (cerebrum, cerebellum, pons), caecum, cervix, colon, duodenum, Peyer's patches, epididymes, femur (bone marrow), heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical, mesenteric), oesophagus, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus.

Statistics:

All data were examined using analysis of variance and covariance for each specified parameter using MIXED procedure in SAS (SAS Institute Inc. SAS/STAT Software: Changes and Enhancements through release 6.11. Cary, NC: SAS Institute INC., 1996)

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

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 treatment-related mortalities. There were a number of findings seen only after first day of dosing in both sexes at 150 and 250 mg/kg/day e.g. subdued and discharge from eye and salivation occurred sporadically in these groups.

BODY WEIGHT AND WEIGHT GAIN: At 250 mg/kg/day decreased bodyweight compared to controls of up to 11% and 6% for males and females respectively. A decrease was also seen at 150 mg/kg/day but was less marked in males. There was no effect on bodyweight at 15 mg/kg/day.

FOOD CONSUMPTION: Slightly decreased in males at 250 mg/kg/day compared to controls during weeks 3 and 4.

HAEMATOLOGY: Minimal decreases in haemoglobin, haematocrit and red blood cell count in 250 mg/kg/day females. There was also an increase in white blood cell count and most differential parameters in 3/5 females at this dose.

Prothrombin time was increased in all male treated groups and females at 150 and 250 mg/kg/day, but was considered not to be treatment-related as the change was slight and showed little evidence of a dose-response. Platelet counts were also slightly increased in both sexes at these doses.

CLINICAL CHEMISTRY: Minimal increase in plasma urea and plasma alkaline phosphatase activity in males at 250 mg/kg/day and a few other statistically significant changes but none were considered to be of any biological or toxicological significance as changes were small and no evidence of a dose-response.

URINALYSIS: A decrease in urinary volume with concomitant rise in specific gravity together with decreased urinary pH in males at 150 and 250 mg/kg/day.

NEUROBEHAVIOUR:
- clinical observations: on day 28, salivation was noted in approximately half the males and females at 150 and 250 mg/kg/day and one male had reduced splay reflex. Similar findings at day 29 but only females at 150 mg/kg/day were affected. One female at 250 mg/kg/day and one male at 150 mg/kg/day had reduced splay reflex.
- time to tail flick: no treatment-related effects.
- landing foot splay: slightly decreased in females at 150 and 250 mg/kg/day on day 28 but not day 29. No effects for females at 15 mg/kg/day and any male groups.
- fore- and hind-limb grip strength: No treatment-related effects on hind-limb grip strength. Fore-limb grip strength in females at 250 mg/kg/day slightly reduced on days 28 and 29.
- motor activity: On day 28 males at 250 mg/kg/day were generally less active than controls but on day 29 these animals showed an increase in activity compared to controls form time interval 16-20 minutes until end of observation period. No treatment-related changes were seen in females.

ORGAN WEIGHTS: Males at 150 and 250 mg/kg/day had statistically significantly reduced testis and epididymis weights. Rats of both sexes at 150 and 250 mg/kg/day had increased liver weight particularly after adjustment for terminal bodyweight. Females at 250 mg/kg/day had increased kidney weight after adjustment for terminal bodyweight.

GROSS PATHOLOGY: Treatment-related increase in incidence of reduced testis and reduced epididymis in males at 150 and 250 mg/kg/day.

HISTOPATHOLOGY: In males dosed at 250 or 150 mg/kg/day there was a treatment-related increase in sperm precursor cells in the epididymis and/or reduced sperm, together with bilateral tubular degeneration of the semeniferous tubules of the testis.

In males and females given 250 mg/kg/day there was a treatment-related increased eosinophilia of the centrilobular hepatocytes of the liver with a small increase in males at 150 mg/kg/day.

In males and females given 250 mg/kg/day, 2/5 rats showed minimal focal chronic pneumonitis of the lung and one female showed focal degeneration of the pons.

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reductions in bodyweight and histopathological changes in testis and epididymis at 150 mg/kg/day

Critical effects observed:

not specified

Table 1: Intergroup comparison of bodyweights (g) - selected timepoints; adjusted mean values shown for days 2-29)

	Dose level of NMST (mg/kg/day)
	Males				Females
day	0	15	150	250	0	15	150	250
1	184.8	182.6	181.8	191.6	150.0	151.6	151.4	151.0
2	189.3	187.5	186.2	185.2*	157.2	156.2	153.4*	154.2*
7	232.9	229.2	222.9*	222.3*	172.7	170.1	168.0*	171.6
14	284.3	281.2	271.4*	270.6*	197.7	192.1	187.8	190.8
21	331.0	320.4	312.0*	307.2**	211.7	207.0	202.3*	206.8
29	357.1	349.3	336.8	320.9**	226.9	221.1	218.3	219.8
* 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 food consumption (g/rat/day)

	Dose level of NMST (mg/kg/day)
	Males				Females
week	0	15	150	250	0	15	150	250
1	27.3	26.1	24.2	24.5	18.8	19.4	17.5	18.1
2	32.0	30.7	28.3	33.1	21.5	21.4	19.8	20.1
3	33.6	32.2	31.6	27.5	20.3	21.0	20.5	19.9
4	28.4	27.0	27.1	25.5	19.0	20.0	19.1	18.0
no statistical analysis, all group means based on 1 observation

 

Table 3: Intergroup comparison of selected haematology parameters

	Dose level of NMST (mg/kg/day)
	Males				Females
Parameter	0	15	150	250	0	15	150	250
haemoglobin	15.2	15.1	15.4	15.6	14.6	14.6	14.4	13.8**
haematocrit	0.446	0.446	0.462	0.463	0.430	0.426	0.428	0.411
red blood cell count	7.97	8.06	8.21	8.22	7.72	7.71	7.74	7.37
white blood cell count	7.06	6.29	7.50	8.12	5.05	5.89	6.42	7.29**
prothrombin time	18.1	18.9*	18.9*	19.3**	18.9	19.3	20.4**	20.4**
platelet count	873	896	1001*	996	907	919	1033*	1082**
* 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 4: Intergroup comparison of selected urinalysis parameters

	Dose level of NMST (mg/kg/day)
	Males				Females
Parameter	0	15	150	250	0	15	150	250
volume (mL)	10.88	9.00	7.70*	7.60*	4.20	3.30	7.00	6.90
specific gravity	1.033	1.035	1.041*	1.041*	1.038	1.038	1.035	1.037
pH	6.69	6.69	6.14**	5.97**	6.14	6.06	6.07	6.14

 

Table 5: Intergroup comparison of selected organ weights (g)

		Dose level of NMST (mg/kg/day)
		Males				Females
organ		0	15	150	250	0	15	150	250
testis	actual	3.25	3.08	1.78**	1.18**	-	-	-	-
	adjusted	3.19	3.06	1.79**	1.24**	-	-	-	-
epididymis	actual	0.862	0.813	0.753*	0.663**	-	-	-	-
	adjusted	0.865	0.815	0.752*	0.660**	-	-	-	-
liver	actual	16.6	15.5	17.2	17.4	9.5	9.6	11.6*	12.6**
	adjusted	15.5	15.1	17.5*	18.5**	9.3	9.6	11.8**	12.7**
kidney	actual	2.75	2.71	2.49	2.65	1.82	1.86	1.92	1.97
	adjusted	2.64	2.67	2.52	2.77	1.79	1.85	1.94	1.99*
* 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)

 

Conclusions:

Oral administration of 250 mg/kg/day of the substance for 28 consecutive days produced evidence of toxicity shown by reductions in bodyweight and marked decreases in organ weight and histopathological changes in the testis and epididymis. Minor histopathological changes were noted in the liver, lungs and brain. Histopathological changes were noted in the testis and epididymis at 150 mg/kg/day. The NOEL was 15 mg/kg/day.

Executive summary:

Groups of 5 male and 5 female Alpk:APfSD (Wistar-derived) rats were dosed orally by gavage with 0 (control), 15, 150 or 250 mg/kg/day (using 1% CMC as control substance and vehicle) for 28 consecutive days. At a dose level of 250 mg/kg/day, bodyweights were decreased and there were a few sporadic clinical changes. There were effects on some organ weights: increases in liver (both sexes) and female kidney and marked decreases in testis and epididymis. Marked histopathological changes were noted in the testis and epididymis and minor changes in liver, lung and brain. There were minor changes in some haematological parameters (females) and some urine clinical chemistry parameters (males). Changes were noted in motor activity (males) and in some functional observational battery parameters (females) but the sporadic natures of these changes did not suggest a neurotoxic effect of substance administration. The effects on testis and epididymis and some changes in haematological parameters were seen at a lower incidence and/or severity at a dose level of 150 mg/kg/day. The no observed effect level (NOEL) was 15 mg /kg/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available data is reliable and adequate for the purposes of risk assessment and classification and labelling.

System:

male reproductive system

Organ:

testes

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

Additional information

Oral Repeat-dose Studies

Subacute study

Groups of 5 male and 5 female rats were dosed orally by gavage with 0 (control), 15, 150 or 250 mg test substance/kg/day for 28 consecutive days.

At a dose level of 250 mg/kg/day, bodyweights were decreased in both sexes. Liver weight was increased in both sexes and testis and epididymis weights were reduced. Minor changes were noted in some haematological parameters (females) and some urine clinical chemistry parameters (males). Histopathological changes were evident in the testis, epididymis, and liver. Changes were noted in motor activity (males) and in some functional observational battery parameters (females).

At 150 mg/kg clear reduction in testis and epididymis weight and testicular tubular atrophy was evident.

The no observed effect level (NOEL) was 15 mg test substance/kg/day

Subchronic study

Groups of twelve male and twelve female rats were dosed orally, by gavage, with 0 (control), 5, 15 or 100 mg test substance/kg/day in for 91 consecutive days.  

Oral administration of 100 mg test substance/kg/day resulted in marked degenerative changes in the testes and reduction of spermatozoa in the epididymides. There were slight perturbations in some haematological and clinical chemistry parameters. At 15 mg/kg/day one animal showed minimal unilateral degeneration of testicular tubules and at 5 mg/kg/day 2 animals showed minimal unilateral and one animal showed minimal bilateral degeneration of testicular tubules. Due to the marked toxicity of the substance to the testes at 100 mg/kg the minor variations noted in the testes structure at 15 and 5 mg/kg could not be dismissed in this study. The no observed adverse effect level (NOAEL) for females in this study was 15 mg/kg/day. A NOEL was not established for males.

Alternative routes

It is considered that further repeat-dose testing by the dermal route is not required for the test substance. Toxicity was observed at lower doses in the acute oral toxicity test. The systemic toxicity, including target organ toxicity, has been clearly defined in the two available oral repeat-dose studies. The nature of the target organ effect means that occupational exposure thresholds and risk management measures will be set to control dermal exposure in the workplace.

It is considered that further repeat-dose testing by the inhalation route is not required for the test substance. The systemic toxicity, including target organ toxicity, has been clearly defined in the two available oral repeat-dose studies. The nature of the target organ effect means that occupational exposure thresholds will be set to control inhalation exposure in the workplace. The physical properties of the test substance as supplied suggest that it will not represent a hazard; it is minimally inhalable and almost non-respirable. After being milled twice and generated in dry air, the smallest particle size achievable was 6.06 µm. With particles of this size the majority of any inhaled dose will deposit it the head region and be swallowed, presenting an effective oral dose. In a repeat-dose study it will not be possible to use dry air, because of potential adverse effects on the respiratory tract of the animals. In this case the higher humidity will likely increase the tendency of particles to clump, thus increasing the average particle size and leading to technical difficulties in generating a suitable atmosphere.

Overall, given the low acute dermal and inhalation toxicity of the test substance, together with the fact that the systemic toxicity of the substance is well understood, further testing via dermal or inhalation routes is not justified.

Conclusions

Both 28 day (Rattray 1999) and 90 day (Rattray 2005) studies with the test substance have confirmed that the target organ is the testis. Clear reductions in organ weight and significant histopathological changes (bilateral tubular degeneration in all males) have been noted at dose levels of 100mg/kg bw/day and above.

Minor histopathological changes were seen in the testes at 15 and 5 mg/kg in the 90 day study but such changes were not seen at dose levels of 2 and 15 mg/kg in a one generation study (Milburn 2007). The dosing period for animals in the one generation study is similar to that of the 90 day study and the group size (26) is larger than that used in the 90 day study (12).

Given the minimal nature and lack of a clear dose response in the histopathological findings at 15 and 5 mg/kg in the 90 day study and a lack of reproducibility, in that no such findings were noted in the one generation reproduction study, it is concluded that the findings at 5 and 15 mg/kg in the 90 day study are likely not attributable to treatment with the test substance. A NOEL for testicular effects is concluded to be 15 mg/test substance/kg bw/day.

 

The available data is reliable and adequate for the purposes of risk assessment and classification and labelling.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Minor histopathological changes were seen in the testes at 15 and 5 mg/kg in the 90 day study (Rattray 2005) but such changes were not seen at dose levels of 2 and 15 mg/kg in a one generation study (Milburn 2007). The dosing period for animals in the one generation study is similar to that of the 90 day study and the group size (26) is larger than that used in the 90 day study (12).
Given the minimal nature and lack of a clear dose-response relationship in the histopathological findings at 15 and 5 mg/kg in the 90 day study and a lack of reproducibility, in that no such findings were noted in the one generation reproduction study, it is concluded that the findings at 5 and 15 mg/kg in the 90 day study are likely not attributable to treatment with the test substance. A NOEL for testicular effects is concluded to be 15 mg/test substance/kg bw/day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
It is considered that further repeat-dose testing by the inhalation route is not required for the test substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
It is considered that further repeat-dose testing by the inhalation route is not required for the test substance.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
It is considered that further repeat-dose testing by the dermal route is not required for the test substance.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
It is considered that further repeat-dose testing by the dermal route is not required for the test substance.

Justification for classification or non-classification

No specific or marked changes were observed under the test conditions in non-reproductive organs (see Toxicity to Reproduction) which would lead to classification.