Tetramethylammonium hydroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22JUL2015 - 11DEC2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Version / remarks:

(September 1998)

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)

Version / remarks:

(May 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(August 1998)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetramethylammonium chloride

EC Number:

200-880-8

EC Name:

Tetramethylammonium chloride

Cas Number:

75-57-0

Molecular formula:

C4H12N.Cl

IUPAC Name:

N,N,N-trimethylmethanaminium chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): TMAC – tetramethylammonium chloride
- Substance type: Organic
- Physical state: White crystalline powder
- Storage condition of test material: At room temperature protected from light
- pH: 6-8

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approx. 7 weeks old
- Weight at study initiation: 145-170 g (males); 122-149 g (females)
- Housing: Group housing of 5 animals per sex in Macrolon cages except during locomotor activity monitoring, when animals were housed individually in a Hi-temp polycarbonate cage
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except overnight (for a maximum of 24 hours) before blood samples were collected
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70 (deviations from the daily mean relative humidity occurred, however laboratory historical data did not indicate an effect of the deviations)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 10SEP2015 to 11 December 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations (w/w) were prepared daily within 5 hours prior to dosing. After confirmation of stability, formulations (w/w) were prepared weekly and were used within 8 days after preparation. The formulations were homogenized to a visually acceptable level. No correction was made for purity/composition of the test substance.


DOSE VOLUME: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). During pretest stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator in the dark was also determined (highest and lowest concentration). Long term stability in the freezer was determined over 34 days. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels are based on results of a 14-day oral range finding study with TMAC – tetramethylammonium chloride by daily gavage in the rat. Three females were exposed to either 30 or 100 mg/ kg bw/ day (5mL/kg bw/ day). Clinical signs were recorded daily, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Mortality was recorded at least twice daily. Body weighhts and food consumption were recorded weekly. All animals were subjected to an external, thoracic and abdominal examination after sacrifice.
No mortality occurred at 30 mg/kg bw/ day, at 100 mg/kg bw/ day two animals were found dead and one animal was sacrificed on day 1. The peak effect of occurrence of clinical signs occurred at 1 hour after dosing. This time point was taken into account for conduct of clinical observations and functional observation tests in the main study. No findings were recorded for clinical appearance, body weight and food consumption (only recorded for 30 mg/ kg bw/ day). No abnormalities were noted for all animals at macroscopic examination at sacrifice.

- Selection of animals for selected measurements:
Functional observations were performed on 5 animals/sex/group. Ophthalmoscopic examination were examined in all animals at pre-test, at week 13 in control and high dose group animals. Histopathology was performed on all tissues collected at the scheduled sacrifice from all animals in control and high dose groups, furthermore on all tissues from two males which died spontaneously, and on all gross lesions.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily: immediately after dosing and 1 hour (+/- 15 min) after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption was recorded weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and week 13
- Dose groups that were examined: all (at pre-test), control and high dose in week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observation battery)
- Time schedule for examinations: week 13
- Dose groups that were examined: all
- Battery of functions tested: grip strength/ motor activity /total movements and ambulations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY: Yes
According to test guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were noted in control rats. In animals at 30 mg/kg bw/day, clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis. In addition, abdominal swelling was noted at this dose level in females only. The animals at 10 mg/kg bw/day showed lethargy, hunched posture, piloerection and/or ptosis at a lower incidence and severity compared to animals at 30 mg/kg bw/day. At 3 mg/kg bw/day, hunched posture and piloerection were noted at low incidence in males only. Salivation seen after dosing among the animals at 10 and 30 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). No additional clinical signs during weekly arena observations were noted during the observation period that were considered to be related to treatment. No clinical signs were noted in the deceased rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males treated at 30 mg/kg bw/ day were found dead at day 57 and day 80, respectively (with autolysis in several organs or missing organs (head region) due to cannibalism).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated at 30 mg/kg bw/day showed lower body weight (statistically significant from week 6 to end of study, appr. -12% for both absolute and relative values at study termination compared to controls) and body weight gain (statistically significant in week 2 and from week 7 to end of study). Body weights and body weight gain of female animals remained in the same range as controls over the study period. (See table 1-4 under 'Any other information on results incl. tables')

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Absolute food consumption of males treated at 30 mg/kg bw/day was found to be lower compared to controls during the second half of the treatment period. No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females. (See table 5-8 under 'Any other information on results incl tables')

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

(See table 9 and 10 under 'Any other information on results incl. tables')

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No substance-related adverse effects were seen in heamatological parameters for exposed males compared to control group. A statistically significant decrease of prothrombin time in males dosed at 3 mg/kg bw/day was considered to be an incidental effect and not to be substance-related. In high dose females, the percentage lymphocytes was found to be decreased (-5.5%), with an increased percentage of monocytes (+29%), both effects were statistically significant. In absence of a clear dose-relationship of the effects, this was not found to be adverse. (See table 11 and 12 under 'Any other information on results incl. tables')

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males exposed to 30 mg/kg bw/day showed statistically significant increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels (respectively +67% and +20% compared to controls). ALAT levels were also increased in high dose females (+86%), and found together with increased alkaline phosphatase levels (+82%, both statistically significant).
Furthermore, concentration bilirubin was increased in high dose males (+23%) and females (+19%). Glucose levels were decreased in high dose males (-13%). A dose-related increase in bile acids was observed for all exposed males (+50%, +150% and +719% for males dosed at 3, 10 and 30 mg/kg bw/day, respectively, statistically significant only at highest dose). This increase was also seen in females with +38%, +83% and +1560% at respectively 3, 10 and 30 mg/kg bw/day (statistically significant only at highest dose). Furthermore, effects on ionic balance were noted in males (increased sodium levels at 3 and 10 mg/kg bw/day, dose related increased potassium (only statistically signficant at 30 mg/kg bw/day) and increased inorganic phophate levels at 30 mg/kg bw/ day) and females (increased potassium at highest dose). In high dose females, also effects were noted on total protein and albumin (both decreased with -11%, statistically significant effect). Cholesterol levels increased for all exposed females (+5%, +5% and +24% (statistically significant only at high dose). (See table 13 and 14 under 'Any other information on results incl. tables')

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements (-73.5%) and ambulations (-83%), no such effect was seen in males. For males, grip strenth was decreased for all exposed rats compared to control group (appr. -23% and -21%, -27% and -17%, -29% and -20% for respectively fore and hind grip at 3, 10 and 30 mg/kg bw/day), however these effects were not statistically significant. Grip strenth was not affected in females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females dosed at 30 mg/kg bw/day were found to have increased liver weights (appr. +23% and +25% for absolute and relative values respectively, both statistically significant). In parallel, relative liver weight was also slightly increased in males in the highest dose group (+7%, statistically significant). Lower thymus weights were noted in females (18% for absolute and 17% for relative weight, both statistically significant) at 30 mg/kg bw/day.
Other statistically significant changes in organ weights at the end of the treatment were considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain or were in males considered to be the result of a test item-related effect on final body weight. These observations included effects on relative testes weights which were affected in a dose related manner compared to control males with +3%, +11% and +13% respectively for rats exposed to 3, 10 and 30 mg/kg bw/day (statistically significant only for two highest dose groups). Further observations included decreased weight of the brain in males only at 30 mg/kg bw/day (appr. -4% for absolute and +7.5% relative to body weight, both statistically significant). Relative kidneys weights were slightly increased for males in the highest dose group (+11%, statistically significant). At 10 mg/kg bw/ day, absolute and relative ovary weights were increased, but in absence of dose-relationship this was found to be not substance-related. No other findings were noted in males and females. (See table 15-18 under 'Any other information on results incl. tables')

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Emaciation was noted in one female at 30 mg/kg bw/day at macroscopic examination. Swelling of the abdomen was noted for several rats. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the two animals deceased before scheduled sacrifice was only possible to a limited agree due to the status. Findings consisted of many dark red foci of the thymus (correlated with congestion/hemorrhage) and many dark red foci of the thymus in one of the two rats. Therefore conclusion on cause of pre-scheduled deaths was not possible.
In all exposed females microscopic findings were noted in the liver, consisting of hepatocellular hypertrophy at minimal degree. The effects were found in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity (minimal) and in absence of effects in other parameters this observation was considered to be non-adverse at 3 and 10 mg/kg bw/day. In the thymus of 2/10 females at 30 mg/kg bw/day atrophy at minimal degree was observed. Based on the low severity, this effect was found to be non-adverse. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. (See table 19-21 under 'Any other information on results incl. tables')

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

No test substance was detected in the control group formulations. The concentrations analyzed in the formulations with the test substance were in agreement with the target concentrations (i.e. mean accuracies between 95.1% and 96.5%). The formulations of high and low dose group were prepared homogeneous (i.e. coefficient of variation ≤ 1.8%). Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 hours and for 8 days at room temperature (i.e. relative difference ≤ 3.2%). The long term storage samples were stable at ≤-70°C for 34 days.

 

 

SUMMARY TABLES

Table 1. Body weights (gram) summary (males)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAY 1	MEAN	157	156	157	156
WEEK 1	ST.DEV	6.1	5.8	7.7	5.9
	N	10	10	10	10
DAY 8	MEAN	201	200	201	193
WEEK 2	ST.DEV	7.1	6.8	9.7	6.9
	N	10	10	10	10
DAY 15	MEAN	244	240	245	234
WEEK 3	ST.DEV	7.9	10.9	11.0	11.8
	N	10	10	10	10
DAY 22	MEAN	275	273	279	265
WEEK 4	ST.DEV	9.2	12.0	12.5	14.8
	N	10	10	10	10
DAY 29	MEAN	300	299	306	288
WEEK 5	ST.DEV	11.0	15.4	11.8	19.7
	N	10	10	10	10
DAY 36	MEAN	322	320	327	302 *
WEEK 6	ST.DEV	14.7	16.3	13.5	25.3
	N	10	10	10	10
DAY 43	MEAN	340	337	344	314 *
WEEK 7	ST.DEV	17.0	18.2	17.3	27.8
	N	10	10	10	10
DAY 50	MEAN	354	349	356	323 *
WEEK 8	ST.DEV	16.8	20.0	19.4	32.0
	N	10	10	10	10
DAY 57	MEAN	367	362	367	330 **
WEEK 9	ST.DEV	17.4	22.3	20.5	33.4
	N	10	10	10	10
DAY 64	MEAN	379	371	374	341 **
WEEK 10	ST.DEV	19.7	24.8	20.0	34.6
	N	10	10	10	9
DAY 71	MEAN	385	379	380	347 **
WEEK 11	ST.DEV	19.1	25.9	19.7	32.9
	N	10	10	10	9
DAY 78	MEAN	396	385	389	353 **
WEEK 12	ST.DEV	19.7	28.0	20.7	33.4
	N	10	10	10	9
DAY 85	MEAN	405	394	396	363 **
WEEK 13	ST.DEV	19.3	27.4	21.8	34.7
	N	10	10	10	8
DAY 91	MEAN	410	399	403	367 **
WEEK 13	ST.DEV	19.4	30.4	20.4	33.2
	N	10	10	10	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 2. Body weigths (gram) summary (Females)

TREATMENT		GROUP 1 CONTROL	GROUP 2  3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAY 1	MEAN	136	132	131	134
WEEK 1	ST.DEV	7.1	6.2	6.8	5.8
	N	10	10	10	10
DAY 8	MEAN	155	152	149	154
WEEK 2	ST.DEV	9.2	8.6	6.3	4.6
	N	10	10	10	10
DAY 15	MEAN	171	171	167	174
WEEK 3	ST.DEV	10.9	8.7	9.1	5.8
	N	10	10	10	10
DAY 22	MEAN	188	184	181	189
WEEK 4	ST.DEV	10.9	11.7	12.9	8.0
	N	10	10	10	10
DAY 29	MEAN	202	192	194	203
WEEK 5	ST.DEV	11.8	11.2	13.1	7.4
	N	10	10	10	10
DAY 36	MEAN	211	201	202	208
WEEK 6	ST.DEV	12.1	11.5	12.0	6.4
	N	10	10	10	10
DAY 43	MEAN	218	211	212	217
WEEK 7	ST.DEV	11.9	13.0	11.2	7.8
	N	10	10	10	10
DAY 50	MEAN	224	215	217	224
WEEK 8	ST.DEV	12.2	15.1	15.4	10.7
	N	10	10	10	10
DAY 57	MEAN	231	220	225	231
WEEK 9	ST.DEV	12.0	13.8	15.0	8.1
	N	10	10	10	10
DAY 64	MEAN	234	225	226	234
WEEK 10	ST.DEV	11.1	11.8	13.3	8.3
	N	10	10	10	10
DAY 71	MEAN	233	227	229	234
WEEK 11	ST.DEV	12.7	14.9	13.2	8.7
	N	10	10	10	10
DAY 78	MEAN	238	230	233	240
WEEK 12	ST.DEV	11.8	15.9	17.3	11.7
	N	10	10	10	10
DAY 85	MEAN	242	233	239	243
WEEK 13	ST.DEV	12.0	12.2	17.7	10.0
	N	10	10	10	10
DAY 91	MEAN	242	239	241	242
WEEK 13	ST.DEV	13.3	12.9	15.9	10.2
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3. Body weight gain (%) summary (males)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0.0	0.0	0.0	0.0
	N	10	10	10	10
DAY 8	MEAN	28	28	28	24 **
WEEK 2	ST.DEV	1.8	2.1	1.2	3.3
	N	10	10	10	10
DAY 15	MEAN	55	54	56	50
WEEK 3	ST.DEV	3.2	4.9	2.8	8.3
	N	10	10	10	10
DAY 22	MEAN	75	75	78	70
WEEK 4	ST.DEV	5.6	6.5	3.8	9.3
	N	10	10	10	10
DAY 29	MEAN	91	91	95	84
WEEK 5	ST.DEV	7.1	8.4	5.1	11.4
	N	10	10	10	10
DAY 36	MEAN	105	105	109	93
WEEK 6	ST.DEV	8.9	9.8	6.3	16.2
	N	10	10	10	10
DAY 43	MEAN	116	116	119	101 *
WEEK 7	ST.DEV	10.6	10.7	6.5	17.5
	N	10	10	10	10
DAY 50	MEAN	125	123	127	107 *
WEEK 8	ST.DEV	10.7	11.4	8.3	20.4
	N	10	10	10	10
DAY 57	MEAN	133	132	134	112 **
WEEK 9	ST.DEV	11.9	12.9	9.8	21.4
	N	10	10	10	10
DAY 64	MEAN	141	138	139	120 *
WEEK 10	ST.DEV	13.4	14.1	9.9	21.1
	N	10	10	10	9
DAY 71	MEAN	145	142	143	124 **
WEEK 11	ST.DEV	13.4	14.8	10.1	20.1
	N	10	10	10	9
DAY 78	MEAN	152	147	148	127 **
WEEK 12	ST.DEV	14.3	15.6	10.9	20.2
	N	10	10	10	9
DAY 85	MEAN	158	152	153	133 **
WEEK 13	ST.DEV	14.2	15.3	12.6	20.7
	N	10	10	10	8
DAY 91	MEAN	161	155	157	136 **
WEEK 13	ST.DEV	14.1	17.4	12.8	20.8
	N	10	10	10	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 4. Body weight gain (%) summary (females)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0.0	0.0	0.0	0.0
	N	10	10	10	10
DAY 8	MEAN	14	16	14	15
WEEK 2	ST.DEV	2.7	3.6	5.1	2.7
	N	10	10	10	10
DAY 15	MEAN	26	30	28	30
WEEK 3	ST.DEV	4.9	5.1	4.0	3.9
	N	10	10	10	10
DAY 22	MEAN	38	40	39	41
WEEK 4	ST.DEV	4.0	8.6	6.7	5.6
	N	10	10	10	10
DAY 29	MEAN	49	46	49	51
WEEK 5	ST.DEV	4.8	7.5	7.5	6.2
	N	10	10	10	10
DAY 36	MEAN	56	53	54	55
WEEK 6	ST.DEV	4.9	6.8	9.2	7.9
	N	10	10	10	10
DAY 43	MEAN	61	60	63	62
WEEK 7	ST.DEV	6.5	9.7	6.1	7.5
	N	10	10	10	10
DAY 50	MEAN	65	63	66	67
WEEK 8	ST.DEV	6.6	12.1	8.9	9.5
	N	10	10	10	10
DAY 57	MEAN	70	68	72	73
WEEK 9	ST.DEV	6.6	10.3	9.3	9.4
	N	10	10	10	10
DAY 64	MEAN	72	71	73	75
WEEK 10	ST.DEV	6.9	8.6	10.9	9.9
	N	10	10	10	10
DAY 71	MEAN	72	73	75	75
WEEK 11	ST.DEV	8.8	12.2	8.9	10.2
	N	10	10	10	10
DAY 78	MEAN	75	75	78	80
WEEK 12	ST.DEV	7.4	12.2	11.5	10.3
	N	10	10	10	10
DAY 85	MEAN	78	78	83	82
WEEK 13	ST.DEV	7.6	8.9	11.3	10.9
	N	10	10	10	10
DAY 91	MEAN	78	82	84	81
WEEK 13	ST.DEV	9.6	9.2	10.2	10.9
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. Food consumption (g/animal/day) summary (males)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAYS 1-8	MEAN	20	20	20	17
WEEKS 1-2	ST.DEV	0.2	0.2	0.2	0.5
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	22	22	23	21
WEEKS 2-3	ST.DEV	0.8	0.3	0.4	1.4
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	24	24	25	23
WEEKS 3-4	ST.DEV	0.3	0.2	0.5	0.0
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	24	24	25	24
WEEKS 4-5	ST.DEV	0.2	0.0	0.3	1.0
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	24	24	26	23
WEEKS 5-6	ST.DEV	0.5	0.5	0.2	0.6
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	23	23	26	22
WEEKS 6-7	ST.DEV	0.7	0.4	0.0	0.1
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	24	23	25	21
WEEKS 7-8	ST.DEV	1.0	0.3	0.5	1.1
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	23	22	23	20
WEEKS 8-9	ST.DEV	0.9	0.2	1.0	1.3
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	24	23	24	21
WEEKS 9-10	ST.DEV	1.0	0.1	0.4	1.7
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	22	22	23	20
WEEKS 10-11	ST.DEV	0.8	0.1	0.6	1.8
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	23	23	24	22
WEEKS 11-12	ST.DEV	0.8	0.2	1.0	1.1
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	23	23	24	20
WEEKS 12-13	ST.DEV	0.8	0.1	1.3	2.4
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	24	23	24	22
WEEK 13	ST.DEV	0.7	0.6	0.5	0.5
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	23	23	24	21

 

Table 6. Food consumption (g/animal/day) summary (females)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAYS 1-8	MEAN	15	15	14	13
WEEKS 1-2	ST.DEV	0.3	1.1	0.0	0.3
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	16	15	15	15
WEEKS 2-3	ST.DEV	0.4	1.2	0.0	0.2
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	17	17	16	16
WEEKS 3-4	ST.DEV	0.6	1.5	0.1	0.0
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	18	17	17	17
WEEKS 4-5	ST.DEV	0.3	1.5	0.2	0.4
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	18	17	17	16
WEEKS 5-6	ST.DEV	0.0	1.6	0.2	0.8
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	17	17	16	15
WEEKS 6-7	ST.DEV	0.0	1.9	0.0	0.1
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	17	17	17	17
WEEKS 7-8	ST.DEV	0.4	1.9	0.3	1.0
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	17	16	16	15
WEEKS 8-9	ST.DEV	0.1	1.4	0.6	0.6
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	17	17	17	17
WEEKS 9-10	ST.DEV	0.1	1.1	0.4	0.8
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	16	16	16	16
WEEKS 10-11	ST.DEV	0.2	1.8	0.5	0.4
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	17	17	16	18
WEEKS 11-12	ST.DEV	0.0	1.3	0.6	1.5
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	17	17	17	16
WEEKS 12-13	ST.DEV	0.5	1.2	1.0	0.9
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	17	17	17	17
WEEK 13	ST.DEV	1.0	1.2	0.7	0.7
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	17	16	16	16

 

Table 7. Relative food consumption (g/kg body weight/day) summary (males)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAYS 1-8	MEAN	100	100	102	90
WEEKS 1-2	ST.DEV	0.9	1.0	1.1	2.6
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	91	92	93	88
WEEKS 2-3	ST.DEV	3.5	0.6	1.6	3.9
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	86	86	89	88
WEEKS 3-4	ST.DEV	1.3	0.5	2.7	0.7
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	80	80	83	82
WEEKS 4-5	ST.DEV	0.8	1.0	1.7	4.1
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	75	74	79	75
WEEKS 5-6	ST.DEV	1.6	1.9	1.0	0.6
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	69	69	75	70
WEEKS 6-7	ST.DEV	2.3	1.4	0.5	1.4
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	66	67	71	66
WEEKS 7-8	ST.DEV	2.8	1.0	2.2	1.4
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	62	62	63	61
WEEKS 8-9	ST.DEV	2.9	0.0	3.2	2.3
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	62	62	63	60
WEEKS 9-10	ST.DEV	3.0	0.4	1.8	4.8
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	58	59	60	59
WEEKS 10-11	ST.DEV	2.4	0.4	2.0	4.9
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	59	59	61	62
WEEKS 11-12	ST.DEV	2.5	0.1	2.8	2.4
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	58	58	59	56
WEEKS 12-13	ST.DEV	2.6	0.7	3.1	6.7
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	58	58	60	59
WEEK 13	ST.DEV	2.3	1.8	0.9	1.4
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	71	71	74	70

 

Table 8. Relative food consumption (g/kg body weight/day) summary (females)

TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
DAYS 1-8	MEAN	98	99	97	86
WEEKS 1-2	ST.DEV	0.7	5.8	0.4	0.9
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	92	91	91	87
WEEKS 2-3	ST.DEV	1.8	4.0	0.7	1.2
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	92	90	89	87
WEEKS 3-4	ST.DEV	0.9	4.2	0.2	0.2
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	87	88	86	84
WEEKS 4-5	ST.DEV	0.7	3.1	1.4	1.6
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	83	84	83	79
WEEKS 5-6	ST.DEV	0.8	4.5	0.3	3.0
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	78	79	78	71
WEEKS 6-7	ST.DEV	0.3	4.8	0.3	0.3
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	78	77	77	74
WEEKS 7-8	ST.DEV	0.4	4.3	1.1	4.2
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	72	74	72	66
WEEKS 8-9	ST.DEV	1.1	1.6	3.1	2.2
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	74	76	73	73
WEEKS 9-10	ST.DEV	0.3	1.2	1.8	3.0
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	70	69	69	67
WEEKS 10-11	ST.DEV	0.7	3.4	2.0	1.1
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	72	72	71	74
WEEKS 11-12	ST.DEV	0.6	1.7	1.9	6.3
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	71	72	72	67
WEEKS 12-13	ST.DEV	1.9	1.4	2.9	3.4
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	70	72	69	70
WEEK 13	ST.DEV	3.6	1.8	2.2	2.8
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	80	80	79	76

 

Table 9. Ophthalmoscopic examinations summary (males)

	GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
PRETEST
No findings	6/10	10/10	7/10	6/10
Focal Corneal Edema	3/10	0/10	1/10	2/10
Focal Corneal Opacity	3/10	0/10	3/10	3/10
Pinpoint Corneal Opacities	1/10	0/10	0/10	0/10
END OF TREATMENT
No Findings	10/10			8/8

 

Table 10. Ophthalmoscopic examinations summary (females) 

	GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
PRETEST
No Findings	8/10	8/10	8/10	10/10
Focal Corneal Edema	0/10	0/10	2/10	0/10
Focal Corneal Opacity	1/10	1/10	1/10	0/10
Haemorrhage From Hyaloid Vessel	1/10	1/10	0/10	0/10
END OF TREATMENT
No Findings	10/10			10/10

 

Table 11. Haematology summary (males) 

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
WBC	MEAN	7.1	7.3	6.5	8.6
10E9/L	ST.DEV	1.5	2.0	1.2	1.4
	N	9	10	8	7
Neutrophils	MEAN	16.5	17.6	16.3	19.4
%WBC	ST.DEV	3.7	4.0	4.4	3.3
	N	9	10	8	7
Lymphocytes	MEAN	80.2	79.2	80.5	76.7
%WBC	ST.DEV	4.0	4.6	4.6	3.1
	N	9	10	8	7
Monocytes	MEAN	1.8	1.5	1.5	1.6
%WBC	ST.DEV	0.7	0.4	0.3	0.5
	N	9	10	8	7
Eosinophils	MEAN	1.3	1.6	1.5	2.1
%WBC	ST.DEV	0.5	0.6	0.5	0.9
	N	9	10	8	7
Basophils	MEAN	0.2	0.2	0.2	0.2
%WBC	ST.DEV	0.1	0.1	0.1	0.1
	N	9	10	8	7
Red blood cells	MEAN	8.97	8.65	8.67	9.01
10E12/L	ST.DEV	0.63	0.40	0.51	0.15
	N	10	10	9	8
Reticulocytes	MEAN	1.9	1.7	1.8	1.7
%RBC	ST.DEV	0.3	0.2	0.4	0.3
	N	10	10	9	8
RDW	MEAN	12.7	13.3	13.3	12.1
%	ST.DEV	1.2	1.2	1.4	0.5
	N	10	10	9	8
Haemoglobin	MEAN	9.9	9.8	9.8	10.0
mmol/L	ST.DEV	0.4	0.2	0.4	0.2
	N	10	10	9	8
Haematocrit	MEAN	0.455	0.446	0.450	0.463
L/L	ST.DEV	0.023	0.012	0.020	0.010
	N	10	10	9	8
MCV	MEAN	50.8	51.6	51.9	51.3
fL	ST.DEV	2.1	1.7	1.4	1.2
	N	10	10	9	8
MCH	MEAN	1.10	1.13	1.13	1.10
fmol	ST.DEV	0.06	0.06	0.05	0.02
	N	10	10	9	8
MCHC	MEAN	21.69	21.98	21.71	21.52
mmol/L	ST.DEV	0.44	0.54	0.55	0.21
	N	10	10	9	8
Platelets	MEAN	757	710	685	753
10E9/L	ST.DEV	76	84	61	83
	N	10	10	9	8
PT	MEAN	17.0	16.3 *	16.4	17.2
s	ST.DEV	0.5	0.5	0.7	0.8
	N	10	10	10	8
APTT	MEAN	18.4	17.3	17.1	16.8
s	ST.DEV	1.5	2.2	3.1	2.5
	N	10	10	10	8

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 12. Haematology summary (females)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
WBC	MEAN	4.8	3.7	4.8	5.5
10E9/L	ST.DEV	2.1	0.6	1.0	1.1
	N	10	10	9	9
Neutrophils	MEAN	12.3	16.9	13.5	15.9
%WBC	ST.DEV	3.6	5.2	3.4	3.7
	N	10	8	9	9
Lymphocytes	MEAN	84.3	79.5	82.7	79.7 +
%WBC	ST.DEV	4.0	5.6	3.4	3.3
	N	10	8	9	9
Monocytes	MEAN	1.7	2.1	1.6	2.2 +
%WBC	ST.DEV	0.5	0.7	0.4	0.3
	N	10	8	9	9
Eosinophils	MEAN	1.5	1.4	2.0	2.0
%WBC	ST.DEV	0.7	0.2	0.8	0.5
	N	10	8	9	9
Basophils	MEAN	0.2	0.2	0.1	0.2
%WBC	ST.DEV	0.1	0.1	0.1	0.1
	N	10	8	9	9
Red blood cells	MEAN	7.90	7.93	7.91	8.01
10E12/L	ST.DEV	0.35	0.46	0.34	0.34
	N	10	10	10	9
Reticulocytes	MEAN	2.5	2.5	2.2	2.2
%RBC	ST.DEV	0.4	0.4	0.4	0.4
	N	10	10	10	9
RDW	MEAN	13.2	12.7	11.3	11.4
%	ST.DEV	4.7	3.8	0.6	0.5
	N	10	10	10	9
Haemoglobin	MEAN	9.1	9.0	9.1	9.2
mmol/L	ST.DEV	0.3	0.4	0.3	0.3
	N	10	10	10	9
Haematocrit	MEAN	0.420	0.421	0.425	0.428
L/L	ST.DEV	0.019	0.017	0.019	0.014
	N	10	10	10	9
MCV	MEAN	53.1	53.1	53.6	53.3
fL	ST.DEV	1.3	1.2	1.2	0.9
	N	10	10	10	9
MCH	MEAN	1.15	1.14	1.15	1.15
fmol	ST.DEV	0.04	0.03	0.03	0.03
	N	10	10	10	9
MCHC	MEAN	21.70	21.52	21.51	21.49
mmol/L	ST.DEV	0.49	0.30	0.45	0.34
	N	10	10	10	9
Platelets	MEAN	751	712	763	817
10E9/L	ST.DEV	105	124	73	102
	N	10	10	10	9
PT	MEAN	16.3	15.8	15.9	16.1
s	ST.DEV	0.4	0.5	0.8	0.6
	N	9	10	10	9
APTT	MEAN	16.8	16.1	16.7	15.4
s	ST.DEV	1.7	1.6	2.4	1.9
	N	9	10	10	9

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 13. Clinical biochemistry summary (males) 

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
ALAT	MEAN	46.0	46.6	47.7	77.1 **
U/L	ST.DEV	9.4	13.0	9.9	26.4
	N	10	10	10	8
ASAT	MEAN	76.3	78.3	78.5	91.6 *
U/L	ST.DEV	8.2	9.8	8.8	17.1
	N	10	10	10	8
ALP	MEAN	127	130	120	188 *
U/L	ST.DEV	52	42	35	68
	N	10	10	10	8
Total protein	MEAN	64.3	63.4	62.3	62.6
g/L	ST.DEV	2.1	2.8	2.1	3.0
	N	10	10	10	8
Albumin	MEAN	31.4	31.5	31.2	31.4
g/L	ST.DEV	1.1	1.2	0.8	0.9
	N	10	10	10	8
Total bilirubin	MEAN	2.1	2.1	2.4	2.6 *
umol/L	ST.DEV	0.3	0.3	0.3	0.4
	N	10	10	10	8
Urea	MEAN	6.0	6.2	5.9	6.9
mmol/L	ST.DEV	1.0	1.1	1.1	1.6
	N	10	10	10	8
Creatinine	MEAN	37.4	39.0	37.5	37.9
umol/L	ST.DEV	0.8	1.9	0.8	2.5
	N	10	10	10	8
Glucose	MEAN	9.10	9.01	9.05	7.96 *
mmol/L	ST.DEV	1.18	0.71	0.85	0.76
	N	10	10	10	8
Cholesterol	MEAN	2.05	2.06	1.77	2.00
mmol/L	ST.DEV	0.44	0.43	0.28	0.30
	N	10	10	10	8
Bile Acids	MEAN	19.2	28.4	47.5	157.2 **
umol/L	ST.DEV	7.4	13.4	25.8	95.4
	N	10	10	10	8
Sodium	MEAN	142.5	143.7 **	144.4 **	142.7
mmol/L	ST.DEV	0.7	0.8	0.6	0.9
	N	10	10	10	8
Potassium	MEAN	3.85	3.90	3.99	4.15 **
mmol/L	ST.DEV	0.13	0.20	0.15	0.20
	N	10	10	10	8
Chloride	MEAN	104	105	105 *	104
mmol/L	ST.DEV	1	1	1	1
	N	10	10	10	8
Calcium	MEAN	2.56	2.54	2.54	2.60
mmol/L	ST.DEV	0.03	0.06	0.05	0.03
	N	10	10	10	8
Inorg.Phos	MEAN	1.78	1.62	1.75	2.00 *
mmol/L	ST.DEV	0.11	0.14	0.14	0.21
	N	10	10	10	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 14. Clinical biochemistry summary (females)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
ALAT	MEAN	50.4	47.8	46.5	93.2 **
U/L	ST.DEV	18.4	13.8	13.3	22.0
	N	10	10	10	10
ASAT	MEAN	88.3	78.7	79.5	85.9
U/L	ST.DEV	26.8	9.7	7.2	11.9
	N	10	10	10	10
ALP	MEAN	57	51	77	104 **
U/L	ST.DEV	22	18	31	50
	N	10	10	10	10
Total protein	MEAN	68.6	67.2	65.8	60.8 **
g/L	ST.DEV	3.2	4.7	3.1	3.1
	N	10	10	10	10
Albumin	MEAN	36.2	35.0	34.8	31.9 **
g/L	ST.DEV	1.9	2.5	2.1	1.6
	N	10	10	10	10
Total bilirubin	MEAN	2.6	2.4	2.6	3.1 **
umol/L	ST.DEV	0.3	0.6	0.3	0.4
	N	10	10	10	10
Urea	MEAN	7.6	7.1	7.3	8.6
mmol/L	ST.DEV	1.0	1.2	1.6	1.2
	N	10	10	10	10
Creatinine	MEAN	43.0	41.5	42.8	41.9
umol/L	ST.DEV	2.6	4.1	4.5	3.1
	N	10	10	10	10
Glucose	MEAN	7.61	7.28	7.69	7.69
mmol/L	ST.DEV	1.00	0.70	0.94	0.73
	N	10	10	10	10
Cholesterol	MEAN	1.52	1.66	1.61	1.88 *
mmol/L	ST.DEV	0.16	0.33	0.32	0.26
	N	10	10	10	10
Bile Acids	MEAN	24.2	33.2	43.9	400.0 **
umol/L	ST.DEV	12.2	18.4	17.7	148.0
	N	10	10	10	10
Sodium	MEAN	141.1	141.7	141.6	140.6
mmol/L	ST.DEV	1.8	1.3	1.2	1.3
	N	10	10	10	10
Potassium	MEAN	3.55	3.45	3.69	3.95 **
mmol/L	ST.DEV	0.17	0.17	0.32	0.25
	N	10	10	10	10
Chloride	MEAN	104	105 *	105 *	104
mmol/L	ST.DEV	2	1	1	2
	N	10	10	10	10
Calcium	MEAN	2.66	2.61	2.60	2.62
mmol/L	ST.DEV	0.06	0.09	0.07	0.09
	N	10	10	10	10
Inorg.Phos	MEAN	1.54	1.53	1.68	1.76
mmol/L	ST.DEV	0.18	0.23	0.21	0.33
	N	10	10	10	10

 

Table 15. Organ weights (gram) summary (males)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
BODY W.	MEAN	387	373	374	342 **
(GRAM)	ST.DEV	21	28	20	32
	N	10	10	10	8
BRAIN	MEAN	2.03	1.98	2.01	1.95 *
(GRAM)	ST.DEV	0.06	0.04	0.06	0.06
	N	10	10	10	8
HEART	MEAN	0.993	0.972	0.989	0.918
(GRAM)	ST.DEV	0.057	0.084	0.070	0.095
	N	10	10	10	8
LIVER	MEAN	9.02	8.60	8.69	8.53
(GRAM)	ST.DEV	0.89	0.83	0.57	0.79
	N	10	10	10	8
THYROIDS	MEAN	0.017	0.015	0.017	0.014
(GRAM)	ST.DEV	0.001	0.003	0.003	0.002
	N	10	10	10	8
THYMUS	MEAN	0.332	0.330	0.317	0.273
(GRAM)	ST.DEV	0.063	0.069	0.067	0.032
	N	10	10	10	8
KIDNEYS	MEAN	2.37	2.31	2.35	2.31
(GRAM)	ST.DEV	0.25	0.23	0.21	0.22
	N	10	10	10	8
ADRENALS	MEAN	0.053	0.054	0.050	0.051
(GRAM)	ST.DEV	0.007	0.004	0.008	0.007
	N	10	10	10	8
SPLEEN	MEAN	0.590	0.577	0.547	0.546
(GRAM)	ST.DEV	0.091	0.068	0.114	0.077
	N	10	10	10	8
TESTES	MEAN	3.47	3.48	3.75	3.48
(GRAM)	ST.DEV	0.26	0.24	0.32	0.27
	N	10	10	10	8
PROSTATE	MEAN	0.621	0.667	0.586	0.517
(GRAM)	ST.DEV	0.111	0.152	0.119	0.075
	N	10	10	10	8
EPIDIDYMIDES	MEAN	1.204	1.163	1.207	1.144
(GRAM)	ST.DEV	0.093	0.082	0.091	0.032
	N	10	10	10	8
SEMINAL VES	MEAN	1.529	1.459	1.657	1.343
(GRAM)	ST.DEV	0.208	0.159	0.218	0.145
	N	10	10	10	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 16. Organ/body weight ratios (%) summary (males)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
BODY W.	MEAN	387	373	374	342 **
(GRAM)	ST.DEV	21	28	20	32
	N	10	10	10	8
BRAIN	MEAN	0.53	0.53	0.54	0.57 *
(%)	ST.DEV	0.03	0.03	0.02	0.04
	N	10	10	10	8
HEART	MEAN	0.257	0.261	0.265	0.269
(%)	ST.DEV	0.013	0.017	0.022	0.025
	N	10	10	10	8
LIVER	MEAN	2.33	2.30	2.32	2.50 *
(%)	ST.DEV	0.15	0.15	0.12	0.16
	N	10	10	10	8
THYROIDS	MEAN	0.004	0.004	0.005	0.004
(%)	ST.DEV	0.000	0.001	0.001	0.001
	N	10	10	10	8
THYMUS	MEAN	0.086	0.088	0.085	0.080
(%)	ST.DEV	0.017	0.015	0.017	0.005
	N	10	10	10	8
KIDNEYS	MEAN	0.61	0.62	0.63	0.68 *
(%)	ST.DEV	0.05	0.05	0.06	0.04
	N	10	10	10	8
ADRENALS	MEAN	0.014	0.015	0.013	0.015
(%)	ST.DEV	0.002	0.001	0.002	0.002
	N	10	10	10	8
SPLEEN	MEAN	0.153	0.155	0.146	0.160
(%)	ST.DEV	0.022	0.012	0.028	0.013
	N	10	10	10	8
TESTES	MEAN	0.90	0.93	1.00 **	1.02 **
(%)	ST.DEV	0.07	0.07	0.07	0.08
	N	10	10	10	8
PROSTATE	MEAN	0.161	0.179	0.156	0.152
(%)	ST.DEV	0.031	0.037	0.029	0.026
	N	10	10	10	8
EPIDIDYMIDES	MEAN	0.312	0.313	0.323	0.337
(%)	ST.DEV	0.033	0.031	0.025	0.031
	N	10	10	10	8
SEMINAL VES	MEAN	0.396	0.394	0.443	0.398
(%)	ST.DEV	0.046	0.059	0.055	0.071
	N	10	10	10	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 17. Organ weights (gram) summary (females)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
BODY W.	MEAN	230	222	224	226
(GRAM)	ST.DEV	11	13	16	9
	N	10	10	10	10
BRAIN	MEAN	1.90	1.85	1.89	1.85
(GRAM)	ST.DEV	0.07	0.05	0.07	0.07
	N	9	10	9	10
HEART	MEAN	0.729	0.703	0.703	0.691
(GRAM)	ST.DEV	0.049	0.035	0.045	0.037
	N	10	10	10	10
LIVER	MEAN	5.75	5.63	6.09	7.07 **
(GRAM)	ST.DEV	0.42	0.40	0.60	0.94
	N	10	10	10	10
THYROIDS	MEAN	0.014	0.013	0.014	0.013
(GRAM)	ST.DEV	0.003	0.003	0.002	0.003
	N	10	10	9	10
THYMUS	MEAN	0.303	0.301	0.320	0.248 *
(GRAM)	ST.DEV	0.035	0.054	0.047	0.043
	N	10	10	10	10
KIDNEYS	MEAN	1.57	1.56	1.62	1.61
(GRAM)	ST.DEV	0.09	0.06	0.13	0.11
	N	10	10	10	10
ADRENALS	MEAN	0.064	0.060	0.064	0.060
(GRAM)	ST.DEV	0.009	0.007	0.010	0.008
	N	10	10	10	10
SPLEEN	MEAN	0.412	0.424	0.423	0.430
(GRAM)	ST.DEV	0.061	0.047	0.053	0.062
	N	10	10	10	10
OVARIES	MEAN	0.140	0.142	0.161 *	0.144
(GRAM)	ST.DEV	0.018	0.018	0.020	0.016
	N	10	10	10	10
UTERUS	MEAN	0.954	0.774	0.728	0.915
(GRAM)	ST.DEV	0.593	0.320	0.233	0.433
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 18. Organ/body weight ratios (%) summary (females)

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 3 MG/KG	GROUP 3 10 MG/KG	GROUP 4 30 MG/KG
BODY W.	MEAN	230	222	224	226
(GRAM)	ST.DEV	11	13	16	9
	N	10	10	10	10
BRAIN	MEAN	0.82	0.84	0.85	0.82
(%)	ST.DEV	0.05	0.06	0.06	0.04
	N	9	10	9	10
HEART	MEAN	0.318	0.317	0.315	0.305
(%)	ST.DEV	0.023	0.014	0.015	0.017
	N	10	10	10	10
LIVER	MEAN	2.50	2.54	2.73	3.12 **
(%)	ST.DEV	0.16	0.20	0.24	0.39
	N	10	10	10	10
THYROIDS	MEAN	0.006	0.006	0.006	0.006
(%)	ST.DEV	0.002	0.002	0.001	0.001
	N	10	10	9	10
THYMUS	MEAN	0.132	0.136	0.144	0.109 *
(%)	ST.DEV	0.015	0.024	0.025	0.018
	N	10	10	10	10
KIDNEYS	MEAN	0.68	0.70	0.73	0.71
(%)	ST.DEV	0.03	0.05	0.06	0.04
	N	10	10	10	10
ADRENALS	MEAN	0.028	0.027	0.029	0.027
(%)	ST.DEV	0.004	0.003	0.005	0.003
	N	10	10	10	10
SPLEEN	MEAN	0.179	0.192	0.189	0.190
(%)	ST.DEV	0.025	0.025	0.023	0.025
	N	10	10	10	10
OVARIES	MEAN	0.061	0.064	0.072 *	0.064
(%)	ST.DEV	0.008	0.008	0.011	0.006
	N	10	10	10	10
UTERUS	MEAN	0.415	0.351	0.331	0.403
(%)	ST.DEV	0.255	0.150	0.118	0.185
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

HISTOPHATOLOGICAL FINDINGS

Table 19. Mean percent weight differences from control groups

Females
Dose level (mg/kg/day):	3	10	30
Liver
Absolute	-2	+6	+23**
Relative to body weight	+2	+9	+25**
Thymus
Absolute	-1	+6	-18*
Relative to body weight	+3	+9	-17*

*: P≤0.05, **: P≤0.01

 

Table 20. Summary Test Item-Related Microscopic Findings: Liver (females)

Females
Dose level (mg/kg/day):	0	3	10	30
Liver^a Hypertrophy hepatocellular Minimal	10	10	10	10
	0	4	4	8

a = Number of organs examined from each group.

 

Table 21. Summary Test Item-Related Microscopic Findings: Thymus (females)

Females
Dose level (mg/kg/day):	0	3	10	30
Thymus^a Atrophy Minimal	10	10	10	10
	0	0	0	2

a = Number of organs examined from each group.

Applicant's summary and conclusion

Conclusions:

In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, the NOAEL for TMAC was determined to be 10 mg/kg bw/day based on adverse effects on liver at 30 mg/kg bw/ day.

Executive summary:

A 90-day oral repeated dose toxicity study was conducted according to OECD/EC guidelines and GLP principles. Male and female rats were exposed to 0, 3, 10 and 30 mg/kg bw/day via oral gavage. Two males exposed to 30 mg/kg bw/day died during the study, on exposure days 57 and 80, respectively. In absence of clinical signs, effects on body weight and/or food consumption and observations during necropsy, no cause of death could be established. In the surviving exposed rats clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis, especially at the highest dose. Severity and incidence increased with dose. Males treated at 30 mg/kg bw/day showed statistically significant lower body weight (gain). Hematological parameters were not affected by the test substance. Rats exposed to 30 mg/kg bw/day showed increase in ALAT (males and females), ASAT (males only) levels, increased ALP levels (females only), bilirubin and bile acid levels. Cholesterol levels decreased for high dose females only. Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements and ambulations, no other functional effects were observed. Females dosed at 30 mg/kg bw/day were found to have statistically significantly increased relative and absolute liver weights and lower thymus weights. Histopathology revealed hepatocellular hypertrophy in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity at 3 and 10 mg/kg/day and in absence of effects in other parameters this observation was considered to be non-adverse at these dose levels. In the thymus of females at 30 mg/kg bw/day atrophy at minimal degree was observed in 2/10 females with low severity.

Taken all data together, the NOAEL of TMAC was determined to be 10 mg/kg bw/day based on adverse effects at 30 mg/kg bw/day.