Fatty acid chlorides, C18 unsatd., reaction products with sodium N-methyltaurinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015 - 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-[methyloleoylamino]ethane-1-sulphonate

EC Number:

205-285-7

EC Name:

Sodium 2-[methyloleoylamino]ethane-1-sulphonate

Cas Number:

137-20-2

Molecular formula:

C21H41NO4S.Na

IUPAC Name:

sodium 2-[methyl(oleoyl)amino]ethanesulfonate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animal room was air-conditioned with adequate (above 10) air changes per hour. The experimental room was continuously monitored for tem¬pera¬ture and relative humidity. The ranges for room tem¬pera¬ture and relative humidity were 20.9°C to 23.1°C and 59 to 68%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Once daily by oral gavage for 90 consecutive days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity analysis as well as dose formulation analysis.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

4 dose groups plus a control recovery group and a high dose recovery group

Examinations

Observations and examinations performed and frequency:

Viability/ Mortality, clinical signs (daily), Ophthalmoscopy (Before start of treatment and towards end of the treatment period), body weights and feed consumption (once weekly) during treatment and recovery period. urinalysis and clinical pathology (all animals from tretament and recovery groups).

Sacrifice and pathology:

Macroscopic and microscopic observations (end of treatment and recovery period) as well as histopathology of all preserved organs (control and high dose groups). Gross lesions (all groups) and stomach from low, intermediate, high and recovery groups.

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
•            Data was summarized in tabular form. Statistical analysis was performed using statplus program.
•            All the data was checked for normality with Shapiro-Wilk W test
•            Data for each group of animals was subjected to analysis of variance (ANOVA). Values were given as mean ± standard deviation (SD).
•            t-test was used to compare the difference between treated and control groups. Statistical significances of differences were calculated with one-way analyses of variance. P≤0.05 (5% level of significance) was considered to represent the significance in the respective parameters

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Minor clinical signs of toxicity (dullness) observed in some animals of the mid and high dose group. Reversed during the recovery period.
In intermediate dose group (group 3), all the animals appeared normal up to day 65. Dullness was observed in from day 66 (Animal No. 046 and 056, 1 male and 1 female), from day 68 (Animal No. 059, 1female), from day 69 (Animal No. 053 and 054, 2 males) until day 90 (last day of observation).
In high dose group (group 4), all the animals appeared normal up to day 51. Dullness and Burrowing were observed in from day 52 (Animal No. 069, 070 and 079, 080, 2 males and 2 females), from day 53 (Animal No. 065, 066, 067, 068 and 075, 076, 077, 078, 4 males and 4 females), from day 60 (Animal No. 061, 062, 063, 064 and 071, 072, 073, 074, 4 males and 4 females) until day 90. Dullness alone was observed on day 91 of observation period (before terminal sacrifice) in all the animals.
In high dose recovery group group 4R, all the animals appeared normal up to day 51. Dullness and Burrowing were observed in from day 52 (Animal No. 109, 110 and 119, 120, 2 males and 2 females), from day 53 (Animal No. 101, 102, 103, 104 and 111, 112, 113, 114, 4 male and 4 female), from day 54 (Animal No. 105, 106, 107, 108 and 115, 116, 117, 118, 4 male, 4 female) until day 90. Dullness alone was observed from day 91 to 94. All the animals recovered from clinical signs and appeared normal from day 95 to until day 119 (last day of observation).

Mortality:

no mortality observed

Description (incidence):

Animals treated in study with test item at dose levels of 100, 300 and 750 mg/kg body weight did not reveal any mortality in both males and females throughout the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect on body weight and bodyweight gain (%) was detected for treated animals when compared with vehicle control.
The body weight and body weight gain (%) was generally decreased in males at high dose group (G4) compared to control group from day 15 to 90. In females there was no decrease in body weight or body weight gain (%) in any of the treated animals of low, intermediate and high dose group (G2 to G4). However, high dose recovery females as well as males showed decreased body weight and body weight gain (%) when compared with respective vehicle control. Concurrently a significantly reduced food consumption was observed in high dose males and females. Based hereupon, the reduced body weights and body weight gains may be explained by lower food intake due to local irritative effects of the test item at the port of entry as indicated in the forestomach of the test animals. Thus, these findings are not considered to be adverse.
Detailed data see the tables in section "Any other information on results inc. tables"

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean feed consumption was slightly but statistically significantly decreased in males and females at high dose (G4) when compared with vehicle control (G1). This lower food intake is considered due to local irritative effects of the test item at the port of entry as indicated in the forestomach of the test animals. Thus, these findings are not considered to be adverse.

Food efficiency:

not examined

Description (incidence and severity):

not applicable because no drinking water study.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Opthalmological examiniation did not reveal any abnormalities in the vehicle control and high dose treated animals at the end of treatment period.

Haematological findings:

no effects observed

Description (incidence and severity):

Erythrocyte count (RBC), hemoglobin (Hb), hematocrit (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (thrombocyte) count (PLT), total leukocyte count (WBC), differential leukocyte count (DC), prothrombin time (PT) and activated partial thromboplastin time (APTT) did not show any toxicologically relevant finding in both sexes. In recovery group at end of week-17, no significant changes were observed in females whereas, in males, MCHC significantly increased in high dose recovery (G4R) when compared with vehicle control recovery group (G1R).
The changes observed in the hematological parameters are marginal, not clinically significant and could not be attributed to the test item administration as these values were within biological variation.
Detailed data see the tables in section "Any other information on results inc. tables"

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical parameters like glucose (GLU), urea, creatinine (CREA), cholesterol (CHOL), total triglycerides (TRIGL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, sodium (Na), potassium (K), chloride (Cl), total protein (TPO), albumin (ALB), globulin (GLB) and A/G ratio did not show any toxicologically relevant findings in both the sexes in treatment and recovery group with the exception of statistically significant increase in alanine transaminase (ALT) which was noticed in high dose (G4) male animals when compared to vehicle control group (G1). Total proteins (TPO) in high dose (G4) male was increased when compared to vehicle control group (G1) at end of week 13 only.
In recovery group animals at the end of week-17, significant increase in glucose (GLU), potassium (K) aspartate aminotransferase (AST) in males was observed. In high dose recovery females (G4R) a decrease in aspartate aminotransferase (AST) was observed as compared to vehicle control recovery group (G1R).
Although statistically significant, all observed changes fall within the historical control range, were not clinically significant and did not correlate with any microscopic finding observed in high dose animals. Hence, the test item had no effect on the biochemical parameters of treated animals up to the high dose of 750 mg/kg body weight.
Detailed data see the tables in section "Any other information on results inc. tables"

Urinalysis findings:

no effects observed

Description (incidence and severity):

Treatment had no significant effect on urinary parameters in any of the groups with exception of decreased urinary volume in high dose recovery group (G4R) whern compared with vehicle control (G1R). These changes were not clinically significant and do not correlate with any microscopic finding. Hence, the test item had no effect on the urine parameters of treated animals upto the dose of 750 mg/kg body weight.
Detailed data see the tables in section "Any other information on results inc. tables"

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No functional behavioural effects have been observed during the course of the study.

Immunological findings:

no effects observed

Description (incidence and severity):

Despite inflammatory related findings, no effects in immunological competent organs have been detected.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant test item-related differences in absolute and relative organ weights were observed.
In main study groups, there was no significance observed in absolute organ weights of male animals. Sporadically observed increased in absolute and relative organ weights of female animals showed no dose response relationship and are not considered treatment related.
In recovery groups, absolute organ weights of heart, spleen and thymus of male animals from G4R (750 mg/kg) was significantly lower than that of control recovery group G1R (0 mg/kg) male animals. Relative weights of liver and brain in male animals of group G4R (750 mg/kg) was significantly higher than those of group G1R. Regarding female recovery animals there was no significance noted in absolute organ weight when compared to controls. Relative organ weights of heart, spleen and kidneys of females from 750 mg/kg recovery group G4R was significantly higher than that of control recovery group G1R female animals.
Despite these differences, microscopically no test item-related change was observed in these organs. Hence, the changes observed in organ weight were not considered to be biologically significant.
Detailed data see the tables in section "Any other information on results inc. tables"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy performed at the end of the treatment period revealed slightly thickened forestomach (9 males, 4 female) and red foci in glandular stomach (7 male, 1 female) in group G4 (750 mg/kg) wherase only slightly thickened forestomach was observed in 3 males and 1 female animal from G3 (300 mg/kg) group. No gross findings were observed on completion of treatment in animals of Group G2 (100 mg/kg) as well as in male and females recovery animals of G4R (750 mg/kg).
These findings are of no relevance for humans as discussed in the executive summary and are considered a response to the observed port of entry effects in the forestomach of test animals. In addition, these effects were reversed after the treatment free recovery period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed forestomach and glandular stomach findings in high dose animals comparable to the observed gross pathological changes. In high dose animals, forestomach submucosal hemorrhages (2 male, 1 female), polymorphonuclear (4 male) and/or mononuclear cells infiltrate (4 male, 3 female), mucosal necrosis (4 male, 1 female), hyperkeratosis (8 male, 4 female) and hyperplasia (9 male, 6 female) of squamous epithelium were observed.
Animals treated with test item at 300 mg/kg (G3) revealed glandular stomach hemorrhage (2 male), forestomach submucosal hemorrhage (2 male), polymorphonuclear and mononuclear cells infiltrate (2 male, 1 female), mucosal necrosis (2 male, 1 female), hyperkeratosis (3 male) and hyperplasia of squamous epithelium (3 female).
These changes are not considered having relevance for humans, were reversible as demonstrated in the high dose recovery animals and are considered as port of entry effects due to the local irritant effect of the test item at mentioned doses and treatment period (see also discussion section in the executive summary).
No abnormality attributable to the test item in group G2 (100 mg/kg) and recovery group G4R (recovery group, 750 mg/kg) was revealed on microscopic examination.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No indications of neoplastic histopathological findings revealed

Other effects:

no effects observed

Details on results:

No other effects of relevance observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

The only significant effects observed were related to the forestomach of rats. This finding is not considered relevant for humans (details see discussion section).

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

other: Forestomach

Any other information on results incl. tables

Observed forestomach lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions and are considered to be `site of first contact` findings due to local irritative effects. Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. This view is also reflected by the endpoint specific guidance given by ECHA (Chapter R.7.2.1). The forestomach changes in rats are thus considered to be of minor or no relevance for human health hazard / risk assessment (for a detailled assessment see discussion section in the executive summary).

Further data in tabular form:

BODY WEIGHTS (G)– SUMMARY

Males

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	133.0	133.6	134.7	134.3
	SD	3.80	3.77	3.00	2.80
	Min	128.1	127.7	129.4	130.4
	Max	138.6	138.9	138.5	138.2
	N	10	10	10	10
Day 8	Mean	181.6	183.1	183.2	183.6
	SD	5.01	3.62	4.53	4.28
	Min	173.2	176.5	175.9	172.9
	Max	187.4	188.2	188.7	187.1
	N	10	10	10	10
Day 15	Mean	309.8	239.7	236.5	231.5*
	SD	14.06	4.72	10.58	8.46
	Min	284.0	229.7	214.0	216.7
	Max	328.3	246.6	246.1	243.6
	N	10	10	10	10

 

*Significant at p ≤ 0.05 level with group 1

 

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 22	Mean	279.6	273.4	275.7	258.3*!
	SD	11.11	8.24	20.37	13.92
	Min	255.7	261.5	243.1	233.5
	Max	291.7	283.7	314.0	277.3
	N	10	10	10	10
Day 29	Mean	309.8	300.6	300.9	286.2*
	SD	14.06	15.96	21.31	11.74
	Min	284.0	282.6	266.4	266.1
	Max	328.3	326.8	343.3	301.3
	N	10	10	10	10
Day 36	Mean	334.3	323.5	323.0	303.5*
	SD	18.05	20.67	22.96	16.11
	Min	302.1	298.8	292.4	284.0
	Max	366.7	357.6	368.2	327.8
	N	10	10	10	10
Day 43	Mean	349.8	340.2	337.1	319.5*
	SD	18.89	22.73	25.35	17.15
	Min	321.8	319.1	305.7	294.4
	Max	386.0	380.6	394.0	350.1
	N	10	10	10	10

*Significant at p ≤ 0.05 level with group 1

*!Significant at p ≤ 0.05 level with group 1, 3

 

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 50	Mean	366.1	353.7	351.4	334.7*
	SD	21.14	22.38	31.47	18.38
	Min	335.2	333.4	314.6	319.6
	Max	403.9	390.1	419.8	371.2
	N	10	10	10	10
Day 57	Mean	376.4	365.8	362.3	343.0
	SD	24.26	27.05	34.04	22.43
	Min	345.3	339.2	326.8	320.2
	Max	422.4	416.8	438.7	381.8
	N	10	10	10	10
Day 64	Mean	389.9	378.3	374.5	351.8*
	SD	25.71	30.97	31.65	22.21
	Min	355.6	341.1	338.5	319.8
	Max	437.6	427.7	445.9	390.8
	N	10	10	10	10
Day 71	Mean	395.1	384.0	382.1	356.3*
	SD	27.08	30.62	32.33	24.64
	Min	357.4	343.0	339.4	317.1
	Max	441.9	430.7	451.4	403.8
	N	10	10	10	10

*Significant at p ≤ 0.05 level with group 1

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 78	Mean	405.4	394.3	392.8	361.4*
	SD	28.36	32.56	34.48	26.63
	Min	371.4	352.7	348.0	317.8
	Max	457.3	449.3	462.2	416.9
	N	10	10	10	10
Day 85	Mean	413.7	401.9	401.0	367.2*
	SD	27.94	32.39	34.64	27.61
	Min	377.8	362.9	361.8	316.8
	Max	460.8	456.7	471.2	421.3
	N	10	10	10	10
Day 90	Mean	420.6	407.7	407.5	371.4*
	SD	28.16	32.08	34.55	29.04
	Min	383.2	367.5	370.8	316.2
	Max	468.7	459.9	476.8	427.4
	N	10	10	10	10

 

*Significant at p ≤ 0.05 level with group 1

BODY WEIGHTS (G)– SUMMARY

 

FEMales

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	131.8	132.1	132.4	132.0
	SD	4.54	4.79	4.27	4.62
	Min	125.3	125.4	126.9	125.4
	Max	139.2	139.5	139.4	138.7
	N	10	10	10	10
Day 8	Mean	156.9	160.3	158.4	158.9
	SD	7.27	8.94	4.55	11.56
	Min	144.6	151.2	146.3	132.5
	Max	165.7	178.3	162.7	170.1
	N	10	10	10	10
Day 15	Mean	177.0	181.4	178.7	179.0
	SD	12.37	5.44	5.34	12.82
	Min	155.2	170.8	170.6	149.1
	Max	189.2	186.7	186.5	193.9
	N	10	10	10	10

 

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 22	Mean	191.7	188.7	190.7	188.7
	SD	13.44	8.57	10.14	14.41
	Min	162.5	175.8	178.4	158.0
	Max	206.7	201.4	212.6	208.2
	N	10	10	10	10
Day 29	Mean	201.7	201.8	199.2	199.8
	SD	14.20	9.22	13.16	15.26
	Min	173.2	185.9	178.6	171.3
	Max	222.6	216.4	228.2	220.8
	N	10	10	10	10
Day 36	Mean	207.0	208.8	203.9	207.6
	SD	13.88	9.24	14.69	15.96
	Min	179.4	194.6	181.3	175.3
	Max	227.1	223.4	237.7	227.9
	N	10	10	10	10
Day 43	Mean	213.0	214.8	210.0	212.7
	SD	14.78	9.74	17.12	15.70
	Min	183.4	198.3	186.7	178.6
	Max	236.4	227.7	250.6	233.6
	N	10	10	10	10

 

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 50	Mean	218.3	220.7	215.8	221.4
	SD	15.64	10.72	17.54	14.85
	Min	187.4	202.4	188.6	190.6
	Max	243.7	234.8	256.6	246.4
	N	10	10	10	10
Day 57	Mean	220.6	223.1	219.1	223.7
	SD	15.79	10.77	17.71	15.09
	Min	189.0	203.4	189.8	192.3
	Max	245.6	236.0	259.6	248.7
	N	10	10	10	10
Day 64	Mean	223.8	226.2	223.0	224.5
	SD	15.65	11.30	17.76	15.40
	Min	191.2	205.4	193.3	190.4
	Max	247.5	240.8	260.8	243.9
	N	10	10	10	10
Day 71	Mean	226.3	228.5	223.0	223.0
	SD	15.87	12.30	17.76	16.91
	Min	193.6	206.7	193.3	189.4
	Max	252.0	249.2	260.8	248.3
	N	10	10	10	10

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT		Group 1	Group 2	Group 3	Group 4
Day 78	Mean	229.3	230.8	226.2	223.0
	SD	15.89	12.75	17.41	18.37
	Min	194.6	207.2	198.9	187.4
	Max	254.4	251.3	263.4	251.5
	N	10	10	10	10
Day 85	Mean	232.0	232.6	228.5	222.4
	SD	16.40	12.58	17.08	19.69
	Min	196.0	210.3	205.6	185.3
	Max	258.2	252.7	267.3	254.4
	N	10	10	10	10
Day 90	Mean	234.4	235.0	230.7	222.7
	SD	16.48	12.83	17.40	21.53
	Min	197.8	214.2	208.6	183.8
	Max	261.3	255.3	270.2	257.6
	N	10	10	10	10

 

 

BODY WEIGHTS (G) – SUMMARY

MALES  

RECOVERY		Group 1R	Group 4R
Day 1	Mean	135.4	135.5
	SD	2.21	1.70
	Min	131.6	132.7
	Max	138.9	137.9
	N	10	10
Day 8	Mean	183.8	183.6
	SD	4.43	3.44
	Min	176.2	179.5
	Max	189.9	189.8
	N	10	10
Day 15	Mean	239.5	239.4
	SD	5.07	5.89
	Min	227.5	228.4
	Max	246.8	249.5
	N	10	10
Day 22	Mean	275.1	272.8
	SD	8.44	18.28
	Min	261.1	253.1
	Max	287.0	318.0
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY		Group 1R	Group 4R
Day 29	Mean	300.3	296.8
	SD	8.48	24.62
	Min	284.5	257.4
	Max	310.5	355.5
	N	10	10
Day 36	Mean	322.5	314.4
	SD	11.56	22.22
	Min	304.3	271.6
	Max	337.2	360.5
	N	10	10
Day 43	Mean	342.2	326.6
	SD	12.11	19.17
	Min	324.6	290.6
	Max	355.7	365.6
	N	10	10
Day 50	Mean	356.5	343.1
	SD	12.26	19.64
	Min	332.5	301.8
	Max	370.5	377.6
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY		Group 1R	Group 4R
Day 57	Mean	368.0	346.0
	SD	14.52	20.68
	Min	345.8	306.5
	Max	385.6	371.5
	N	10	10
Day 64	Mean	382.4	351.9
	SD	16.99	22.28
	Min	362.0	317.2
	Max	411.2	378.3
	N	10	10
Day 71	Mean	391.7	355.3
	SD	18.44	25.13
	Min	371.6	317.5
	Max	423.4	385.1
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY		Group 1R	Group 4R
Day 78	Mean	402.1	361.0
	SD	20.04	28.46
	Min	383.3	317.6
	Max	440.2	391.3
	N	10	10
Day 85	Mean	409.4	364.3
	SD	18.78	30.03
	Min	390.4	316.7
	Max	445.5	395.6
	N	10	10
Day 91	Mean	419.9	368.6
	SD	21.49	33.51
	Min	396.8	315.7
	Max	460.3	401.2
	N	10	10
Day 98	Mean	425.6	373.3
	SD	20.19	34.06
	Min	405.7	318.4
	Max	463.1	405.5
	N	10	10

 

 

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

                                                                                                                          

RECOVERY		Group 1R	Group 4R
Day 105	Mean	431.8	380.7
	SD	21.12	33.85
	Min	410.3	322.3
	Max	475.2	412.7
	N	10	10
Day 112	Mean	437.8	387.9
	SD	21.11	33.81
	Min	418.3	329.4
	Max	481.3	421.3
	N	10	10
Day 118	Mean	444.8	395.4
	SD	21.44	33.84
	Min	424.9	336.4
	Max	490.5	429.5
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY

FEMALES  

RECOVERY		Group 1R	Group 4R
Day 1	Mean	132.6	132.7
	SD	4.30	4.00
	Min	126.7	127.9
	Max	138.6	137.9
	N	10	10
Day 8	Mean	165.6	158.6
	SD	5.12	5.52
	Min	157.2	148.2
	Max	172.4	166.2
	N	10	10
Day 15	Mean	183.4	179.7
	SD	6.21	7.37
	Min	174.9	166.7
	Max	192.2	186.2
	N	10	10
Day 22	Mean	192.4	185.5
	SD	9.55	9.27
	Min	180.3	167.7
	Max	206.2	200.6
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY		Group 1R	Group 4R
Day 29	Mean	202.5	196.7
	SD	8.22	8.09
	Min	191.0	183.3
	Max	213.3	208.8
	N	10	10
Day 36	Mean	209.8	201.8
	SD	9.17	8.53
	Min	199.3	185.5
	Max	226.6	214.3
	N	10	10
Day 43	Mean	214.2	207.0
	SD	8.37	9.26
	Min	203.4	187.3
	Max	229.7	218.3
	N	10	10
Day 50	Mean	219.6	212.7
	SD	9.09	9.75
	Min	207.4	191.2
	Max	234.2	224.4
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY		Group 1R	Group 4R
Day 57	Mean	224.0	213.1
	SD	8.39	9.78
	Min	209.7	193.0
	Max	236.7	226.8
	N	10	10
Day 64	Mean	228.0	215.6
	SD	9.34	10.74
	Min	211.6	201.6
	Max	241.5	232.6
	N	10	10
Day 71	Mean	230.1	214.7
	SD	9.25	11.56
	Min	212.6	199.9
	Max	243.2	235.8
	N	10	10
Day 78	Mean	232.8	213.9
	SD	9.52	12.54
	Min	214.7	197.5
	Max	247.6	238.5
	N	10	10

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY		Group 1R	Group 4R
Day 85	Mean	235.6	213.5
	SD	9.28	13.08
	Min	216.7	197.3
	Max	249.2	239.1
	N	10	10
Day 91	Mean	239.1	213.2
	SD	9.75	13.87
	Min	220.6	196.9
	Max	254.2	240.1
	N	10	10
Day 98	Mean	241.4	215.5
	SD	9.97	13.74
	Min	222.4	197.2
	Max	255.6	242.5
	N	10	10
Day 105	Mean	244.4	218.7
	SD	10.18	14.07
	Min	225.3	199.2
	Max	258.0	245.3
	N	10	10

*Significant at p ≤ 0.05 level with group 1R

BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY		Group 1R	Group 4R
Day 112	Mean	248.0	222.9
	SD	9.98	14.28
	Min	229.7	202.4
	Max	261.4	249.5
	N	10	10
Day 118	Mean	252.2	227.0
	SD	10.46	14.48
	Min	232.4	205.3
	Max	266.4	253.4
	N	10	10

*Significant at p ≤ 0.05 level with group 1R

 

SPERM COUNT SUMMARY – MALES 

TREATMENT		Total Count
		Mean x 20 x 10^6
Group 1	Mean	194
	SD	27.014
Group 2	Mean	194
	SD	25.289
Group 3	Mean	193
	SD	50.711
Group 4	Mean	197
	SD	49.389

 WEEK -17

RECOVERY		Total Count
		Mean x 20 x 10^6
Group 1R	Mean	241
	SD	71.237
Group 4R	Mean	213
	SD	74.643

 

HEMATOLOGY SUMMARY – MALES

Week -13

 

TREATMENT		RBC	Hb	PCV	MCV	MCH	MCHC	PLT
		x10^6Cells	g/dL	%	fL	pg	g/dL	x10^3cells
Group 1	Mean	8.53	15.81	44.62	52.41	18.58	35.42	684.60
	SD	0.490	0.495	1.652	1.629	1.389	1.650	113.256
Group 2	Mean	8.36	15.37	43.18	51.67	18.52	35.87	688.00
	SD	0.769	0.732	3.896	1.191	1.956	3.933	108.056
Group 3	Mean	8.47	14.97	43.76	51.67	17.71	34.25	734.70
	SD	0.665	1.142	2.659	1.649	0.555	1.449	88.043
Group 4	Mean	8.18	15.58	44.14	54.05^#!	19.10	35.33	712.90
	SD	0.720	0.879	3.231	1.635	1.155	1.380	106.371

 

 

TREATMENT		WBC	DC (%)					PT	APTT
		x10^3cells	NEUT	LYMPH	MONO	EOS	BASO	Sec	Sec
Group 1	Mean	5.13	19.85	74.05	3.23	1.81	0.31	15.41	15.54
	SD	1.192	4.037	4.641	1.136	0.530	0.074	0.889	1.932
Group 2	Mean	4.59	25.42	67.73	3.69	1.76	0.24	15.89	13.62
	SD	1.120	9.648	9.646	1.050	0.341	0.126	0.805	2.604
Group 3	Mean	4.33	21.15	72.37	3.34	1.91	0.27	15.74	14.96
	SD	1.058	9.774	9.596	0.854	0.832	0.082	0.752	3.630
Group 4	Mean	5.57	23.09	70.27	3.35	1.69	0.27	15.44	14.57
	SD	1.420	7.890	8.140	1.125	0.593	0.095	0.550	1.657

^#!Significant at p ≤ 0.05 level with group 2, 3

 

  

HEMATOLOGY SUMMARY – FEMALES

Week -13

 

TREATMENT		RBC	Hb	PCV	MCV	MCH	MCHC	PLT
		x10^6Cells	g/dL	%	fL	pg	g/dL	x10^3cells
Group 1	Mean	7.88	15.12	42.54	53.97	19.21	35.60	752.70
	SD	0.284	0.365	1.746	1.429	0.814	1.306	158.800
Group 2	Mean	7.95	14.88	42.77	53.78	18.71	34.79	758.40
	SD	0.299	0.439	1.398	1.111	0.443	0.285	145.489
Group 3	Mean	8.05	15.10	43.57	54.20	18.80	34.73	714.60
	SD	0.435	0.665	2.358	2.744	0.800	1.570	100.334
Group 4	Mean	7.93	14.94	43.15	54.45	18.87	34.68	791.70
	SD	0.575	0.769	2.466	1.522	0.721	1.258	55.428

 

 

TREATMENT		WBC	DC (%)					PT	APTT
		x10^3cells	NEUT	LYMPH	MONO	EOS	BASO	Sec	Sec
Group 1	Mean	3.13	16.39	78.36	2.53	1.59	0.23	15.46	13.99
	SD	0.587	2.800	3.351	0.645	0.453	0.125	0.648	1.223
Group 2	Mean	2.98	21.87	72.63	2.65	1.82	0.28	15.21	15.68
	SD	0.913	9.926	10.328	0.597	0.637	0.193	0.409	4.099
Group 3	Mean	3.15	17.99	76.14	3.04	1.84	0.27	15.34	14.19
	SD	0.808	4.405	5.491	0.828	0.665	0.082	0.738	2.059
Group 4	Mean	3.41	20.03	74.10	2.83	1.70	0.28	15.43	15.55
	SD	1.460	6.631	7.158	0.672	0.720	0.140	0.891	3.137

 

 

HEMATOLOGY SUMMARY – MALES

Week -17

 

RECOVERY		RBC	Hb	PCV	MCV	MCH	MCHC	PLT
		x10^6Cells	g/dL	%	fL	pg	g/dL	x10^3cells
Group 1R	Mean	8.49	15.69	44.91	52.98	18.50	34.95	653.70
	SD	0.244	0.314	1.290	1.991	0.516	0.610	54.718
Group 4R	Mean	8.51	15.81	44.63	52.43	18.59	35.43*	625.10
	SD	0.378	1.041	3.054	2.439	0.844	0.211	134.622

 

 

 

RECOVERY		WBC	DC (%)					PT	APTT
		x10^3cells	NEUT	LYMPH	MONO	EOS	BASO	Sec	Sec
Group 1R	Mean	5.41	21.68	72.61	2.70	1.75	0.30	15.43	13.25
	SD	0.982	6.820	7.035	0.952	0.420	0.067	0.926	1.362
Group 4R	Mean	4.46	18.96	74.86	3.18	1.50	0.37	14.89	13.28
	SD	1.063	5.807	6.646	0.981	0.455	0.095	0.601	1.522

 

 

^*Significant at p ≤ 0.05 level with group 1R

HEMATOLOGY SUMMARY – FEMALES

Week -17

 

RECOVERY		RBC	Hb	PCV	MCV	MCH	MCHC	PLT
		x10^6Cells	g/dL	%	fL	pg	g/dL	x10^3cells
Group 1R	Mean	7.81	15.03	42.69	54.72	19.27	35.22	722.70
	SD	0.256	0.231	1.000	2.205	0.750	0.571	130.177
Group 4R	Mean	7.77	15.04	42.62	54.87	19.36	35.31	707.50
	SD	0.611	1.343	3.646	2.635	0.943	0.448	163.011

 

 

 

 

RECOVERY		WBC	DC (%)					PT	APTT
		x10^3cells	NEUT	LYMPH	MONO	EOS	BASO	Sec	Sec
Group 1R	Mean	2.61	19.35	74.98	2.64	2.11	0.17	15.01	12.51
	SD	0.666	3.654	4.363	0.824	0.881	0.067	0.601	1.455
Group 4R	Mean	2.51	15.00	79.44	2.29	2.28	0.23	14.78	13.32
	SD	0.457	7.487	9.049	0.557	1.802	0.067	0.880	1.219

*Significant at p ≤ 0.05 level with group 1R

 

CLINICAL BIOCHEMISTRY SUMMARY – MALES

WEEK -13

 

TREATMENT		GLU	UREA	CREA	CHOL	TRIGL	AST	ALT	ALP
		mmol/L	mmol /L	µmol /L	mmol/L	mmol /L	U/L	U/L	U/L
Group 1	Mean	8.18	7.04	36.22	2.12	0.57	72.35	33.52	63.06
	SD	0.764	0.708	4.062	0.375	0.114	23.667	6.992	13.658
Group 2	Mean	8.29	7.29	38.20	2.13	0.61	62.83	28.26	52.61
	SD	0.864	1.402	7.395	0.320	0.121	3.604	4.733#	12.913
Group 3	Mean	8.25	6.82	36.73	2.30	0.56	68.40	39.21	54.57
	SD	1.166	1.231	5.849	0.447	0.194	11.151	6.218	17.782
Group 4	Mean	8.02	7.33	38.02	2.11	0.60	70.73	55.26*#!	63.10
	SD	1.234	1.243	4.450	0.293	0.105	7.720	8.678	15.241

 

 

 

TREATMENT		BIL	Na	K	Cl	TPO	ALB	GLB	A/G
		µmol /L	mmol /L	mmol/L	mmol/L	g/L	g/L	g/L	_
Group 1	Mean	1.49	147.99	3.55	111.40	67.66	43.00	24.66	1.81
	SD	0.829	2.976	0.286	3.142	6.763	1.520	6.222	0.293
Group 2	Mean	1.47	146.23	3.51	110.13	66.58	44.17	22.41	1.99
	SD	0.634	2.066	0.377	2.307	3.909	4.441	1.629	0.304
Group 3	Mean	1.39	147.28	3.71	111.01	67.29	43.72	23.57	1.87
	SD	0.603	3.739	0.339	3.293	5.423	5.085	1.980	0.257
Group 4	Mean	1.18	146.69	3.58	110.08	64.77	43.93	20.84	2.13
	SD	0.581	1.227	0.392	1.598	1.470	1.784	1.928	0.271

^*Significant at p ≤ 0.05 level with group 1,^#Significant at p ≤ 0.05 level with group 2,^!Significant at p ≤ 0.05 level with group 13

 

CLINICAL BIOCHEMISTRY SUMMARY – FEMALES  

WEEK -13

TREATMENT		GLU	UREA	CREA	CHOL	TRIGL	AST	ALT	ALP
		mmol/L	mmol/L	µmol /L	mmol/L	mmol/L	U/L	U/L	U/L
Group 1	Mean	7.32	7.88	40.25	2.16	0.51	62.95	28.47	26.14
	SD	0.721	1.267	3.665	0.270	0.123	19.452	6.219	7.245
Group 2	Mean	7.96	7.65	41.61	2.06	0.51	65.51	27.10	32.64
	SD	1.074	1.039	5.728	0.301	0.089	9.856	7.517	12.331
Group 3	Mean	7.61	7.68	38.56	1.86	0.48	62.31	31.02	34.51
	SD	1.603	0.872	6.578	0.343	0.143	8.117	7.142	15.659
Group 4	Mean	7.87	7.49	36.87	1.77	0.54	67.95	62.58	42.02
	SD	1.268	1.044	4.073	0.402	0.178	20.950	61.769	33.439

 

 

 

 

 

TREATMENT		BLI	Na	K	Cl	TPO	ALB	GLB	A/G
		µmol /L	mmol /L	mmol/L	mmol/L	g/L	g/L	g/L	_
Group 1	Mean	1.69	147.27	3.39	111.24	72.83	54.06	18.77	2.91
	SD	0.861	1.709	0.359	2.459	1.889	1.942	1.829	0.329
Group 2	Mean	1.27	146.49	3.46	110.38	70.65	51.34	19.31	2.71
	SD	0.581	2.402	0.351	2.438	3.750	2.915	2.682	0.444
Group 3	Mean	1.66	147.11	3.42	110.40	69.60	51.57	18.03	2.89
	SD	0.674	2.657	0.317	3.552	4.531	5.363	1.518	0.465
Group 4	Mean	1.44	146.83	3.65	110.78	68.21*	51.16	17.05	3.05
	SD	0.724	2.314	0.333	2.086	2.546	3.738	2.018	0.519

*Significant at p ≤ 0.05 level with group 1

 

 

CLINICAL BIOCHEMISTRY SUMMARY – MALES 

WEEK -17

RECOVERY		GLU	UREA	CREA	CHOL	TRIGL	AST	ALT	ALP
		mmol/L	mmol /L	µmol /L	mmol/L	mmol/L	U/L	U/L	U/L
Group 1R	Mean	8.17	6.67	37.69	1.82	0.58	58.42	27.95	52.80
	SD	0.773	0.591	3.793	0.249	0.135	4.709	4.776	8.127
Group 4R	Mean	9.12*	6.99	38.27	2.00	0.68	70.55*	32.02	55.73
	SD	0.868	0.720	5.430	0.256	0.185	15.368	5.961	7.811

 

   

 

RECOVERY		BLI	Na	K	Cl	TPO	ALB	GLB	A/G
		µmol /L	mmol /L	mmol/L	mmol/L	g/L	g/L	g/L	_
Group 1R	Mean	1.15	146.84	3.55	110.34	63.42	41.04	22.39	1.84
	SD	0.636	1.055	0.144	0.458	2.189	1.432	1.694	0.160
Group 4R	Mean	0.81	145.84	4.32*	109.58	65.84	42.69	23.15	1.85
	SD	0.277	1.283	0.899	1.457	4.003	2.061	2.161	0.115

 

*Significant at p ≤ 0.05 level with group 1R.

  

 

 

CLINICAL BIOCHEMISTRY SUMMARY – FEMALES

WEEK -17

RECOVERY		GLU	UREA	CREA	CHOL	TRIGL	AST	ALT	ALP
		mmol/L	mmol/L	µmol /L	mmol/L	mmol/L	U/L	U/L	U/L
Group 1R	Mean	8.21	8.01	38.51	2.03	0.60	105.08	47.37	26.00
	SD	0.604	0.993	6.709	0.477	0.217	59.888	43.679	8.220
Group 4R	Mean	8.36	7.94	41.11	2.26	0.51	61.51*	22.69	25.66
	SD	1.483	0.881	4.879	0.455	0.069	10.059	6.971	9.801

 

 

 

 

 

RECOVERY		BLI	Na	K	Cl	TPO	ALB	GLB	A/G
		µmol /L	mmol/L	mmol/L	mmol/L	g/L	g/L	g/L	_
Group 1R	Mean	0.77	145.12	3.77	109.80	68.50	48.87	19.63	2.49
	SD	0.680	1.199	1.080	1.560	3.901	3.413	1.141	0.193
Group 4R	Mean	1.36	145.06	3.71	109.81	69.56	51.09	18.47	2.81
	SD	0.753	1.556	0.350	1.754	4.496	4.551	2.154	0.508

 

*Significant at p ≤ 0.05 level with group 1R.

URINE ANALYSIS – SUMMARY - MALES

Week 13

TREATMENT		Volume	SG	pH	Pro	GLU	KET	UBG	BIL	Color	Clarity
		mL	-	-	mg/dl	-	mg/dl	mg/dl	mg/dl	-	-
Group 1	Mean	5.00	1.01	8.50	30.00	0.00	5.50	0.00	0.20	NA	NA
	SD	0.931	0.003	0.850	15.811	0.000	5.503	0.000	0.422	NA	NA
Group 2	Mean	4.93	1.01	8.60	35.00	0.00	4.50	0.00	0.20	NA	NA
	SD	0.452	0.003	0.843	21.082	0.000	1.581	0.000	0.422	NA	NA
Group 3	Mean	4.92	1.01	8.60	30.00	0.00	4.00	0.00	0.00	NA	NA
	SD	0.713	0.003	0.699	25.820	0.000	4.743	0.000	0.000	NA	NA
Group 4	Mean	4.58	1.01	8.80	20.00	0.00	1.50	0.00	0.00	NA	NA
	SD	0.666	0.003	0.422	30.732	0.000	2.415	0.000	0.000	NA	NA

 

TREATMENT		ERY	LEU
		/µL	/µL
Group 1	Mean	2.00	47.50
	SD	4.216	36.228
Group 2	Mean	4.50	102.50
	SD	8.317	144.554
Group 3	Mean	1.00	62.50
	SD	3.162	39.528
Group 4	Mean	0.00	55.00
	SD	0.000	38.730

 

Key:NA – Not Applicable
URINE ANALYSIS – SUMMARY – FEMALES

Week 13

TREATMENT		Volume	SG	pH	Pro	GLU	KET	UBG	BIL	Color	Clarity
		mL	-	-	mg/dl	-	mg/dl	mg/dl	mg/dl	-	-
Group 1	Mean	4.31	1.01	8.30	22.50	0.00	2.50	0.00	0.00	NA	NA
	SD	0.695	0.003	0.949	7.906	0.000	2.635	0.000	0.000	NA	NA
Group 2	Mean	3.95	1.01	9.00	40.00	0.00	3.00	0.00	0.00	NA	NA
	SD	0.595	0.002	0.000	31.623	0.000	4.830	0.000	0.000	NA	NA
Group 3	Mean	3.88	1.01	8.90	45.00	0.00	3.00	0.00	0.00	NA	NA
	SD	0.403	0.003	0.316	25.820	0.000	2.582	0.000	0.000	NA	NA
Group 4	Mean	4.15	1.02	8.50	25.00	0.00	5.00	0.00	0.20	NA	NA
	SD	0.403	0.002	0.850	20.412	0.000	5.774	0.000	0.422	NA	NA

 

TREATMENT		ERY	LEU
		/µL	/µL
Group 1	Mean	1.00	47.50
	SD	3.162	36.228
Group 2	Mean	2.50	107.50
	SD	7.906	143.880
Group 3	Mean	0.00	87.50
	SD	0.000	148.254
Group 4	Mean	3.50	82.50
	SD	8.182	150.946

 

Key:NA – Not Applicable
URINE ANALYSIS – SUMMARY–MALES

Week 17

RECOVERY		Volume	SG	pH	Pro	GLU	KET	UBG	BIL	Color	Clarity
		mL	-	-	mg/dl	-	mg/dl	mg/dl	mg/dl	-	-
Group 1R	Mean	5.71	1.01	8.70	27.50	0.00	4.00	0.00	0.10	NA	NA
	SD	0.509	0.002	0.483	18.447	0.000	2.108	0.000	0.316	NA	NA
Group 4R	Mean	5.16*	1.01	9.00	35.00	0.00	2.50	0.00	0.00	NA	NA
	SD	0.427	0.002	0.000	45.947	0.000	2.635	0.000	0.000	NA	NA

 

 

 

RECOVERY		ERY	LEU
		/µL	/µL
Group 1R	Mean	0.00	70.00
	SD	0.000	38.730
Group 4R	Mean	0.00	117.50
	SD	0.000	138.969

Key: NA = Not Applicable

* Significant at p ≤ 0.05 with Group 1R

 

URINE ANALYSIS – SUMMARY - FEMALES

Week 17

RECOVERY		Volume	SG	pH	Pro	GLU	KET	UBG	BIL	Color	Clarity
		mL	-	-	mg/dl	-	mg/dl	mg/dl	mg/dl	-	-
Group 1R	Mean	4.82	1.01	8.90	27.50	0.00	3.00	0.00	0.10	NA	NA
	SD	0.478	0.002	0.316	18.447	0.000	2.582	0.000	0.316	NA	NA
Group 4R	Mean	4.78	1.02	9.00	70.00	0.00	2.50	0.00	0.00	NA	NA
	SD	0.399	0.002	0.000	152.662	0.000	2.635	0.000	0.000	NA	NA

 

 

RECOVERY		ERY	LEU
		/µL	/µL
Group 1R	Mean	0.00	70.00
	SD	0.000	38.730
Group 4R	Mean	1.00	82.50
	SD	3.162	37.361

Key: NA = Not Applicable

ORGAN WEIGHTS (GRAM) – SUMMARY - MALES

 

TREATMENT		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight	Mean	404.67	394.99	394.06	357.37	424.29	379.75
	SD	404.32	30.92	34.88	27.68	25.20	33.61
	N	10	10	10	10	10	10
Adrenals	Mean	0.0812	0.0799	0.0744	0.0745	0.0788	0.1467
	SD	0.0298	0.0147	0.0076	0.0110	0.0063	0.2240
	N	10	10	10	10	10	10
Kidneys	Mean	2.2282	2.1630	2.1143	2.0029	2.2439	2.0790
	SD	0.2602	0.1850	0.2035	0.2037	0.1990	0.1653
	N	10	10	10	10	10	10
Liver	Mean	11.3176	11.5933	11.1981	10.9633	11.3923	11.0422
	SD	1.3017	1.3157	1.4083	1.0043	0.7604	0.8848
	N	10	10	10	10	10	10
Heart	Mean	1.2417	1.2417	1.2311	1.1959	1.2562	1.1550*
	SD	0.1432	0.0957	0.1214	0.1340	0.0830	0.1072
	N	10	10	10	10	10	10
Thymus	Mean	0.5052	0.5677	0.5077	0.4598	0.5765	0.4816*
	SD	0.0996	0.0934	0.0637	0.1274	0.0557	0.1030
	N	10	10	10	10	10	10

 

^*Significant at p ≤ 0.05 level with group 1R

ORGAN WEIGHTS (GRAM) – SUMMARY - MALES (CONTD.)

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Spleen	Mean	0.6606	0.7199	0.6965	0.7091	0.7916	0.7072*
	SD	0.1052	0.0691	0.0770	0.1261	0.0654	0.1026
	N	10	10	10	10	10	10
Testes	Mean	3.9899	3.8488	4.0070	3.8737	3.9596	3.7811
	SD	0.2702	0.2606	0.2590	0.3286	0.3038	0.2269
	N	10	10	10	10	10	10
Right Epididymides	Mean	0.7738	0.7674	0.7697	0.7233	0.8974	0.8727
	SD	0.1062	0.0478	0.0563	0.1205	0.0995	0.0651
	N	10	10	10	10	10	10
Brain	Mean	2.1784	2.1707	2.1753	2.0792	2.1835	2.1791*
	SD	0.1091	0.0803	0.0698	0.0763	0.0754	0.0879
	N	10	10	10	10	10	10
Left Epididymides	Mean	0.7633	0.7156	0.7117	0.6859	0.8071	0.7532
	SD	0.1152	0.0625	0.0484	0.0813	0.0962	0.0811
	N	10	10	10	10	10	10
Left Cauda	Mean	0.2058	0.2018	0.2078	0.1805	0.1920	0.1898
	SD	0.0188	0.0229	0.0328	0.0312	0.0266	0.0227
	N	10	10	10	10	10	10

 

^*Significant at p ≤ 0.05 level with group 1R

 

 

 

ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight	Mean	224.39	222.26	220.24	212.35	237.58	217.68
	SD	17.35	12.47	17.54	22.10	9.57	14.02
	N	10	10	10	10	10	10
Adrenals	Mean	0.0803	0.0991^*	0.0933	0.0838^#	0.1916	0.0771
	SD	0.0138	0.0090	0.0104	0.0146	0.3362	0.0127
	N	10	10	10	10	10	10
Kidneys	Mean	1.4152	1.5144	1.4906	1.4668	1.4768	1.4914
	SD	0.1328	0.1503	0.1379	0.1439	0.1120	0.1238
	N	10	10	10	10	10	10
Liver	Mean	7.0592	7.3385	7.0130	7.2293	6.8651	6.8447
	SD	0.8370	0.9889	0.9861	0.6709	0.9525	0.8560
	N	10	10	10	10	10	10
Heart	Mean	0.8300	0.9344	0.9142	0.8206	0.8252	0.8551
	SD	0.0943	0.1176	0.0915	0.0748	0.0738	0.0891
	N	10	10	10	10	10	10
Thymus	Mean	0.4052	0.4440	0.3833	0.3998	0.4233	0.3761
	SD	0.0766	0.0796	0.0843	0.1019	0.0751	0.0767
	N	10	10	10	10	10	10

^*Significant at p ≤ 0.05 level with group 1 and 1R

^#Significant at p ≤ 0.05 level with group 2

 

 

ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES (CONTD.)

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Spleen	Mean	0.5175	0.4881	0.4808	0.4883	0.5048	0.5355
	SD	0.1033	0.0748	0.1370	0.0861	0.0435	0.0724
	N	10	10	10	10	10	10
Ovaries	Mean	0.3317	0.1641	0.2220	0.1497	0.1657	0.1429
	SD	0.5704	0.0355	0.1829	0.0377	0.0497	0.0316
	N	10	10	10	10	10	10
Uterus	Mean	0.8555	0.9271	0.7668	0.8594	0.8009	0.8334
	SD	0.2900	0.4389	0.3018	0.3363	0.2646	0.1427
	N	10	10	10	10	10	10
Brain	Mean	2.0013	1.9595	2.0287	1.9679	1.9716	1.9480
	SD	0.0987	0.0898	0.0696	0.0639	0.0984	0.1230
	N	10	10	10	10	10	10

 

ORGAN WEIGHTS RATIO (%) – SUMMARY- MALES

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight	Mean	404.67	394.99	394.06	357.37	424.29	379.75
	SD	27.921	30.92	34.88	27.68	25.20	33.61
	N	10	10	10	10	10	10
Adrenals	Mean	0.0201	0.0203	0.0191	0.0209	0.0186	0.0397
	SD	0.0074	0.0037	0.0030	0.0029	0.0018	0.0619
	N	10	10	10	10	10	10
Kidneys	Mean	0.5500	0.5488	0.5382	0.5615	0.5288	0.5513
	SD	0.0432	0.0417	0.0500	0.0506	0.0329	0.0677
	N	10	10	10	10	10	10
Liver	Mean	2.7904	2.9397	2.8419	3.0726	2.6885	2.9231
	SD	0.1656	0.3013	0.2507	0.2412	0.1646	0.2902
	N	10	10	10	10	10	10
Heart	Mean	0.3067	0.3151	0.3142	0.3361	0.2970	0.3063
	SD	0.0265	0.0231	0.0380	0.0430	0.0264	0.0391
	N	10	10	10	10	10	10

ORGAN WEIGHTS RATIO (%) – SUMMARY MALES (CONTD.)

 

Organs		TREATMENT (WEEK- 13)				RECOVERY (WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Thymus	Mean	0.1248	0.1444	0.1297	0.1291	0.1361	0.1276
	SD	0.0228	0.0267	0.0199	0.0359	0.0132	0.0291
	N	10	10	10	10	10	10
Spleen	Mean	0.1630	0.1444	0.1777	0.1981*	0.1870	0.1881
	SD	0.0217	0.0267	0.0222	0.0300	0.0180	0.0342
	N	10	10	10	10	10	10
Testes	Mean	0.9875	0.9768	1.0220	1.0870^*#	0.9356	1.0033
	SD	0.0560	0.0607	0.0902	0.0953	0.0843	0.1138
	N	10	10	10	10	10	10
Right Epididymides	Mean	0.1908	0.1954	0.1971	0.2028	0.2119	0.2307
	SD	0.0186	0.0208	0.0253	0.0337	0.0245	0.0182
	N	10	10	10	10	10	10
Brain	Mean	0.5399	0.5523	0.5555	0.5843	0.5159	0.5786
	SD	0.0356	0.0437	0.0471	0.0416	0.0285	0.0639
	N	10	10	10	10	10	10
Left Epididymides	Mean	0.1886	0.1821	0.1819	0.1924	0.1900	0.1992
	SD	0.0236	0.0208	0.0196	0.0225	0.0164	0.0222
	N	10	10	10	10	10	10
Left Cauda	Mean	0.0510	0.0512	0.0533	0.0505	0.0453	0.0504
	SD	0.0049	0.0058	0.0105	0.0077	0.0057	0.0078
	N	10	10	10	10	10	10

^*Significant at p ≤ 0.05 level with group 1,^*#Significant at p ≤ 0.05 level with group 1, 2

ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight	Mean	224.39	222.26	220.24	212.35	237.58	217.68
	SD	17.35	12.47	17.54	22.10	9.57	14.02
	N	10	10	10	10	10	10
Adrenals	Mean	0.0359	0.0447*	0.0428	0.0400	0.0842	0.0355
	SD	0.0064	0.0043	0.0069	0.0087	0.1533	0.0064
	N	10	10	10	10	10	10
Kidneys	Mean	0.6324	0.6817	0.6767	0.6967	0.6234	0.6862*
	SD	0.0573	0.0573	0.0271	0.0945	0.0640	0.0558
	N	10	10	10	10	10	10
Liver	Mean	3.1419	3.3062	3.1786	3.4421	2.8872	3.1418
	SD	0.2361	0.4396	0.3089	0.5090	0.3586	0.3101
	N	10	10	10	10	10	10
Heart	Mean	0.3695	0.4195*	0.4169	0.3893	0.3477	0.3928*
	SD	0.0227	0.0371	0.0476	0.0459	0.0320	0.0322
	N	10	10	10	10	10	10
Thymus	Mean	0.1808	0.1999	0.1755	0.1893	0.1778	0.1728
	SD	0.0324	0.0357	0.0445	0.0505	0.0284	0.0344
	N	10	10	10	10	10	10

*Significant at p ≤ 0.05 level with group 1 & 1R
ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES (CONTD.)

 

Organs		TREATMENT (WEEK- 13)				RECOVERY(WEEK-17)
		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Spleen	Mean	0.2305	0.2205	0.2192	0.2316	0.2126	0.2462*
	SD	0.0408	0.0392	0.0636	0.0441	0.0182	0.0314
	N	10	10	10	10	10	10
Ovaries	Mean	0.1487	0.0745	0.1010	0.0712	0.0701	0.0660
	SD	0.2579	0.0188	0.0830	0.0193	0.0219	0.0148
	N	10	10	10	10	10	10
Uterus	Mean	0.3818	0.4245	0.3543	0.4044	0.3376	0.3829
	SD	0.1290	0.2232	0.1606	0.1534	0.1126	0.0608
	N	0.3818	10	10	10	10	10
Brain	Mean	0.8971	0.8833	0.9251	0.9360	0.8322	0.8977*
	SD	0.0856	0.0493	0.0636	0.1017	0.0714	0.0723
	N	0.8971	10	10	10	10	10

*Significant at p ≤ 0.05 level with group 1&1R

 

   SUMMARY OF INCIDENCE OF MACROSCOPIC FINDING

MALE                                                                                                                     

GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
ANIMALS EXAMINED	10	10	10	10	10	10
ANIMALS WITHOUT ABNORMALITY	10	10	08	01	10	10
ANIMALS AFFECTED	00	00	02	09	00	00
STOMACH: Thickened, forestomach	00	00	02	09	00	00
STOMACH: Red foci, glandular stomach	00	00	00	06	00	00

 

FEMALE

GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
ANIMALS EXAMINED	10	10	10	10	10	10
ANIMALS WITHOUT ABNORMALITY	08	06	07	04	08	06
ANIMALS AFFECTED	02	04	03	06	02	04
UTERUS: Distended with watery content	02	04	02	02	02	04
STOMACH: Thickened, forestomach	00	00	01	04	00	00
STOMACH: Red foci, glandular stomach	00	00	00	01	00	00

  

 

 

 

SUMMARY OF INCIDENCE OF MICROSCOPIC FINDINGS

SEX: MALE
GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
NUMBER OF ANIMALS	10	10	10	10	10	10
ADRENAL GLANDS	(10)	-	-	(10)	-	-
Dilatation, sinusoidal	01			00
Vacuolation	01			00
AORTA	0 (10)	-	-	0 (10)	-	-
AXILLARY LYMPH NODES	0 (10)	-	-	0 (10)	-	-
BONE MARROW (STERNUM)	0 (10)	-	-	0 (10)	-	-
BRAIN (3 LEVELS)	0 (10)	-	-	0 (10)	-	-
CECUM	0 (10)	-	-	0 (10)	-	-
COLON	0 (10)	-	-	0 (10)	-	-
DUODENUM	0 (10)	-	-	0 (10)	-	-
EPIDIDYMIDES	0 (10)	-	-	0 (10)	-	-
HEART	(10)	-	-	(10)	-	-
Infiltrate, mononuclear cells	01			00
Necrosis, myocardium	01			00
ILEUM WITH PEYER’S PATCHES	0 (10)	-	-	0 (10)	-	-
JEJUNUM	0 (10)	-	-	0 (10)	-	-
KIDNEYS	0 (10)	-	-	0 (10)	-	-
LIVER	0 (10)	-	-	0 (10)	-	-
LUNGS	(10)	-	-	(10)	-	-
Infiltrate, mononuclear cells	01			0
Histiocytosis	01			0
MESENTERIC LYMPH NODES	0 (10)	-	-	0 (10)	-	-
OESOPHAGUS	0 (10)	-	-	0 (10)	-	-

Key: Number in parenthesis indicates number of animals examined.

 

SEX: MALE
GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
NUMBER OF ANIMALS	10	10	10	10	10	10
PANCREAS	0 (10)	-	-	0 (10)	-	(1)
RECTUM	0 (10)	-	-	0 (10)	-	-
SKELETAL MUSCLE	0 (10)	-	-	0 (10)	-	-
SPINAL CORD (CERVICAL, MID THORACIC, LUMBAR)	0 (10)	-	-	0 (10)	-	-
SPLEEN	0 (10)	-	-	0 (10)	-	-
STOMACH: FORESTOMACH	(10)	(10)	(10)	(10)	(10)	(10)
Hemorrhage, submucosal	00	00	02	02	00	00
Infiltrate, polymorphonuclear and mononuclear cells	00	00	02	04	00	00
Infiltrate, polymorphonuclear	00	00	00	04	00	00
Necrosis, mucosal	00	00	02	04	00	00
Hyperkeratosis	00	00	03	08	00	00
Hyperplasia	00	00	03	09	00	00
STOMACH: GLANDULAR	(10)	(10)	(10)	(10)	(10)	(10)
Hemorrhage, mucosal	00	00	02	07	00	00
TESTES	0 (10)	-	-	0 (10)	-	-
THYMUS	(10)	-	-	(10)	-	-
Apoptotic necrosis	04	-	-	05	-	-
Hemorrhage	01	-	-	01	-	-
THYROID WITH PARATHYROID	0 (10)	-	-	(10)	-	-
TRACHEA	0 (10)	-	-	0 (10)	-	-
URINARY BLADDER	0 (10)	-	-	0 (10)	-	-

Key: Number in parenthesis indicates number of animals examined.

 

 

SEX: FEMALE
GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
NUMBER OF ANIMALS	10	10	10	10	10	10
ADRENAL GLANDS	(10)	-	-	(10)	-	-
Dilatation	02			01
AORTA	0 (10)	-	-	0 (10)	-	-
AXILLARY LYMPH NODES	0 (10)	-	-	0 (10)	-	-
BONE MARROW (STERNUM)	0 (10)	-	-	0 (10)	-	-
BRAIN (3 LEVELS)	0 (10)	-	-	0 (10)	-	-
CECUM	0 (10)	-	-	0 (10)	-	-
COLON	0 (10)	-	-	0 (10)	-	-
DUODENUM	0 (10)	-	-	0 (10)	-	-
HEART	0 (10)	-	-	0 (10)	-	-
ILEUM WITH PEYER’S PATCHES	0 (10)	-	-	0 (10)	-	-
JEJUNUM	0 (10)	-	-	0 (10)	-	-
KIDNEYS	(10)	-	-	(10)	-	-
Mineralization	01			00
LIVER	0 (10)	-	-	(10)	-	-
LUNGS	(10)	-	-	(10)	-	-
Infiltrate, mononuclear cells	01			01
Histiocytosis	01			00
Hemorrhage, alveolar	00			01
MESENTERIC LYMPH NODES	0 (10)	-	-	0 (10)	-	-
OESOPHAGUS	0 (10)	-	-	0 (10)	-	-

Key: Number in parenthesis indicates number of animals examined.

 

SEX: FEMALE
GROUP	G1	G2	G3	G4	G1R	G4R
DOSE (mg/kg bw)	(0)	(100)	(300)	(750)	(0)	(750)
NUMBER OF ANIMALS	10	10	10	10	10	10
OVARIES	0 (10)	-	-	0 (10)	-	-
PANCREAS	(10)	-	-	(10)	-	-
Vacuolation	01			00
RECTUM	0 (10)	-	-	0 (10)	-	-
SKELETAL MUSCLE	0 (10)	-	-	0 (10)	-	-
SPINAL CORD (CERVICAL, MID THORACIC, LUMBAR)	0 (10)	-	-	0 (10)	-	-
SPLEEN	0 (10)	-	-	0 (10)	-	-
STOMACH: FORESTOMACH	(10)	(10)	(10)	(10)	(10)	(10)
Infiltrate, polymorphonuclear and  mononuclear cells	00	00	01	03	00	00
Necrosis	00	00	01	01	00	00
Hyperkeratosis	00	00	0	04	00	00
Hyperplasia	00	00	0	06	00	00
Congestion	00	00	0	01	00	00
Hemorrhage	00	00	0	01	00	00
STOMACH: GLANDULAR	(10)	(10)	(10)	(10)	(10)	(10)
Hemorrhage	00	00	00	02	00	00
THYMUS	(10)	-	-	(10)	-	-
Apoptotic Necrosis	01			06
Hemorrhage	02			01
Hyperplasia, epithelial	00			02
THYROID	0 (10)	-	-	0 (10)	-	-
TRACHEA	0 (10)	-	-	0 (10)	-	-
URINARY BLADDER	0 (10)	-	-	0 (10)	-	-
UTERUS	(10)	(04)	(02)	(10)	(02)	(04)
Dilatation, luminal	02	04	02	02	02	04

Key: Number in parenthesis indicates number of animals examined.

Applicant's summary and conclusion

Conclusions:

The administration of HOSTAPON TPHC to Wistar rats by oral gavage, at dose levels of 100, 300 and 750 mg/kg/day, resulted in no treatment-related changes in the body weights, feed consumption, haematology, clinical biochenistry and organ weights. Treatment related effects of clinical signs, macroscopic findings and microscopic effects observed in high dose group (750 mg/kg/day) were not considered adverse and were reversible as demonstrated in the recovery animals. Therefore, a ‘No Observed Adverse Effect Level` (NOAEL) is considered to be highest dose employed in the study i.e.,750 mg/kg/day.

Executive summary:

This study was conducted based on OECD Tèst Guideline 408 `Repeated Dose 90-Day Oral Toxicity Study in Rodents` and followed current principles of GLP. The purpose of this study was to assess the toxicity of HOSTAPON TPHC when administered once daily to Wistar rats by oral gavage for a period of 90 consecutive days. The reversibility of treatment-related changes was assessed after a treatment free 28 day recovery period. The doses selected for main study were 100, 300 and 750 mg/kg body weight. The test item was formulated in distilled water and administered once daily for 90 days to Wistar rats at dose levels of 100 (low dose, G2), 300 (intermediate dose, G3) and 750 (high dose, G4) mg/kg body weight/day. Vehicle control group 0 mg/kg (G1) was administered with distilled water for 90 days. Each group consisting of 10 males and 10 females were used for this study. In addition, 10 males and 10 females each were allocated to G1R (Control Recovery) and G4R (High Dose Recovery) respectively.

No mortalities were observed during the treatment period of 90 days and recovery period of 28 days. No clinical signs of toxicity were observed in control (G1) and low dose group (G2) animals during the study. Dullness was observed in few animals of intermediate group (G3). In addition to dullness, burrowing was observed in all animals of high dose group (G4) and high dose recovery group (G4R). All animals recovered from these signs after treatment withdrawal. This indicates that these clinical signs attribute to treatment. No adverse effects on food consumption were detected for treated animals when compared with vehicle control. No adverse effect on body weight and bodyweight gain (%) was detected for treated animals when compared with vehicle control. Opthalmological examiniation did not reveal any abnormalities in the vehicle control and high dose treated animals at the end of treatment period. No toxicologically releavant findings were noted in hematology, clinical biochemistry and urine alaysis parameters at the end of treatment and recovery period. No significant test item-related differences in absolute and relative organ weights were observed. Post-mortem examinations revealed thickened fore stomach and reddish foci in glandular stomach of animals of high dose (750 mg/kg) and intermediate dose (300 mg/kg) groups. Based upon the total sperm count, testes weights, gross and microscopic examination it was found that test item had no effect on the spermatogenic function of the testes under the present conditions of the study. Macroscopic examination revealed relevant findings to that of gross pathological changes as hemorrhage in forestomach and glandular stomach. Histopathologically, in forestomach polymorphonuclear and/or mononuclear cells infiltrate, necrosis, hyperkeratosis and hyperplasia of squamous epithelium were observed. These changes were reversible as demonstrated in the recovery animals.  The histopathological changes observed in the forestomach of the treated animals may be owed to the local irritant effect of the test item at mentioned doses and treatment period and considered as port of entry effects and are considered to be of minor or no relevance for human health hazard / risk assessment as elaborated hereafter in more detail.

Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. In assessing the relevance of such findings in rodents aspects like the method of administration (e. g. gavage versus feeding) and the applicability of the forestomach to human organs (e. g. tissue concordance) have to be considered. Direct doses to the forestomach as it is the case via oral gavage, promotes the irritation of the epithelial lining of the forestomach due to a physical trauma by the gavage needle and/or chemical-induced irritation associated with the deposition of high doses of test material which then comes directly in contact with the forestomach epithelium for long periods of time. Beside the absence of a forestomach in humans, both aspects are neither representative of natural pathways of exposure nor relevant to human exposure conditions. Local irritation to the forestomach mucosa as such may cause local injury that stimulates compensatory cell proliferation. As an

example, forestomach tumors from repeated gavage dosing of ethyl acrylate were demonstrated to be an effect related specifically to the gavage administration rather than a substance specific effect (NTP, 2000).

Tissue concordance issues are also of importance when discussing specific toxicological responses of chemicals. In this context, dose-response profiles for a chemical are tissue specific (Klaassen, 1996). When assessing the applicability of rodent forestomach effects to humans, it is therefore important to consider that humans do not have a functional analogue of a rodent forestomach. Although the human esophagus and glandular stomach are histologically similar organs, there are fundamental functional and anatomical differences to the rodent forestomach. As the rodent forestomach has primarily storage and predigestion functions, tissue doses and tissue exposure times are not equivalent to a. m. organs in humans. Additionally, the forestomach mucosa in rodents has potent regenerative power, as it is to be expected for tissues which are exposed for long periods to irritant stimuli like acids and/or other aggressive food constituents. Abnormal continuous stimulation of this regenerative capacity by simple mechanical and chemical irritation can result in extensive hyperplasia of the rodent forestomach (Harrison, 1992). Structural differences like the lack of a protective lining in combination with higher pH levels after dosing may also contribute to a greater susceptibility and vulnerability of the rodent forestomach. In fact, several attempts to demonstrate similar reactivity for the esophageal and stomach mucosa in various species were unsuccessful, suggesting that these organs are less sensitive than the rodent forestomach (Wilbourn et al., 1986; Wester and Kroe, 1988). This view was also reached by the European Food Safety Authority (EFSA) during a re-evaluation of butylated hydroxyanisole (BHA), an antioxidant for which long-term gavage studies have demonstrated proliferative changes in the rodent forestomach. In its assessment, the EFSA expert panel concluded that forestomach hyperplasia in rodents may no longer be considered relevant for human risk assessment (EFSA, 2011). This view is also reflected by the endpoint specific guidance given by ECHA (Chapter R.7.2.1).

Based on the findings from this guideline conform 90-day subchronic oral toxicity study, the `no observed adverse effect level` (NOAEL) is considered to be 750 mg/kg body weight per day. This result is in good agreement with findings from a subacute 28 -day oral toxicity study in rats, where a NOAEL of 1000 mg/kg body weight per day was revealed.