6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Endpoint summary

Description of key information

No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived for 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2016 to 30 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

yes

Remarks:

see "Any other information on materials and methods incl. tables" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Internal identification number: 207570/A
Identification: LOWINOX® TBP-6
Appearance: White powder
Batch: C034J0059
Purity/Composition: 99.8 %
Test item storage: At room temperature
Stable under storage conditions until: 12 May 2017 (expiry date)

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality).

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Source F0:Charles River Deutschland, Sulzfeld, Germany.

Age at start pretest: Females - approximately 11 weeks.

Age at start F0-treatment: Males - approximately 10 weeks / Females: approximately 13 weeks.

Number of F0-animals: 48 females and 40 males.

At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study.

Acclimatization F0: At least 5 days prior to start of pretest (females) or treatment (males).

Health inspection F0: At least upon receipt of the animals.

Randomization F0: Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Identification F0: During pretest (females) and treatment (males and females): by earmark and tattoo. Reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo.

Mating procedures
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
 
Parturition
The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 424 pups.

Identification of pups
On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Culling
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (see section 0). Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Animal Husbandry
Room number: Room A0.07 (A0.23 for motor activity measurements).
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod is between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).

Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).

Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).

Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Lactation
Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30 40 minutes.

General
Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. (Test Facility Study No. 513607 was used for DCS).

Vehicle:

propylene glycol

Details on oral exposure:

Treatment - Parental Animals
Method
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. (Test Facility Study No. 513607 was used for DCS).
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Treatment period: Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-45 days.
Routinely, females that are littering are left undisturbed. In this study, female nos. 52, 55 and 59 (Group 2) and 73 (Group 4) were not dosed on one occasion as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Treatment - Pups
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of doses was conducted using a Liquid chromatography diode array detector (LC-DAD)

Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90-110%. It demonstrated that the analytical method was adequate for the determination of the test item in the test samples.

Test samples
In the Group 1 formulation, no test item was detected.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-45 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dosing group

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg (Test Facility Study No. 513610), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

Positive control:

None.

Observations and examinations performed and frequency:

Parental Animals
Mortality / Viability: At least twice daily.

Clinical signs: Clinical observations (detailed clinical signs and arena) were conducted from start of treatment onwards up to the day prior to necropsy at least at 1 hour (± 30 min)) after dosing based on the peak period of anticipated effects. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head. The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and started at 1 hour (± 30 min) after dosing.

Body weights: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Pups
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.

Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.

Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
Sex: Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see "Any other information on materials and methods incl. tables"

HISTOPATHOLOGY: Yes, see "Any other information on materials and methods incl. tables"

Other examinations:

Parental animals
Estrous cycle determination
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Pups:
Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; 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.
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.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Slight salivation was noted after dosing, starting after about two weeks of treatment, at all dose levels, most frequently at 150 and 500 mg/kg. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
One female at 500 mg/kg (no. 76) showed lethargy, shallow respiration and piloerection towards the end of the gestation period (on one or two days). Based on the incidental occurrence of these signs of distress and the absence of abnormalities in her body weight development and food consumption, these findings were considered not to be toxicologically relevant.
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Pups:
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of the clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

Parental animals: No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Parental animals: Body weight and body weight gain were not affected by treatment.

Pups: Body weights of pups were considered not to be affected by treatment.
Compared to controls, mean body weights of male and female pups at 500 mg/kg were higher from birth until PND 13. The relative differences gradually decreased from about 20% at PND 1 (statistically significant) to about 10% at PND 13 (no longer statistically significant). The higher means at 500 mg/kg were particularly due to the higher (exceeding normal limits) weights and weight gain of the pups of one small litter (no. 73, two male and two female pups). The higher pup weights in this small litter are consistent with the generally recognized inverse relation between litter size and pup body weight. Pups of the other litters at 500 mg/kg had normal body weights at birth and thereafter (except for those of litter no. 71 which had slightly lower weights at PND 13). For these reasons, the higher mean pup weights at 500 mg/kg were considered to reflect normal biological variation rather than an effect of the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Parental animals: No treatment-related changes in food consumption before or after allowance for body weight were noted.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: No toxicologically relevant changes occurred in haematology parameters of treated rats.
Isolated statistically significant variations noted in haematology parameters in males were unrelated to treatment or not toxicologically relevant due to the lack of a dose-related response or a slightly high concurrent control value.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
The statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the lack of a dose-related response, slight magnitude of the difference from controls (values in treated rats remained within normal limits), and/or a slightly low concurrent control value.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were not affected by treatment.

Pups: Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was similar across the groups, except for a statistically significantly lower hind limb grip strength in males at 500 mg/kg. This finding was not accompanied by a decrease in fore limb grip strength and the values at 500 mg/kg were within the normal range for male rats of this strain and age. Therefore, the lower hind limb grip strength in males at 500 mg/kg was considered not to be toxicologically relevant.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: There were no treatment-related alterations in organ weights.
The statistically significant variations noted in organ weights of males were unrelated to treatment due to the lack of a dose-related pattern

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals: There were no treatment-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Pups: No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted (mostly in pups found dead) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the thyroid gland of males, an increased incidence and severity (up to slight) of follicular cell hypertrophy was present starting at 150 mg/kg/day, and colloid alteration was present at 500 mg/kg (up to slight).
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. This included the recorded findings of control animals which erroneously received the test item.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Description (incidence and severity):

There were 1/10 couples treated at 50 mg/kg (female/male no. 51/11, total litter loss), 1/10 couples treated at 150 mg/kg (female/male no. 69/29, not mated) and 2/10 couples treated at 500 mg/kg (female/male no. 77/37, not pregnant; no. 79/39, total litter loss) that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain the reproductive failure or total litter loss.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.

Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Control female no. 46 had an irregular cycle and extended di-estrus during paring was noted for female no. 69 at 150 mg/kg. These findings were considered to be unrelated to treatment because their incidence was within normal limits and showed no dose-related trend.
 

Details on results:

Accuracy
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the Group 1 formulation.
Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: non-adverse microscopic changes in the thyroids of males at 500 mg/kg

Remarks on result:

not determinable

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

 Microscopic Examination

Test item-related microscopic findings were noted in the thyroid gland of males treated at 150 or 500 mg/kg and are summarized in the table below.

	Males
Dose level (mg/kg bw):	0	50	150	500
Thyroid gland^a	5	5	5	5
Follicular cell hypertrophy
Minimal	1	1	3	1
Slight	-	-	1	4
Colloid alteration
Minimal	-	-	-	1
Slight	-	-	-	3

^a = Number of tissues examined from each group.

                    

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
AT WEEK 4
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP FORE	MEAN	1288	1162	1439	1373
GRAM	ST.DEV	176	120	178	187
	N	5	5	5	5
GRIP HIND	MEAN	612	557	581	497 *
GRAM	ST.DEV	65	57	44	93
	N	5	5	5	5

 

 

FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
LACTATION
HEARING	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL L	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
PUPIL R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
STATIC R	MEDIAN	0	0	0	0
SCORE 0/1	N	5	5	5	5
GRIP FORE	MEAN	840	1025	870	1009
GRAM	ST.DEV	122	206	228	245
	N	5	5	5	5
GRIP HIND	MEAN	613	690	621	680
GRAM	ST.DEV	88	67	66	81
	N	5	5	5	5

 

MOTOR ACTIVITY MALES   AT WEEK 4	TEST SUMMARY
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
Total Movements	MEAN1	3794	2571	2772	3065
	ST.DEV	2378	182	1016	669
	N	5	5	5	5
Ambulations	MEAN1	852	587	625	842
	ST.DEV	594	72	334	280
	N	5	5	5	5

 

 

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

1Group mean of all intervals

2combined

MOTOR ACTIVITY TEST SUMMARY
FEMALES
AT LACTATION
	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
TotalMovements                MEAN1	3492	3270	2229	3235
ST.DEV	1449	1151	413	818
N	5	5	5	5
Ambulations                        MEAN1	848	680	545	773
ST.DEV	355	345	140	284
N	5	5	5	5

 

 

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

1Group mean of all intervals combined

 

BODY WEIGHTS (GRAM) SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING DAY 1	MEAN	298	297	294	300
WEEK 1	ST.DEV	7.4	11.1	13.0	9.4
	N	10	10	10	10
DAY 8	MEAN	319	320	315	321
WEEK 2	ST.DEV	8.5	16.4	17.5	12.6
	N	10	10	10	10
MATING PERIOD DAY 1	MEAN	339	341	335	343
WEEK 1	ST.DEV	9.5	20.6	21.5	16.7
	N	10	10	10	10
DAY 8	MEAN	351	357	347	357
WEEK 2	ST.DEV	10.4	20.4	19.3	17.7
	N	10	10	10	10
DAY 15	MEAN	368	375	365	374
WEEK 3	ST.DEV	12.2	22.7	21.3	20.4
	N	10	10	10	10

 

 

FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
PRE MATING DAY 1	MEAN	221	225	222	226
WEEK 1	ST.DEV	8.2	11.5	7.6	14.3
	N	10	10	10	10
DAY 8	MEAN	222	226	224	228
WEEK 2	ST.DEV	8.9	12.5	9.5	15.8
	N	10	10	10	10
MATING PERIOD DAY 1	MEAN	229	232	231	235
WEEK 1	ST.DEV	9.2	13.9	8.7	17.3
	N	10	10	10	10
DAY 8	MEAN			263
WEEK 2	ST.DEV N			--- 1
DAY 15 WEEK 3	MEAN ST.DEV			268 ---
	N			1
DAY 22	MEAN			257
WEEK 4	ST.DEV N			--- 1
DAY 29	MEAN			260
WEEK 5	ST.DEV N			--- 1

 

 

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

 

F0-GENERATION                                                                                                                                                                                       

 

BODY WEIGHTS (GRAM) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
POST COITUM DAY 0	MEAN	231	235	232	238
	ST.DEV.	10.3	14.5	10.0	15.7
	N	10	10	9	9
DAY 4	MEAN	245	248	248	256
	ST.DEV.	10.9	16.1	11.2	16.0
	N	10	10	9	9
DAY 7	MEAN	253	256	255	264
	ST.DEV.	10.1	16.7	12.0	18.5
	N	10	10	9	9
DAY 11	MEAN	269	271	271	277
	ST.DEV.	9.9	17.6	14.3	20.6
	N	10	10	9	9
DAY 14	MEAN	279	284	283	286
	ST.DEV.	11.3	19.7	15.5	20.9
	N	10	10	9	9
DAY 17	MEAN	299	304	305	309
	ST.DEV.	11.6	25.2	16.9	22.8
	N	10	10	9	9
DAY 20	MEAN	336	341	346	341
	ST.DEV.	16.3	34.2	18.6	28.4
	N	10	10	9	9
LACTATION DAY 1	MEAN	260	263	264	272
	ST.DEV.	9.3	20.4	16.3	21.9
	N	10	10	9	9
DAY 4	MEAN	273	276	276	281
	ST.DEV.	15.5	19.8	15.8	24.0
	N	10	9	9	9
DAY 7	MEAN	284	287	289	292
	ST.DEV.	15.6	18.7	16.5	23.2
	N	10	9	9	8
DAY 13	MEAN	302	299	297	306
	ST.DEV.	16.0	21.5	17.5	21.9
	N	10	9	9	8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

BODY WEIGHT GAIN (%) SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING 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	7	8	7	7
WEEK 2	ST.DEV	2.0	2.2	2.1	1.6
	N	10	10	10	10
MATING PERIOD DAY 1	MEAN	14	15	14	14
WEEK 1	ST.DEV	2.9	3.5	3.0	2.6
	N	10	10	10	10
DAY 8	MEAN	18	20	18	19
WEEK 2	ST.DEV	3.5	3.7	3.1	2.9
	N	10	10	10	10
DAY 15	MEAN	24	26	24	24
WEEK 3	ST.DEV	4.2	4.2	3.4	3.5
	N	10	10	10	10

 

 

FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
PRE MATING 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	0	0	1	1
WEEK 2	ST.DEV	2.5	1.4	1.7	1.8
	N	10	10	10	10
MATING PERIOD DAY 1	MEAN	3	3	4	4
WEEK 1	ST.DEV	2.6	2.5	2.9	3.5
	N	10	10	10	10
DAY 8	MEAN			15
WEEK 2	ST.DEV N			--- 1
DAY 15 WEEK 3	MEAN ST.DEV			17 ---
	N			1
DAY 22	MEAN			12
WEEK 4	ST.DEV N			--- 1
DAY 29	MEAN			14
WEEK 5	ST.DEV N			--- 1

 

 

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

 

F0-GENERATION                                                                                                                                                                                       

 

BODY WEIGHT GAIN (%) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
POST COITUM DAY 0	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	10	10	9	9
DAY 4	MEAN	6	6	7	8
	ST.DEV.	1.8	1.8	1.8	2.8
	N	10	10	9	9
DAY 7	MEAN	9	9	10	11
	ST.DEV.	1.7	1.9	2.2	3.3
	N	10	10	9	9
DAY 11	MEAN	16	15	17	16
	ST.DEV.	2.5	2.1	3.1	3.4
	N	10	10	9	9
DAY 14	MEAN	20	20	22	20
	ST.DEV.	2.1	2.5	3.4	2.8
	N	10	10	9	9
DAY 17	MEAN	30	29	32	30
	ST.DEV.	3.0	4.5	3.5	4.7
	N	10	10	9	9
DAY 20	MEAN	45	45	49	43
	ST.DEV.	3.0	8.3	3.6	7.6
	N	10	10	9	9
LACTATION DAY 1	MEAN	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	10	10	9	9
DAY 4	MEAN	5	5	5	3
	ST.DEV.	3.1	2.9	3.0	3.4
	N	10	9	9	9
DAY 7	MEAN	10	9	10	8
	ST.DEV.	3.1	3.4	4.1	2.4
	N	10	9	9	8
DAY 13	MEAN	16	14	13	13
	ST.DEV.	3.6	4.4	6.8	2.6
	N	10	9	9	8

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 

 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING DAYS 1-8	MEAN	22	22	22	23
WEEKS 1-2	ST.DEV	0.9	0.1	0.1	0.7
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	21	22	22	23
WEEKS 2-3	ST.DEV	0.2	0.1	0.5	1.3
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER PRE MATI...	MEAN	22	22	22	23
MATING PERIOD
DAYS 1-8	MEAN	23	23	23	23
WEEKS 1-2	ST.DEV	0.7	0.2	0.4	1.0
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	22	22	22	23
WEEKS 2-3	ST.DEV	0.5	0.1	0.8	1.0
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER MATING P...	MEAN	22	22	22	23
FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
PRE MATING DAYS 1-8	MEAN	15	15	15	15
WEEKS 1-2	ST.DEV	0.6	0.7	0.7	0.7
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	14	14	15	16
WEEKS 2-3	ST.DEV	0.6	0.4	0.4	0.6
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER PRE MATI...	MEAN	15	15	15	15

 

F0-GENERATION                                                                                                                                                                                       

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
POST COITUM DAYS 0-4	MEAN	16	16	16	19
	ST.DEV.	2.8	2.8	3.0	3.8
	N	10	10	9	9
DAYS 4-7	MEAN	18	18	18	20
	ST.DEV.	1.6	2.2	1.7	3.4
	N	10	10	9	9
DAYS 7-11	MEAN	19	18	19	20
	ST.DEV.	1.3	2.3	2.4	3.2
	N	10	10	9	9
DAYS 11-14	MEAN	21	21	22	22
	ST.DEV.	1.1	2.2	3.0	2.4
	N	10	10	9	9
DAYS 14-17	MEAN	20	20	20	22
	ST.DEV.	0.6	2.4	2.2	2.9
	N	10	10	9	9
DAYS 17-20	MEAN	23	22	24	24
	ST.DEV.	2.2	2.5	2.5	2.8
	N	10	10	9	9
MEAN OF MEANS		19	19	20	21
LACTATION DAYS 1-4	MEAN	25	27	25	26
	ST.DEV.	4.1	3.0	2.8	5.3
	N	6	6	8	9
DAYS 4-7	MEAN	37	38	37	37
	ST.DEV.	4.3	3.5	3.6	5.2
	N	10	9	9	8
DAYS 7-13	MEAN	49	46	46	47
	ST.DEV.	3.3	3.8	4.4	5.4
	N	10	9	9	8
MEAN OF MEANS		37	37	36	36

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PRE MATING DAYS 1-8	MEAN	68	70	69	71
WEEKS 1-2	ST.DEV	1.9	0.2	1.1	2.3
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	67	69	69	70
WEEKS 2-3	ST.DEV	0.0	0.4	2.3	4.1
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER PRE MATI...	MEAN	68	69	69	71
MATING PERIOD
DAYS 1-8	MEAN	64	64	65	65
WEEKS 1-2	ST.DEV	1.3	1.0	0.8	2.7
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	58	58	60	61
WEEKS 2-3	ST.DEV	0.8	0.4	2.0	2.3
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER MATING P...	MEAN	61	61	63	63
FEMALES
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
PRE MATING DAYS 1-8	MEAN	67	66	68	67
WEEKS 1-2	ST.DEV	1.0	1.5	2.0	1.3
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	65	63	67	68
WEEKS 2-3	ST.DEV	0.9	0.3	0.7	1.1
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER PRE MATI...	MEAN	66	64	67	68

 

F0-GENERATION                                                                                                                                                                                       

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
POST COITUM DAYS 0-4	MEAN	66	65	65	73
	ST.DEV.	10.6	9.3	12.7	14.4
	N	10	10	9	9
DAYS 4-7	MEAN	70	69	72	76
	ST.DEV.	6.2	4.7	5.3	9.4
	N	10	10	9	9
DAYS 7-11	MEAN	70	67	71	73
	ST.DEV.	4.4	5.2	6.5	8.5
	N	10	10	9	9
DAYS 11-14	MEAN	74	72	77	76
	ST.DEV.	4.3	4.5	8.7	5.4
	N	10	10	9	9
DAYS 14-17	MEAN	68	66	66	70
	ST.DEV.	2.2	4.5	5.8	6.1
	N	10	10	9	9
DAYS 17-20	MEAN	68	66	69	71
	ST.DEV.	7.0	6.4	7.2	6.8
	N	10	10	9	9
MEAN OF MEANS		69	67	70	73
LACTATION DAYS 1-4	MEAN	93	98	91	92
	ST.DEV.	12.8	8.5	7.8	18.1
	N	6	6	8	9
DAYS 4-7	MEAN	130	133	126	125
	ST.DEV.	12.1	14.5	11.1	13.4
	N	10	9	9	8
DAYS 7-13	MEAN	161	154	155	154
	ST.DEV.	9.9	10.3	12.8	16.0
	N	10	9	9	8
MEAN OF MEANS		128	128	124	124

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 

 

HAEMATOLOGY SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT WBC	MEAN	6.9	7.5	5.7	6.8
10E9/L	ST.DEV	2.0	1.4	0.9	1.3
	N	5	5	5	5
Neutrophils	MEAN	14.3	14.6	13.6	12.1
%WBC	ST.DEV	3.1	3.9	3.4	1.7
	N	5	5	5	4
Lymphocytes	MEAN	82.9	82.7	83.6	85.1
%WBC	ST.DEV	2.7	4.6	3.3	1.9
	N	5	5	5	4
Monocytes	MEAN	1.7	1.8	1.8	1.9
%WBC	ST.DEV	0.3	0.5	0.6	0.3
	N	5	5	5	4
Eosinophils	MEAN	1.0	0.9	1.0	0.9
%WBC	ST.DEV	0.5	0.5	0.2	0.2
	N	5	5	5	4
Basophils	MEAN	0.1	0.1	0.1	0.1
%WBC	ST.DEV	0.1	0.0	0.1	0.0
	N	5	5	5	4
Red blood cells	MEAN	8.88	8.71	8.78	8.62
10E12/L	ST.DEV	0.21	0.29	0.32	0.34
	N	5	5	5	5
Reticulocytes	MEAN	2.2	2.5	2.7	2.7
%RBC	ST.DEV	0.3	0.4	0.3	0.6
	N	5	5	5	5
RDW	MEAN	11.8	12.9	12.1	12.1
%	ST.DEV	0.6	2.1	0.7	0.3
	N	5	5	5	5
Haemoglobin	MEAN	10.0	9.8	10.0	9.7
mmol/L	ST.DEV	0.2	0.2	0.3	0.2
	N	5	5	5	5
Haematocrit	MEAN	0.451	0.447	0.463	0.449
L/L	ST.DEV	0.013	0.007	0.018	0.013
	N	5	5	5	5
MCV	MEAN	50.7	51.4	52.8	52.2
fL	ST.DEV	1.5	1.7	2.1	1.7
	N	5	5	5	5
MCH	MEAN	1.12	1.13	1.14	1.13
fmol	ST.DEV	0.03	0.04	0.04	0.03
	N	5	5	5	5
MCHC	MEAN	22.19	22.05	21.58 *	21.67
mmol/L	ST.DEV	0.33	0.52	0.30	0.22
	N	5	5	5	5
Platelets	MEAN	837	757	755	683 *
10E9/L	ST.DEV	136	20	48	50
	N	5	5	5	5

 

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

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

 

 

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT PT	MEAN	17.9	18.6	18.1	18.4
s	ST.DEV	0.6	0.3	0.5	0.4
	N	5	5	5	5
APTT	MEAN	15.0	17.1	15.4	16.4
s	ST.DEV	1.3	2.3	1.4	2.4
	N	5	5	5	5

 

HAEMATOLOGY SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT WBC	MEAN	7.3	7.2	7.6	8.1
10E9/L	ST.DEV	1.0	1.3	1.3	2.1
	N	5	5	5	5
Neutrophils	MEAN	52.3	45.6	43.5	45.6
%WBC	ST.DEV	9.1	8.4	4.4	6.4
	N	5	5	5	5
Lymphocytes	MEAN	41.5	48.7	50.9	48.8
%WBC	ST.DEV	7.8	8.2	4.2	6.0
	N	5	5	5	5
Monocytes	MEAN	4.2	4.5	4.3	4.9
%WBC	ST.DEV	1.4	1.3	0.7	1.2
	N	5	5	5	5
Eosinophils	MEAN	2.0	1.1	1.1	0.6
%WBC	ST.DEV	1.5	0.5	0.3	0.4
	N	5	5	5	5
Basophils	MEAN	0.1	0.1	0.1	0.1
%WBC	ST.DEV	0.0	0.0	0.0	0.0
	N	5	5	5	5
Red blood cells	MEAN	7.79	7.89	7.70	7.57
10E12/L	ST.DEV	0.50	0.70	0.46	0.58
	N	5	5	5	5
Reticulocytes	MEAN	3.4	3.2	2.9	3.5
%RBC	ST.DEV	0.7	0.5	0.5	0.5
	N	5	5	5	5
RDW	MEAN	13.1	13.7	14.7	13.4
%	ST.DEV	1.1	1.1	1.3	0.5
	N	5	5	5	5
Haemoglobin	MEAN	9.3	9.4	9.2	9.2
mmol/L	ST.DEV	0.9	1.0	0.6	0.7
	N	5	5	5	5
Haematocrit	MEAN	0.433	0.434	0.431	0.442
L/L	ST.DEV	0.036	0.047	0.038	0.047
	N	5	5	5	5
MCV	MEAN	55.6	55.0	56.0	58.3
fL	ST.DEV	1.7	2.2	2.0	1.8
	N	5	5	5	5
MCH	MEAN	1.19	1.19	1.19	1.21
fmol	ST.DEV	0.05	0.04	0.02	0.02
	N	5	5	5	5
MCHC	MEAN	21.43	21.66	21.28	20.77
mmol/L	ST.DEV	0.32	0.40	0.52	0.60
	N	5	5	5	5
Platelets	MEAN	782	793	803	801
10E9/L	ST.DEV	78	36	126	184
	N	5	5	5	5

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT PT	MEAN	17.3	17.4	17.7	17.3
s	ST.DEV	0.5	1.4	0.8	0.6
	N	5	5	5	5
APTT	MEAN	13.1	13.4	12.7	14.2
s	ST.DEV	1.7	2.5	0.7	2.0
	N	5	5	5	5

 

CLINICAL BIOCHEMISTRY SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT ALAT	MEAN	49.8	47.7	43.1	44.6
U/L	ST.DEV	20.5	10.4	9.3	6.8
	N	5	5	5	5
ASAT	MEAN	86.4	82.8	85.2	85.8
U/L	ST.DEV	4.0	4.7	10.0	13.3
	N	5	5	5	5
ALP	MEAN	179	162	151	169
U/L	ST.DEV	62	57	59	39
	N	5	5	5	5
Total protein	MEAN	65.0	64.3	63.0	63.0
g/L	ST.DEV	2.1	1.6	1.2	2.1
	N	5	5	5	5
Albumin	MEAN	34.7	34.1	33.7	33.2 *
g/L	ST.DEV	1.2	0.7	0.6	1.1
	N	5	5	5	5
Total bilirubin	MEAN	2.5	2.0 *	2.1	2.1
umol/L	ST.DEV	0.3	0.2	0.3	0.3
	N	5	5	5	5
Urea	MEAN	8.1	8.0	7.9	7.7
mmol/L	ST.DEV	2.5	1.1	1.5	0.2
	N	5	5	5	5
Creatinine	MEAN	41.1	41.1	41.1	40.3
umol/L	ST.DEV	2.9	1.2	1.5	1.3
	N	5	5	5	5
Glucose	MEAN	7.94	8.26	8.55	8.19
mmol/L	ST.DEV	1.07	1.27	1.19	0.82
	N	5	5	5	5
Cholesterol	MEAN	1.92	1.89	1.85	2.03
mmol/L	ST.DEV	0.16	0.25	0.36	0.40
	N	5	5	5	5
Bile Acids	MEAN	28.7	13.6 *	16.1 *	16.1 *
umol/L	ST.DEV	11.8	2.6	6.5	6.7
	N	5	5	5	5
Sodium	MEAN	141.0	140.6	141.5	141.2
mmol/L	ST.DEV	0.2	0.3	1.5	0.4
	N	5	5	5	5
Potassium	MEAN	3.59	4.02 **	3.75	3.82 *
mmol/L	ST.DEV	0.11	0.13	0.16	0.13
	N	5	5	5	5
Chloride	MEAN	102	103	102	103
mmol/L	ST.DEV	1	1	1	1
	N	5	5	5	5
Calcium	MEAN	2.60	2.58	2.56	2.60
mmol/L	ST.DEV	0.08	0.05	0.03	0.03
	N	5	5	5	5

 

 

 

 

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

 
  - Page 19 -

- Page 19 -

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT Inorg.Phos	MEAN	1.99	2.05	1.94	1.99
mmol/L	ST.DEV	0.20	0.20	0.18	0.21
	N	5	5	5	5
Total T4	MEAN	4.93	4.00	4.06	4.34
ug/dL	ST.DEV	1.58	0.76	0.81	0.92
	N	10	10	10	10

 

 

 

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                              

                                              

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT ALAT	MEAN	75.1	59.9	79.7	76.5
U/L	ST.DEV	17.8	11.6	29.2	13.9
	N	5	5	5	5
ASAT	MEAN	118.1	100.0	112.1	112.1
U/L	ST.DEV	24.7	15.0	29.2	22.4
	N	5	5	5	5
ALP	MEAN	108	125	162	129
U/L	ST.DEV	29	38	67	62
	N	5	5	5	5
Total protein	MEAN	59.0	61.5	62.9	60.8
g/L	ST.DEV	9.7	2.1	1.3	2.3
	N	5	5	5	5
Albumin	MEAN	31.7	32.8	33.0	31.8
g/L	ST.DEV	4.3	0.1	0.6	1.5
	N	5	5	5	5
Total bilirubin	MEAN	2.7	2.3	2.3	2.2
umol/L	ST.DEV	0.3	0.6	0.4	0.4
	N	5	5	5	5
Urea	MEAN	14.2	10.9	11.2	10.2
mmol/L	ST.DEV	4.9	0.6	0.9	2.1
	N	5	5	5	5
Creatinine	MEAN	47.5	45.8	44.4	43.2
umol/L	ST.DEV	3.0	2.5	3.0	3.1
	N	5	5	5	5
Glucose	MEAN	7.03	7.98	7.92	7.96
mmol/L	ST.DEV	1.00	1.07	0.98	0.70
	N	5	5	5	5
Cholesterol	MEAN	1.80	2.15	2.30	2.18
mmol/L	ST.DEV	0.17	0.35	0.39	0.56
	N	5	5	5	5
Bile Acids	MEAN	60.7	58.0	39.3	32.6
umol/L	ST.DEV	60.7	72.3	26.1	7.8
	N	5	5	5	5
Sodium	MEAN	134.6	137.8 **	136.8 *	137.2 **
mmol/L	ST.DEV	0.9	1.7	1.2	0.9
	N	5	5	5	5
Potassium	MEAN	3.62	3.53	3.98	3.79
mmol/L	ST.DEV	0.29	0.46	0.58	0.50
	N	5	5	5	5
Chloride	MEAN	94	96	97	97
mmol/L	ST.DEV	2	3	1	2
	N	5	5	5	5
Calcium	MEAN	2.53	2.63	2.70	2.61
mmol/L	ST.DEV	0.23	0.12	0.10	0.04
	N	5	5	5	5

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT Inorg.Phos	MEAN	3.08	2.91	2.83	2.68
mmol/L	ST.DEV	0.38	0.55	0.53	0.34
	N	5	5	5	5
Total T4	MEAN	---	---	---	---
ug/dL	ST.DEV	---	---	---	---
	N	0	0	0	0

 

MACROSCOPIC FINDINGS SUMMARY MALES	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT Animals examined	10	10	10	10
Animals without findings	7	7	9	10
Animals affected	3	3	1	0
Epididymides
Nodule(s) Cowper's gland	1	1	0	0
Agenesis	0	1	0	0
Thymus Focus/foci	1	1	1	0
Mandibular lymph n
Discolouration	1	0	0	0
FEMALES
	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
INTERCURRENT DEATH
Animals examined		1		1
Animals affected		1		1
General observations
Total litter loss		1		1
END OF TREATMENT Animals examined	10	9	10	9
Animals without findings	7	4	5	5
Animals affected	3	5	5	4
Stomach Focus/foci	3	1	1	1
Uterus Contains fluid	0	0	1	1
Clitoral glands
Focus/foci	0	2	3	1
Thyroid gland Reduced in size	0	1	0	0
Thymus Focus/foci	0	1	0	0
Reduced in size	0	1	0	0
Discolouration	0	1	1	0
Skin Alopecia	0	1	1	1

 

 

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

 

 

 

 

                                   

                                   

 

ORGAN WEIGHTS (GRAM) SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT BODY W.	MEAN	349	356	345	354
(GRAM)	ST.DEV	12	24	19	19
	N	10	10	10	10
BRAIN	MEAN	2.07	2.05	2.01	2.04
(GRAM)	ST.DEV	0.09	0.07	0.05	0.04
	N	5	5	5	5
HEART	MEAN	0.928	1.011	0.895	1.003
(GRAM)	ST.DEV	0.069	0.069	0.023	0.092
	N	5	5	5	5
LIVER	MEAN	9.22	9.22	9.05	9.34
(GRAM)	ST.DEV	0.93	0.93	0.73	1.19
	N	5	5	5	5
THYROIDS	MEAN	0.015	0.015	0.018 *	0.016
(GRAM)	ST.DEV	0.002	0.002	0.003	0.003
	N	10	10	10	10
THYMUS	MEAN	0.334	0.409	0.353	0.365
(GRAM)	ST.DEV	0.071	0.052	0.042	0.125
	N	5	5	5	5
KIDNEYS	MEAN	2.40	2.46	2.39	2.43
(GRAM)	ST.DEV	0.06	0.12	0.19	0.15
	N	5	5	5	5
ADRENALS	MEAN	0.060	0.064	0.072	0.058
(GRAM)	ST.DEV	0.008	0.008	0.006	0.007
	N	5	5	5	5
SPLEEN	MEAN	0.586	0.672	0.640	0.639
(GRAM)	ST.DEV	0.055	0.091	0.110	0.089
	N	5	5	5	5
TESTES	MEAN	3.46	3.57	3.59	3.47
(GRAM)	ST.DEV	0.28	0.32	0.27	0.19
	N	10	10	10	10
PROSTATE GLAND	MEAN	0.893	0.942	1.043	0.916
(GRAM)	ST.DEV	0.140	0.183	0.111	0.104
	N	10	10	10	10
EPIDIDYMIDES	MEAN	1.134	1.096	1.091	1.077
(GRAM)	ST.DEV	0.085	0.118	0.065	0.028
	N	10	10	10	10
SEMINAL VESICLES	MEAN	1.638	1.415 *	1.424 *	1.514
(GRAM)	ST.DEV	0.155	0.164	0.215	0.210
	N	10	10	10	10

 

 

 

 

 

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT BODY W.	MEAN	349	356	345	354
(GRAM)	ST.DEV	12	24	19	19
	N	10	10	10	10
BRAIN	MEAN	0.59	0.58	0.58	0.58
(%)	ST.DEV	0.04	0.04	0.03	0.03
	N	5	5	5	5
HEART	MEAN	0.264	0.285	0.258	0.282
(%)	ST.DEV	0.025	0.021	0.011	0.007
	N	5	5	5	5
LIVER	MEAN	2.62	2.59	2.61	2.62
(%)	ST.DEV	0.17	0.12	0.12	0.15
	N	5	5	5	5
THYROIDS	MEAN	0.004	0.004	0.005 *	0.004
(%)	ST.DEV	0.001	0.001	0.001	0.001
	N	10	10	10	10
THYMUS	MEAN	0.094	0.116	0.102	0.101
(%)	ST.DEV	0.017	0.016	0.008	0.028
	N	5	5	5	5
KIDNEYS	MEAN	0.68	0.69	0.69	0.68
(%)	ST.DEV	0.03	0.03	0.04	0.02
	N	5	5	5	5
ADRENALS	MEAN	0.017	0.018	0.021 *	0.016
(%)	ST.DEV	0.002	0.002	0.003	0.002
	N	5	5	5	5
SPLEEN	MEAN	0.167	0.189	0.184	0.180
(%)	ST.DEV	0.020	0.020	0.025	0.023
	N	5	5	5	5
TESTES	MEAN	0.99	1.01	1.04	0.98
(%)	ST.DEV	0.07	0.09	0.10	0.06
	N	10	10	10	10
PROSTATE GLAND	MEAN	0.257	0.264	0.302 *	0.259
(%)	ST.DEV	0.045	0.046	0.033	0.026
	N	10	10	10	10
EPIDIDYMIDES	MEAN	0.325	0.309	0.317	0.305
(%)	ST.DEV	0.026	0.037	0.027	0.018
	N	10	10	10	10
SEMINAL VESICLES	MEAN	0.471	0.399 *	0.412 *	0.427
(%)	ST.DEV	0.051	0.052	0.051	0.053
	N	10	10	10	10

 

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT BODY W.	MEAN	266	266	263	267
(GRAM)	ST.DEV	12	17	15	28
	N	10	9	10	10
BRAIN	MEAN	1.91	1.91	1.96	1.94
(GRAM)	ST.DEV	0.02	0.05	0.10	0.07
	N	5	5	5	5
HEART	MEAN	0.805	0.841	0.805	0.874
(GRAM)	ST.DEV	0.076	0.072	0.051	0.097
	N	5	5	5	5
LIVER	MEAN	8.79	9.22	9.20	9.99
(GRAM)	ST.DEV	0.34	0.82	0.78	1.02
	N	5	5	5	5
THYROIDS	MEAN	0.014	0.013	0.015	0.015
(GRAM)	ST.DEV	0.002	0.003	0.003	0.002
	N	10	9	10	10
THYMUS	MEAN	0.214	0.182	0.199	0.236
(GRAM)	ST.DEV	0.038	0.052	0.049	0.042
	N	5	5	5	5
KIDNEYS	MEAN	1.80	1.98	1.98	2.03
(GRAM)	ST.DEV	0.10	0.14	0.28	0.23
	N	5	5	5	5
ADRENALS	MEAN	0.075	0.078	0.069	0.076
(GRAM)	ST.DEV	0.009	0.005	0.015	0.004
	N	5	5	5	5
SPLEEN	MEAN	0.518	0.527	0.521	0.570
(GRAM)	ST.DEV	0.089	0.056	0.088	0.078
	N	5	5	5	5
OVARIES	MEAN	0.111	0.121	0.111	0.119
(GRAM)	ST.DEV	0.010	0.024	0.012	0.010
	N	5	5	5	5
UTERUS	MEAN	0.386	0.503	0.464	0.559
(GRAM)	ST.DEV	0.052	0.111	0.084	0.211
	N	5	5	5	5

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
END OF TREATMENT BODY W.	MEAN	266	266	263	267
(GRAM)	ST.DEV	12	17	15	28
	N	10	9	10	10
BRAIN	MEAN	0.72	0.72	0.76	0.69
(%)	ST.DEV	0.03	0.04	0.02	0.04
	N	5	5	5	5
HEART	MEAN	0.305	0.317	0.310	0.308
(%)	ST.DEV	0.025	0.015	0.009	0.024
	N	5	5	5	5
LIVER	MEAN	3.33	3.47	3.55	3.52
(%)	ST.DEV	0.12	0.25	0.25	0.27
	N	5	5	5	5
THYROIDS	MEAN	0.005	0.005	0.006	0.006
(%)	ST.DEV	0.001	0.001	0.001	0.001
	N	10	9	10	10
THYMUS	MEAN	0.080	0.068	0.077	0.083
(%)	ST.DEV	0.011	0.018	0.021	0.010
	N	5	5	5	5
KIDNEYS	MEAN	0.68	0.74	0.76	0.71
(%)	ST.DEV	0.03	0.04	0.08	0.03
	N	5	5	5	5
ADRENALS	MEAN	0.028	0.029	0.027	0.027
(%)	ST.DEV	0.004	0.000	0.005	0.002
	N	5	5	5	5
SPLEEN	MEAN	0.197	0.199	0.201	0.201
(%)	ST.DEV	0.038	0.019	0.031	0.025
	N	5	5	5	5
OVARIES	MEAN	0.042	0.045	0.043	0.042
(%)	ST.DEV	0.005	0.006	0.005	0.002
	N	5	5	5	5
UTERUS	MEAN	0.146	0.188	0.178	0.195
(%)	ST.DEV	0.016	0.031	0.030	0.066
	N	5	5	5	5

 

 

REPRODUCTIONDATASUMMARY	GROUP 1CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
Females paired	10	10	10	10
Females mated	10	10	9	10
Pregnant females	10	10	9	9
Females with total litter loss on Day 1	0	1	0	0
Females with living pups on Day 1	10	9	9	9
Mating index (%)	100	100	90	100
(Females mated / Females paired) * 100
Fertility index (%)	100	100	90	90
(Pregnant females / Females paired) * 100
Conception index (%)	100	100	100	90
(Pregnant females / Females mated) * 100
Gestation index (%)	100	90	100	100
(Females with living pups on Day 1 / Pregnant females) * 100

 

F0-GENERATION - POSTCOITUM                                                                                                                                                         

 

DAY OF THE PAIRING PERIOD	GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
NUMBER OF FEMALES MATED 1	2	1	2	4
2	3	1	3	2
3	3	4	3	1
4	2	4	1	3
MEDIAN PRECOITAL TIME	3	3	2	2
MEAN PRECOITAL TIME	2.5	3.1	2.3	2.3
N	10	10	9	10

 

IMPLANTATION SITES FEMALES	SUMMARY
		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
NECROPSY Implantations	MEAN	12.5	12.1	13.4	10.0
	ST.DEV	2.4	4.4	1.6	4.2
	N	10	10	9	9

 

F0-GENERATION - LACTATION
	GROUP 1 CONTROL	GROUP 2	GROUP 3	GROUP 4
		50 MG/KG	150 MG/KG	500 MG/KG
LITTERS TOTAL	10	10	9	9
DURATION OF GESTATION MEAN (+)	21.2	21.6	21.1	21.7
ST.DEV.	0.4	0.5	0.3	0.7
N	10	10	9	9
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	3	1	2	1
TOTAL	4	1	3	1
MEAN (+)	0.4	0.1	0.3	0.1
ST.DEV.	0.7	0.3	0.7	0.3
N	10	10	9	9
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	51 / 49	52 / 48	45 / 55	53 / 47
TOTAL	113	108	109	85
MEAN (+)	11.3	10.8	12.1	9.4
ST.DEV.	2	4.6	2.3	4.2
N	10	10	9	9
POSTNATAL LOSS
% OF LIVING PUPS	5.3	1.9	5.5	3.5
LITTERS AFFECTED (#)	4	2	4	2
TOTAL (#)	6	2	6	3
MEAN (+)	0.6	0.2	0.7	0.3
ST.DEV.	1	0.4	0.9	0.7
N	10	10	9	9
CULLED PUPS
TOTAL	28	35	32	22
LIVING PUPS DAY 4 P.P.
TOTAL	79	71	71	60
MEAN (+)	7.9	7.1	7.9	6.7
ST.DEV.	0.3	2.5	0.3	2.8
N	10	10	9	9
BREEDING LOSS DAYS 5 - 13 P.P.
% OF LIVING PUPS AT DAY 4 P.P.	0	0	1.4	0
LITTERS AFFECTED (#)	0	0	1	0
TOTAL (#)	0	0	1	0
MEAN (+)	0	0	0.1	0
ST.DEV.	0	0	0.3	0
N	10	10	9	9
LIVING PUPS DAY 13 P.P.
% OF MALES / FEMALES (#)	48 / 52	51 / 49	49 / 51	53 / 47
TOTAL	79	71	70	60
MEAN (+)	7.9	7.1	7.8	6.7
ST.DEV.	0.3	2.5	0.4	2.8
N	10	10	9	9

 

 

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

DEVELOPMENTAL DATA
	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	50 MG/KG	150 MG/KG	500 MG/KG
Total number of offspring born	117	109	112	86
Total number of uterine implantation sites	125	121	121	90
Number of live offspring on Day 1 after littering	113	108	109	85
Number of live offspring on Day 4 (before culling)	107	106	103	82
Number of live offspring on Day 4 (after culling)	79	71	71	60
Number of live offspring on Day 13 after littering	79	71	70	60
Post-implantation survival index (%)	94	90	93	96
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	97	99	97	99
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	95	98	94	96
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%)	100	100	99	100
(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100

BODY WEIGHTS OF PUPS(GRAM)

 

 

F0-GENERATION -LACTATION                                                                                                                                                              

 

DAY	SEX		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
1	M	MEAN	6.0	6.5	6.0	7.1 *
		ST.DEV.	0.7	0.6	0.9	1.3
		N	10	9	9	9
	F	MEAN	5.7	6.2	5.6	6.8 *
		ST.DEV.	0.8	0.7	0.9	1.4
		N	10	9	9	9
	M+F	MEAN	5.9	6.4	5.8	7.0 *
		ST.DEV.	0.7	0.6	0.9	1.4
		N	10	9	9	9
4	M	MEAN	8.7	9.4	8.7	10.5
		ST.DEV.	1.3	1.0	1.3	2.4
		N	10	9	9	8
	F	MEAN	8.4	8.9	8.1	10.2 *
		ST.DEV.	1.3	1.2	1.3	2.3
		N	10	9	9	8
	M+F	MEAN	8.5	9.2	8.4	10.4 *
		ST.DEV.	1.2	1.0	1.3	2.4
		N	10	9	9	8
7	M	MEAN	14.4	15.1	14.3	16.6 *
		ST.DEV.	1.5	1.1	1.6	3.1
		N	10	9	9	8
	F	MEAN	14.0	14.5	13.3	16.2
		ST.DEV.	1.3	1.2	1.7	3.3
		N	10	9	9	8
	M+F	MEAN	14.2	14.8	13.8	16.4
		ST.DEV.	1.3	1.1	1.7	3.2
		N	10	9	9	8
13	M	MEAN	28.5	28.5	27.7	30.9
		ST.DEV.	1.6	1.2	2.1	5.1
		N	10	9	9	8
	F	MEAN	27.7	27.7	26.3	29.8
		ST.DEV.	1.6	1.4	2.4	5.2
		N	10	9	9	8
	M+F	MEAN	28.1	28.1	26.9	30.4
		ST.DEV.	1.5	1.3	2.2	5.1
		N	10	9	9	8

 

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

 

F0-GENERATION -LACTATION                                                                                                                                                              

 

		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
anogenital dist M mm	MEAN	2.48	2.59	2.60	2.70
	ST. DEV.	0.14	0.09	0.13	0.27
	N	10	9	9	9
anogenital dist F mm	MEAN	1.05	0.97	0.94	1.11
	ST.DEV.	0.17	0.02	0.18	0.21
	N	10	9	9	9
Number of nipples	MEAN	0.00	0.00	0.00	0.00
	MEDIAN (+)	0.00	0.00	0.00	0.00
	N	10	9	9	8

 

  

 

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

 

CORRECTED ANOGENITAL DISTANCE SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PND 1 norm anog dist M	MEAN	1.37	1.39	1.43	1.41
mm	ST.DEV	0.07	0.03	0.10	0.06
	N	10	9	9	9
norm anog dist F	MEAN	0.59	0.53	0.53	0.58
mm	ST.DEV	0.10	0.02	0.10	0.09
	N	10	9	9	9

 

CLINICAL BIOCHEMISTRY SUMMARY MALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PUPS (PND 13-15) Total T4	MEAN	6.60	7.36	5.76	6.65
ug/dL	ST.DEV	1.36	2.01	1.13	0.83
	N	10	9	9	8

 

CLINICAL BIOCHEMISTRY SUMMARY FEMALES		GROUP 1 CONTROL	GROUP 2 50 MG/KG	GROUP 3 150 MG/KG	GROUP 4 500 MG/KG
PUPS (PND 13-15) Total T4	MEAN	6.09	6.32	5.62	5.88
ug/dL	ST.DEV	1.31	1.03	1.05	1.01
	N	10	9	9	8

 

Conclusions:

LOWINOX® TBP-6 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females that delivered were treated for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 13 days of lactation (for 50-56 days). Females which failed to deliver healthy offspring were treated for for 38-45 days.

Formulation analysis showed that the formulations were prepared accurately and homogenously. Stability of formulations under (simulated) experimental conditions was confirmed as part of the analytical method development and validation study.

No toxicologically relevant changes were noted in the in-life parameters examined in this study up to 500 mg/kg (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption). Treatment-related in-life findings were limited to slight salivation after dosing at all dose levels, most frequently at 150 and 500 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity.

Microscopic examination showed treatment-related changes in the thyroid of male rats, characterized by an increased incidence and severity of follicular cell hypertrophy starting at 150 mg/kg, and alteration of the colloid at 500 mg/kg. There were no other treatment-related microscopic findings. Furthermore, thyroid weight and the serum level of thyroid hormone T4 were not affected by treatment. Based on this and the low degree (up to slight) of the follicular cell hypertrophy and colloid alteration, these findings were regarded as non-adverse.
No treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® TBP-6 in rats by oral gavage.

The study was based on the following guidelines:

•       OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

•       OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

•       OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

•       OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

•       EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

•       OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

•       OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg (Test Facility Study No. 513610; see APPENDIX 5), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

The test item, LOWINOX® TBP-6, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 38-45 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

There were no toxicologically relevant changes in the in-life parameters up to 500 mg/kg.

Slight salivation was noted after dosing at all dose levels, most frequently at 150 and 500 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity.

Treatment-related microscopic changes were present in the thyroid of male rats, characterized by an increased incidence and severity of follicular cell hypertrophy starting at 150 mg/kg, and alteration of the colloid at 500 mg/kg. These findings were regarded as non-adverse based on their low degree (up to slight) and the absence of other treatment-related findings.

No treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.

In conclusion:

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® TBP-6 in rats by oral gavage.

The study was based on the following guidelines:

•       OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

•       OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

•       OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

•       OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

•       EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

•       OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

•       OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

The test item, LOWINOX® TBP-6, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 38-45 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed once during the study to assess accuracy and homogeneity.

There were no toxicologically relevant changes in the in-life parameters up to 500 mg/kg. Slight salivation was noted after dosing at all dose levels, most frequently at 150 and 500 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity.

Treatment-related microscopic changes were present in the thyroid of male rats, characterized by an increased incidence and severity of follicular cell hypertrophy starting at 150 mg/kg, and alteration of the colloid at 500 mg/kg. These findings were regarded as non-adverse based on their low degree (up to slight) and the absence of other treatment-related findings.

No treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Justification for classification or non-classification

Under the conditions of the study, no treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.  In accordance with CLP Regulation (EC) 1272/2008, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item is not classified for repeated dose toxicity.