Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a one generation reproductive toxicity study the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reason / purpose:
reference to same study
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Wistar Han™: HsdRccHan™: WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd, Oxon, UK.

- Age at study initiation:
Parental animals - approximately six to eight weeks old
F1 generation - not applicable

- Weight at study initiation:
At the start of treatment the Subgroup A males weighed 263 to 305 g, the Subgroup A females weighed 140 to 199 g,
Subgroup B females were introduced to the study. Subgroup B females weighed 169 to 213 g

- Fasting period before study:
Not fasted

- Housing:
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).

- Diet (e.g. ad libitum):
A pelleted diet
(Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK) was used.

- Water (e.g. ad libitum):
The animals were allowed free access to food and water. Mains drinking water was supplied from polycarbonate bottles attached to the cage

- Acclimation period:
The animals were acclimatised for at least 6 days (Subgroup A) and 7 days (Subgroup B) during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21±2ºC

- Humidity (%):
55 ±15%

- Air changes (per hr):
The rate of air exchange was at least fifteen air changes per hour

- Photoperiod (hrs dark / hrs light):
low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: Day 1 To: Day 90
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of Thixatrol Max at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not reported

Details on mating procedure:
Subgroup A males were paired with Subgroup B females on a one male : one female basis within each dose group, for a period of up to twenty-one days. Once mating was confirmed, the male was paired with another female from the same dose group until mating was confirmed. This process was repeated until all females from each dose group were confirmed to have mated or twenty-one days had elapsed. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material formulations were analysed for concentration of Thixatrol Max during the study period.
The concentration of Thixatrol Max in the test material formulations was determined by HPLC using an external standard technique.
The results indicate that the prepared formulations were within +/- 5% of the nominal concentration.
Duration of treatment / exposure:
For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days.
For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.
Frequency of treatment:
For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Details on study schedule:
Subgroup A males were paired with Subgroup B females on a one male : one female basis within each dose group, for a period of up to twenty-one days. Once mating was confirmed, the male was paired with another female from the same dose group until mating was confirmed. This process was repeated until all females from each dose group were confirmed to have mated or twenty-one days had elapsed. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Each pregnant Subgroup B female was observed at approximately 0830, 1230 and 1630 hours and around the period of. expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

On completion of parturition, the number of live and dead offspring was recorded. On Day 1 post parium, all surviving offspring within each litter were individually identified using ink tattoos on the feet (and tails).

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive was recorded daily
iii) The sex of individual offspring was recorded on Days 1,4,7, 14,21 post partum
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring and total litter weights on Day 1,4,7, 14 and 21 post partum
vi) Necropsy findings of offspring

All live offspring were assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post parium and mid-air righting reflex on Day 17 post partum. Pupillary reflex and auditory startle response were performed on Day 21 post partum.

Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
Subgroup A:
10 male and 10 female

Subgroup B
0 Males and 22 Females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of Thixatrol Max at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

- Rationale for animal assignment:
Subgroup A animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure, and Subgroup B females were randomly allocated to tretament groups using random letter tables. The group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
Not applicable.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
For Subgroup B females, individual bodyweights were recorded on Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0,7, 14 and 21 post coitum and on Days 1,4,7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
For Subgroup A females, food consumption was recorded for each cage group at weekly intervals throughout the study.

Dietary intake for Subgroup A males and Subgroup B females was recorded weekly for each cage group until pairing. Following confirmation of mating, dietary intake for Subgroup B females was recorded on Days ato 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4,4 to 7,7 to 14 and 14 to 21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
Oestrous cyclicity (parental animals):
Daily vaginal smears were taken from all Subgroup B females from the start of treatment until pairing. The smears were dried and stained with Giemsa stain. The slides were then examined microscopically and the stage of the oestrus cycle was recorded.
Sperm parameters (parental animals):
The following characteristics were recorded for individual sperm analysis:
Concentration (millions/ml): Based on the total number of motile and non-motile cells identified.

Motility: The ratio of recorded motile cells to the total concentration.

Progressive Velocity (VSL): The straight line distance between the beginning and end of the track divided by time elapsed.
Litter observations:
Litter Data
On completion of parturition, the number of live and dead offspring was recorded. On Day 1 post parium, all surviving offspring within each litter were individually identified using ink tattoos on the feet (and tails).

For each litter the following was recorded: i) Number of offspring born
ii) Number of offspring alive was recorded daily
iii) The sex of individual offspring was recorded on Days 1,4,7, 14,21 post partum
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring and total litter weights on Day 1,4,7, 14 and 21 post partum
vi) Necropsy findings of offspring
Postmortem examinations (parental animals):
On completion of the mating phase all Subgroup A animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. Subgroup B females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 21 post parium.

On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.

All animals (including offspring from Subgroup B females) were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were removed from all Subgroup A animals at the end of the study and dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries (with Oviducts)
Pituitary
Prostate
Seminal vesicles (with coagulating gland)
Spleen
Testes
Thymus
Uterus (with Cervix)

The following organs were weighed from all Subgroup B females at termination:
Pituitary
Ovaries (with Oviducts)
Uterus (with Cervix)

Samples of the following tissues were removed from all Subgroup A animals and preserved in buffered 100/0 formalin, except where stated:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland
Colon
Duodenum
Ovaries (with Oviducts)
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Right Epididymis
Eyes*
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary glands
Muscle (skeletal)
Oesophagus
Spinal cord (cervical, mid-thoracic
and lumbar)
Spleen
Stomach
Right Testis
Thymus
Thyroid/parathyroid
Tongue
Trachea
Urinary bladder
Uterus (with Cervix)
Vagina

Samples of the following tissues were removed from all Subgroup B females and preserved in buffered 10% formalin:
Mammary glands
Ovaries (with Oviducts)
Pituitary
Uterus (with Cervix)
Vagina
Postmortem examinations (offspring):
Any interim death offspring were killed via an intracardiac or intraperitoneal injection of sodium pentobarbitone followed by cervical dislocation. All surviving offspring were terminated via carbon dioxide asphyxiation.

On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value. Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥0.05 (not significant)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p < 0.001 +++ --- ***
p < 0.01 ++ -- **
p < 0.05 + - *
p < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is nondirectional.
Reproductive indices:
Oestrus Cycle
The stage of oestrus were classified according to the following criteria:
Dioestrus (D) - Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P) - Predominantly epithelial cells, usually in significant numbers.
Early Oestrus (E1) - Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Oestrus (E2) - Predominantly cornified cells usually seen as clumps of cells.
Metoestrus (M) - Large numbers of leucocytes with discrete clumps of cornified cells.

The oestrous cycles are classified according to the following criteria:
Normal oestrous - The pattern of daily stages of oestrous show a four to five day cycle, which is generally repeated over 21 days.
Extended oestrous - The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous - The predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle - An irregular length of oestrous cycle is observed over the 21 day evaluation period.

Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.

Mating Performance and Fertility i) Gestation Length ii) Parturition Index

Offspring viability indices:
The following indices were calculated for each group from group mean data:
Live birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index 1(%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
Viability Index 2 (%)= (Number of offspring alive on Day 7/Number of offspring alive on Day 4) x 100
Viability Index 3 (%) = (Number of offspring alive on Day 14/Number of offspring alive on Day 7) x100
Viability Index 4 (%) =(Number of offspring alive on Day 21/Number of offspring alive on Day 14) x100
Viability Index 5 (%) = (Number of offspring alive on Day 21/Number of offspring alive on Day 1) x100

ii) Sex Ratio (% males)
Group mean values calculated from each litter value on Days 1, 4, 7, 14 and 21 using the following formula:
(Number of male offspring/Total number of offspring) x100

iii) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated as follows:
% Pre-implantation loss = (Number of Corpora Lutea - Number of implantation sites/Number of corpora lutea) x100
% Post-implantation loss = [(Group number of implantation sites - Total number of offspring born)/Number of implantation sites] x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinically observable signs of toxicity were detected during the treatment period. Incidents of increased salivation were detected soon after dosing, and up to one hour after dosing for Subgroup A animals of either sex and Subgroup B females treated with 1000 mg/kg/day. The effect was more pronounced in the males. Similar effects were also detected for Subgroup A animals of either sex and Subgroup B females treated with 250 mg/kg/day. The effect was also evident in Subgroup A animals of either sex treated with 50 mg/kg/day. Increased salivation is often recorded following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and, in isolation, is considered not to be indicative of systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse effect on bodyweight change was detected for both Subgroup A and Subgroup B animals during the study period. Subgroup A males treated at all dose level showed statistically significant reductions in bodyweight gain when compared to controls during Week 10 of the study. A convincing dose-related response was not apparent, therefore, the reductions during this week were not considered to represent an adverse effect of treatment. A slight increase in bodyweight gain was detected for Subgroup B females treated with 1000 mg/kg/day females during the gestation and lactation phases of the study, however, statistical analysis of the data did not reveal significant intergroup differences and as such, these slight increases were considered to be of no toxicological importance.

No adverse effects on dietary intake or food efficiency (the ratio of bodyweight to dietary intake) were detected throughout the treatment period for both treated Subgroup A and Subgroup B animals when compared to their concurrent controls. Subgroup A males treated with 1000 and 250 mg/kg/day showed a slight increase in dietary intake during the treatment period, when compared to concurrent controls. These increases, in the absence of significant bodyweight gains in this group, were not considered to represent an "adverse effect of treatment. A slight increase in dietary intake was detected for 1000 mg/kg/day Subgroup 8 females during the pre-mating, gestation and lactation phases of the study and statistical significance was achieved for females treated with 1000 mg/kg/day during the first week of gestation. The significance achieved however was minimal (P<0.05), and as such, was not considered to represent an adverse effect of treatment.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no obvious differences in the oestrous cycles from treated Subgroup B females when compared to their concurrent controls. Extended oestrus (with one animal showing extended early oestrus) was occasionally observed throughout the control and treated groups. In the absence of any significant differences in pre-coital intervals or fertility, extended oestrus was not considered to be attributed to treatment with the test material.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related effects were detected for Subgroup A males following sperm motility, morphology or homogenisation resistance spermatid assessments. A statistically significant reduction in sperm progressive motility values was detected for Subgroup A males treated with 250 mg/kg/day when compared to their concurrent controls. The significance was minimal (P<0.05) and in the absence of a dose-related response or treatment-related effects on fertility, this finding was considered to be of no toxicological importance. Furthermore, cauda epididymal spermatid counts from all treated males were significantly lower than those obtained from the concurrent controls (P<0.05). These reductions correlated with statistically significant increases in epididymides weights throughout the treatment groups when compared to controls (P<0.01). The lower epididymal spermatid counts was considered to be attributable to three higher than expected values in the control group and the higher epididymis weights were considered to be attributed to lower than expected control values, rather than an effect of treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related differences in mating performance for treated Subgroup B females when compared to their concurrent controls. All animals mated, with a majority of animals mating within the first five days of pairing. Statistical analysis of pre-coital intervals did not reveal any significant intergroup differences. Positive evidence of mating was not detected for one control female, two 50 mg/kg/day females and three 1000 mg/kg/day females, however, these animals were pregnant and subsequently produced litters. There were no adverse effects observed on fertility or pregnancy status for treated
animals when compared to controls. There were four non-pregnant females in the control group, four non-pregnant females in the 50 mg/kg/day dose group, two nonpregnant females in the 250 mg/kg/day dose group and one non-pregnant female in the 1000 mg/kg/day dose group. No treatment-related effects on the lengths of the gestation period were detected for treated Subgroup B females when compared to their concurrent controls. The majority of
pregnant females showed gestation lengths between 22% and 23% days. Longer gestation lengths were detected for one control, one 50 mg/kg/day and two
250 mg/kg/day females, however, this did not impact on the litters produced, and was not considered to be related to treatment with the test material. Statistical analysis of the data did not reveal any significant intergroup differences.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects on organ weights were detected for animals from both Subgroup A and Subgroup B. Subgroup A males treated with 1000 and 250 mg/kg/day showed a statistically significant increase in left cauda weights (P<0.01) and left epididymis weights (P<0.05) when compared to - concurrent controls. In the absence of any treatment-related histopathological changes in these organs, these findings were not considered to be of
any toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related macroscopic abnormalities detected at termination of both the Subgroup A and Subgroup B animals. A number of macroscopic abnormalities were detected for animals from Subgroup A. Sloughing of the stomach was evident for one control male, and a yellow mass was
located near the pancreas for another control male. A red mass approximately 2 x 2 cm was attached to the pancreas of one male treated with 50 mg/kg/day, and a further male treated at this dose level showed a small left testis and epididymis. One male treated with 1000 mg/kg/day showed a reddened right eye and haemorrhaging of the left eye. Finally, a reddened thymus was observed from one Subgroup A female treated with 50 mg/kg/day. Macroscopic findings from Subgroup B females were confined to the presence of a dilated left eye with white foci for one female treated with 50 mg/kg/day and the presence of amass in the abdominal region approximately 2 x 2 cm for one female treated with 250 mg/kg/day. Generalised fur loss was also evident at termination
for another 250 mg/kg/day although this animal did not show any abnormal tissues or organs during the post-mortem procedure. Such findings are occasionally observed in laboratory maintained animals and are considered to have arisen incidentally.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects were detected in the tissues examined from both Subgroup A animals and females from Subgroup B. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed. Although group differences in the incidence or severity
of lesions occasionally attained statistical significance, none was considered to be related to treatment. The following conditions warrant specific mention:

SUBGROUP A
Adrenal glands: Cortical vacuolation was seen in a few control and treated animals and was of no toxicological significance in this investigation.
Bone marrow: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups for either sex.

Heart: Focal myocarditis was observed in the majority of control and treated males. This is a common background lesion in laboratory maintained rats, and although the incidence and severity grades observed in this investigation were rather higher than might normally be expected, there was no evidence of an effect of treatment. A single instance was seen among females.

Kidneys: Isolated groups of basophilic tubules were seen occasionally and are commonly encountered in the renal cortex of laboratory maintained rats. Focal corticomedullary mineralisation is commonly seen in the kidneys of laboratory maintained female rats and has no pathological significance at the severities or frequencies reported in this study. Hydronephrosis was also reported for two animals. This is widely considered to be a condition of congenital origin and in any event, it does not arise as a primary toxicological event. Globular accumulations of eosinophilic material, as a consequence of excessive accumulation of Cl2-microglobulin in renal proximal tubular epithelial cells, are occasionally encountered as a spontaneous change in male rats and are without
toxicological significance in this investigation.

Liver: Scattered mononuclear cell foci were observed in the majority of control and treated animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered. Centrilobular lipid-type vacuolation was seen for males without treatment-related group distribution. Isolated instances of hepatocyte enlargement and pigment accumulation were also observed as spontaneous conditions.

Lungs: Minimal severity of bronchus associated lymphoid tissue reported for most animals examined in the study, is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are similarly not suggestive of significant respiratory disease at the severity grades encountered.

Mesenteric Lymph nodes: Dilation of sinusoids was observed in a few animals as spontaneous change.

Oesophagus: Inflammatory cell infiltrates in the peripheral musculature is a commonly observed change that is considered to be related to the physical trauma of gavage dosing. The higher incidence of the lesion seem among high dose males compared with control animals was considered to be fortuitous.

Spleen: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.

Uterus: Dilatation of the uterine horns and keratinisation of the cervical epithelium are commonly observed cyclical conditions in laboratory maintained female rats. The higher incidence of these conditions among high dose females was considered to be of no toxicological significance.

Testis/Epididymis: No treatment-related changes were seen. Interstitial chronic inflammatory cell infiltrates were seen in the epididymis for a few animals and are commonly observed as a spontaneous condition in the epididymis of male rats.

Prostate: Interstitial chronic inflammatory cell infiltrates were observed for a few high dose males. Although the condition was not seen among control animals, this is a frequently observed spontaneous condition in laboratory maintained male rats and the group distribution of incidence in this study is considered to be unrelated to treatment.

SUBGROUPB
Mammary gland: Glandular hyperplasia is invariably observed in relation to pregnancy and lactation and unrelated to treatment with the test material.
Ovary and Oviducts: No significant histopathological changes were seen. An absence of corpora lutea was reported for one high dose female but this was of no toxicological significance.

Uterus: Focal haemorrhage, fibrosis, foam cell accumulations and haemosiderin pigment deposition were all observed with varying severity in the peripheral uterine tissues of previously pregnant animals. These changes are commonly encountered consequences of pregnancy and parturition in the female rat. Uterine dilatation and keratinisation of the cervical epithelium were also seen occasionally as normal cyclical changes. A subendometrial area of haemorrhage was seen for one control animal.

Vagina: No significant pathology was observed. Keratinisation of the epithelial lining and inflammatory cell debris are common vaginal findings in laboratory rats, being either cyclical in nature or consequential to previous pregnancy.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were seen, therefore the NOAEL has been determined to be 1000 mg/kg bw/day for both systemic and reproductive toxicity
Remarks on result:
other: Generation: P/F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
There were no treatment-related differences in the number of corpora lutea, implantation counts, pre- or post-implantation losses for treated animals when compared to controls and statistical analysis of the data did not reveal any significant intergroup differences.

One non-pregnant control female displayed two corpora lutea at termination, although there were no implantation sites present, suggesting a pre-implantation loss for this animal. This is a low incidence finding occasionally observed in studies of this type and as this finding was detected in a control animal, there was no correlation to treatment with the test material.

Litter sizes from treated animals were comparable to litters delivered by control dams. Although the data may suggest slightly larger litters were evident from treated animals when compared to controls, statistical analysis of the data did not reveal any significant intergroup differences. Furthermore, there were no significant differences in the viability of the litters from treated females when compared to those from the controls.

CLINICAL SIGNS (OFFSPRING)
Daily clinical observations of offspring did not reveal any findings considered to be attributed to test material toxicity. The clinical observations observed throughout the control and treated groups were low incidence findings observed in studies of this type and were considered not to be related to treatment with the test material.

BODY WEIGHT (OFFSPRING) & SEXUAL MATURATION (OFFSPRING)
There were no treatment-related effects detected in offspring growth or development from litters of treated animals when compared to litters from the controls.

Litter weights for treated groups were comparable to those from the control group. Meanoffspring bodyweights and bodyweight changes during the lactation period, for bothmales and females for treated litters were also comparable to controls. There were no treatment-related differences in the number of offspring successfully passing the surface righting assessments performed on Day 1 post parium or mid-air righting reflex assessments undertaken on Day 17 post parium. Furthermore, startle reflex and pupil reflex assessments performed on Day 21 post parium did not reveal any intergroup differences between treated and control groups. Statistical analysis of the data did not reveal any significant intergroup differences in the
parameters investigated.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for both interim death and terminal kill offspring throughout the control and treated groups. The incidental findings observed throughout the control and treatment groups were occassionally observed low incidence findings in animals of the age employed and not considered to represent treatment-related changes in the affected tissues or organs.

There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem findings of offspring did not reveal any treatment-related changes at the dose levels assessed in this study.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically significant effects at dose levels of up to 1000 mg/kg/day
Reproductive effects observed:
no
Conclusions:
Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.
Executive summary:

Introduction. The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983). The study was also designed to assess subchronic exposure to the test material to the rat and is based on the OECD Guidelines for Testing of Chemicals No 408 "Subchronic Oral Toxicity - Rodent" (Adopted 21 September 1998).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods. For the Subchronic phase (Subgroup A), the test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™: HsdRccHan™: WIST strain rats, for one hundred and eight consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology, blood chemistry and urinalytical investigations were undertaken for all Subgroup A animals during Week 10. Ophthalmoscopic examination was also performed on Subgroup A control and high dose animals once prior to the start of treatment and during Week 10. During Week 11, sensory reactivity, grip strength and motor activity assessments were performed.

During Week 8, females for the reproductive phase (Subgroup B) were introduced to the study. The test material was administered to gavage to three groups, each of twenty-two female Wistar HanTM: HsdRccHanTM: WIST strain rats,for at least twenty-eight days prior to mating and throughout the mating, gestation and lactation phases of the study at dose levels of 50, 250 and 1000 mg/kg/day. A control group of twenty-two females were dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored for Subgroup B females. Oestrus cycle assessments were also undertaken from the start of treatment until pairing.

After Week 11 of the study, Subgroup A males were paired with Subgroup B females on a one male: one female basis within each dose group, for a maximum of twenty-one days. Pairing was continued until all twenty-two females from each group had mated with a Subgroup A male from the same dose group. On completion of the mating phase, all Subgroup A animals, were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

All pregnant Subgroup B females were allowed to girth birth and maintain their offspring until Day 21 post parium. Offspring development was observed during this time. On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.

Results.

Mortality: There were no unscheduled deaths during the study in both Subgroup A or Subgroup B.

Clinical observations: No clinical:y observable signs of toxicity were detected throughout the treatment period for both Subgroup A and Subgroup B.

Behavioural assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Functional performance tests: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Sensory reactivity assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Bodyweight: No adverse effect on bodyweight development was detected for Subgroup A animals throughout the treatment period, or for Subgroup B females prior to mating, or during the gestation and lactation phases of the study.

Food consumption: No adverse effects on dietary intake or food efficiency were detected for Subgroup A animals during the treatment period, or for Subgroup B females during the pre-mating, gestation and lactation phases of the study.

Water consumption: No intergroup differences were detected for treated animals from both Subgroup A and Subgroup B when compared to their concurrent controls.

Ophthalmoscopy: No ocular effects were detected for high dose Subgroup A animals.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters investigated for Subgroup A animals.

Haematology: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Blood chemistry: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Oestrus cycle assessments: No significant differences in oestrus cycles were detected for treated Subgroup B females when compared to their concurrent controls.

Mating performance: There were no treatment-related differences in mating performance.

Fertility & pregnancy: There were no treatment-related effects detected on fertility or pregnancy status for treated animals when compared to controls.

Gestation length: No treatment-related effects on gestation lengths were detected for treated Subgroup B females when compared to controls.

Litter responses: There were no treatment-related effects detected for litters from treated animals when compared to controls. There were no differences in corpora lutea or pre/post implantation losses for treated animals when compared to controls.

No treatment-related effects were detected for offspring during the daily observations, litter weights or offspring bodyweights. Reflexological assessments did not reveal any significant differences between litters from treated and control dams. No treatmentrelated macroscopic abnormalities were detected at termination.

Semen assessment: No treatment-related effects were detected in sperm motility, morphology or homogenisation resistant spermatid counts from treated Subgroup A males when compared to concurrent controls.

Organ weights: No toxicologically significant intergroup differences were detected for both Subgroup A animals and Subgroup B females.

Necropsy: No treatment-related macroscopic abnormalities were detected for both Subgroup A and Subgroup B animals at terminal kill.

Histopathology: No treatment-related microscopic abnormalities were detected for animals from both Subgroup A and Subgroup B.

Conclusion: Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP study conducted in accordance with appropriate test guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Oral administration of the test substance by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

Embryotoxic / teratogenic effects:no effects. Remark: No toxicologically significant effects were detected.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 8th July 2008 and 10 March 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™: HsdRccHan™: WIST
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd, Oxon, UK.

- Age at study initiation:
Parental animals - approximately six to eight weeks old
F1 generation - not applicable

- Weight at study initiation:
At the start of treatment the Subgroup A males weighed 263 to 305 g, the Subgroup A females weighed 140 to 199 g,
Subgroup B females were introduced to the study. Subgroup B females weighed 169 to 213 g

- Fasting period before study:
Not fasted

- Housing:
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).

- Diet (e.g. ad libitum):
A pelleted diet
(Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK) was used.

- Water (e.g. ad libitum):
The animals were allowed free access to food and water. Mains drinking water was supplied from polycarbonate bottles attached to the cage

- Acclimation period:
The animals were acclimatised for at least 6 days (Subgroup A) and 7 days (Subgroup B) during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21±2ºC

- Humidity (%):
55 ±15%

- Air changes (per hr):
The rate of air exchange was at least fifteen air changes per hour

- Photoperiod (hrs dark / hrs light):
low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: Day 1 To: Day 90
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of Thixatrol Max at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material formulations were analysed for concentration of Thixatrol Max at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.
Details on mating procedure:
Subgroup A males were paired with Subgroup B females on a one male : one female basis within each dose group, for a period of up to twenty-one days. Once mating was confirmed, the male was paired with another female from the same dose group until mating was confirmed. This process was repeated until all females from each dose group were confirmed to have mated or twenty-one days had elapsed. Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals from both Subgroup A and Subgroup B were treated in an identical manner with 10 ml/kg/day of Arachis oil BP. For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Duration of test:
90 days
No. of animals per sex per dose:
Subgroup A:
10 male and 10 female

Subgroup B
0 Males and 22 Females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for three hours. Formulations were therefore prepared daily. Test material formulations were analysed for concentration of Thixatrol Max at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory during the study period. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration.

- Rationale for animal assignment:
Subgroup A animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure, and Subgroup B females were randomly allocated to tretament groups using random letter tables. The group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
For Subgroup B females, individual bodyweights were recorded on Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0,7, 14 and 21 post coitum and on Days 1,4,7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
For Subgroup A females, food consumption was recorded for each cage group at weekly intervals throughout the study.

Dietary intake for Subgroup A males and Subgroup B females was recorded weekly for each cage group until pairing. Following confirmation of mating, dietary intake for Subgroup B females was recorded on Days ato 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4,4 to 7,7 to 14 and 14 to 21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes

- Soft tissue examinations: Yes

- Skeletal examinations: Yes

- Head examinations: Yes
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value. Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥0.05 (not significant)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p < 0.001 +++ --- ***
p < 0.01 ++ -- **
p < 0.05 + - *
p < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is nondirectional.
Indices:
The following indices were calculated for each group from group mean data:
Live birth Index (%) = (Number of offspring alive on Day 1/Number of offspring born) x 100
Viability Index 1(%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
Viability Index 2 (%)= (Number of offspring alive on Day 7/Number of offspring alive on Day 4) x 100
Viability Index 3 (%) = (Number of offspring alive on Day 14/Number of offspring alive on Day 7) x100
Viability Index 4 (%) =(Number of offspring alive on Day 21/Number of offspring alive on Day 14) x100
Viability Index 5 (%) = (Number of offspring alive on Day 21/Number of offspring alive on Day 1) x100

ii) Sex Ratio (% males)
Group mean values calculated from each litter value on Days 1, 4, 7, 14 and 21 using the following formula:
(Number of male offspring/Total number of offspring) x100

iii) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated as follows:
% Pre-implantation loss = (Number of Corpora Lutea - Number of implantation sites/Number of corpora lutea) x100
% Post-implantation loss = [(Group number of implantation sites - Total number of offspring born)/Number of implantation sites] x 100
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: No toxicologically significant effects were detected.

Details on maternal toxic effects:
With regards to reproduction, administration of the test material did not affect mating performance, fertility or reproductive performance in both Subgroup A males or Subgroup B females. There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem findings of offspring did not reveal any treatment-related changes at the dose levels assessed in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No toxicologically significant effects were detected.

Details on embryotoxic / teratogenic effects:
With regards to reproduction, administration of the test material did not affect mating performance, fertility or reproductive performance in both Subgroup A males or Subgroup B females. There were no treatment-related effects detected in litter size or viability, growth or development of litters from treated animals and post-mortem findings of offspring did not reveal any treatment-related changes at the dose levels assessed in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxic / teratogenic effects:no effects. Remark: No toxicologically significant effects were detected.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.
Executive summary:

Introduction. The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983). The study was also designed to assess subchronic exposure to the test material to the rat and is based on the OECD Guidelines for Testing of Chemicals No 408 "Subchronic Oral Toxicity - Rodent" (Adopted 21 September 1998).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods. For the Subchronic phase (Subgroup A), the test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™: HsdRccHan™: WIST strain rats, for one hundred and eight consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology, blood chemistry and urinalytical investigations were undertaken for all Subgroup A animals during Week 10. Ophthalmoscopic examination was also performed on Subgroup A control and high dose animals once prior to the start of treatment and during Week 10. During Week 11, sensory reactivity, grip strength and motor activity assessments were performed.

During Week 8, females for the reproductive phase (Subgroup B) were introduced to the study. The test material was administered to gavage to three groups, each of twenty-two female Wistar HanTM: HsdRccHanTM: WIST strain rats,for at least twenty-eight days prior to mating and throughout the mating, gestation and lactation phases of the study at dose levels of 50, 250 and 1000 mg/kg/day. A control group of twenty-two females were dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored for Subgroup B females. Oestrus cycle assessments were also undertaken from the start of treatment until pairing.

After Week 11 of the study, Subgroup A males were paired with Subgroup B females on a one male: one female basis within each dose group, for a maximum of twenty-one days. Pairing was continued until all twenty-two females from each group had mated with a Subgroup A male from the same dose group. On completion of the mating phase, all Subgroup A animals, were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

All pregnant Subgroup B females were allowed to girth birth and maintain their offspring until Day 21 post parium. Offspring development was observed during this time. On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.

Results.

Mortality: There were no unscheduled deaths during the study in both Subgroup A or Subgroup B.

Clinical observations: No clinical:y observable signs of toxicity were detected throughout the treatment period for both Subgroup A and Subgroup B.

Behavioural assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Functional performance tests: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Sensory reactivity assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Bodyweight: No adverse effect on bodyweight development was detected for Subgroup A animals throughout the treatment period, or for Subgroup B females prior to mating, or during the gestation and lactation phases of the study.

Food consumption: No adverse effects on dietary intake or food efficiency were detected for Subgroup A animals during the treatment period, or for Subgroup B females during the pre-mating, gestation and lactation phases of the study.

Water consumption: No intergroup differences were detected for treated animals from both Subgroup A and Subgroup B when compared to their concurrent controls.

Ophthalmoscopy:No ocular effects were detected for high dose Subgroup A animals.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters investigated for Subgroup A animals.

Haematology:No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Blood chemistry: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Oestrus cycle assessments: No significant differences in oestrus cycles were detected for treated Subgroup B females when compared to their concurrent controls.

Mating performance: There were no treatment-related differences in mating performance.

Fertility & pregnancy: There were no treatment-related effects detected on fertility or pregnancy status for treated animals when compared to controls.

Gestation length: No treatment-related effects on gestation lengths were detected for treated Subgroup B females when compared to controls.

Litter responses: There were no treatment-related effects detected for litters from treated animals when compared to controls. There were no differences in corpora lutea or pre/post implantation losses for treated animals when compared to controls.

No treatment-related effects were detected for offspring during the daily observations, litter weights or offspring bodyweights. Reflexological assessments did not reveal any significant differences between litters from treated and control dams. No treatmentrelated macroscopic abnormalities were detected at termination.

Semen assessment: No treatment-related effects were detected in sperm motility, morphology or homogenisation resistant spermatid counts from treated Subgroup A males when compared to concurrent controls.

Organ weights: No toxicologically significant intergroup differences were detected for both Subgroup A animals and Subgroup B females.

Necropsy: No treatment-related macroscopic abnormalities were detected for both Subgroup A and Subgroup B animals at terminal kill.

Histopathology: No treatment-related microscopic abnormalities were detected for animals from both Subgroup A and Subgroup B.

Conclusion: Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Not classifed for rperoductive or developmental toxicology.

Oral administration of the test substance by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. Embryotoxic / teratogenic effects:no effects. Remark: No toxicologically significant effects were detected