Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 2009 to 28 September 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 the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan (UK) Ltd
- Age at study initiation: 10 - 12 wks
- Weight at study initiation: Males: 260.1 - 359.0 g; Females: 175.6 - 229.8 g
- Fasting period before study: Not specified
- Housing: Single, exclusive room. Animals were housed in groups of up to five (pre pairing and post pairing), one female with one male (pairing) or individually (mated females) in cages that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 1989).
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester). Shortly before littering pregnant females were provided with wood shavings (Datesand Ltd, Manchester) for the duration of the lactation period.
The bedding was considered not to have contained any contaminants at a level which might have affected the integrity or outcome of the study.
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
The diet and water were considered not to have contained any contaminants at a level which might have affected the integrity or outcome of the study.
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%. On one occasion during the acclimatisation period the relative humidity was above the specified protocol range (72%).
- Air changes (per hr): minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800 h) and 12 hours darkness.


IN-LIFE DATES: From: 8 July 2009 To: 4 September 2009
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily.
The test article was formulated as a suspension in 0.5% w/v aqueous carboxymethylcellulose following dispensary SOPs.
The formulations were prepared immediately before use, stirred on arrival at the animal room and were stirred continuously before and throughout dosing.

DIET PREPARATION
Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data provided
- Concentration in vehicle: 0, 50, 100 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): No data supplied
- Purity: No data supplied
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until confirmation of mating
- Proof of pregnancy: vaginal plug or sperm in vaginal washing referred to as day 0 of pregnancy
- After - days of unsuccessful pairing replacement of first male by another male with proven fertility - no data provided
- Further matings after two unsuccessful attempts: no data provided
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: none specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A Charged Aerosol Detector (CAD) was used for analyses.

Formulations prepared at 1.0 and 200.0 mg/mL were analysed to determine homogeneity and stability. Formulations were to be considered homogeneous if the coefficient of variation (CV) of the results is <= 6.0%. In addition all the homogeneity results should be within ± 10% of the mean. Formulations are normally considered to be stable if the mean of the results at each time point are ± 10% of the mean at 0 hour. Results were within these criteria over the 15 day room temperature storage period.

Homogeneity and Stability
Samples were removed in triplicate from the top and bottom of each formulation. These were analysed for test article concentration to determine homogeneity. The formulations were split into 2 aliquots. One aliquot was stored at room temperature (10 to 30°C). After 1, 8 and 15 days the formulations were analysed to determine stability.
The other aliquot was stored refrigerated (1 to 10°C) to be analysed if degradation was observed in the room temperature formulations.

Achieved concentration
Three samples were removed from the test article formulations. Two of the samples were analysed with the third to be analysed only if the samples results are outside the target range (90 to 110% of nominal concentration). Two samples were taken from control formulations. One of these samples was analysed.

Formulations prepared for use at the start middle and end of dosing were analysed to determine achieved concentration. The target range for preparation of liquid formulations is 90 to 110% of nominal. Results were within this range, with the exception of Groups 2 and 4 on Day 1 and Group 4 on Day 22. Test article was not detected in the Group 1 control samples.

Formulations prepared for use at the start middle and end of dosing were analysed to determine achieved concentration. The target range for preparation of liquid formulations is 90 to 110% of nominal. Results were within the range of 91 to 109 %, with the exception of Groups 2 and 4 on Day 1 which were in the range of 63 to 116 % and Group 4 on Day 22 which was found to be in the range of 86 to 91%. Repeat samples were taken from the formulations prepared on Day 6 and found to be in the range of 100 to 108 %. Test article was not detected in the Group 1 control samples.
Results for homogeneity and stability of the formulations at 1.00 and 200 mg/mL were all within the acceptance criteria of a CV ≤ 6 %, and concentrations were within 10 % of the mean over a period of 16 days.
Duration of treatment / exposure:
Two weeks until males paired with females. Males were treated throughout the pairing period until the day before necropsy. Females were treated throughout the pairing period and until Day 4 post partum, inclusive. Dosing was occasionally deferred or omitted if the animal was in or near parturition. Males were treated for at least 8 weeks prior to necropsy.
Frequency of treatment:
Daily
Details on study schedule:
No further details
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 male and 10 female per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level was based on a 90 day dietary rat toxicity study performed by the Sponsor where a dose level of 10,000 ppm (approximately equivalent to 830 mg/kg/day) elicited toxicity in the form of histopathological changes to the urinary bladder and kidney in both sexes. Therefore a high dose level of 1000 mg/kg/day was chosen for this study. The low dose level of 250 mg/kg/day was expected to be a no observed effect level (NOEL), and the intermediate dose level of 500 mg/kg/day was the geometric mean of the low and high dose levels.
Individual dose volumes were adjusted according to the latest recorded body weight.
- Rationale for animal assignment (if not random): The animals were assigned to treatment groups during the acclimatisation period using a total randomisation procedure. Treatment group positions in the room were assigned using a set of random number permutations.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATION: Yes
- Time schedule: As below
- Cage side observations checked:
All animals were examined at least once daily for signs of ill health or overt toxicity. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded and a detailed individual record was maintained of the clinical condition of each animal on the days of body weight recording.
In addition, the animals were observed immediately after dosing and at 0.5, 1, 2 and 4 hours post dose for signs of reaction to treatment. On 16 July 2009 the 4 hour observation was performed between 2.5 and 3 hours post dose, so as to be completed within the working day. However, from Day 23 of dosing animals were observed immediately after dosing, 0.5, and 1 hour post dose.
All animals were examined twice daily to detect any which were dead or moribund.

BODY WEIGHT: Yes
- Time schedule for examinations:
Male body weights were recorded before treatment on the first day of dosing and at weekly intervals thereafter. Female body weights were recorded weekly prior to pairing and until confirmation of mating, on Days 0, 7, 14 and 20 of gestation and on Days 1 and 4 post partum.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined : Yes
The food consumed by each cage of animals was determined weekly during the pre pairing periods.
Individual food intake of mated females was recorded for Days 0 to 6, 7 to 13 and 14 to 19 of gestation, and Days 1 to 3 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Oestrous cyclicity (parental animals):
Daily vaginal washings were taken from each female and the stage of oestrus was recorded from the start of treatment until the confirmation of mating.
During the pairing procedure one male was housed with one female. Mating was confirmed by the presence of a vaginal plug in situ or sperm in a vaginal washing. On confirmation of mating vaginal washing was discontinued and the male was removed and rehoused. The day on which mating was confirmed was designated Day 0 of gestation.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight
Litter observations:
The females were allowed to litter and the date of parturition and the duration of gestation were recorded.
The following data were recorded for each litter to Day 4 post-partum:

number of pups born (live and dead)
daily live litter size and sex (reported on Days 1, and 4)
daily clinical observations
individual pup weights on Days 1 and 4 post partum
necropsy findings of dead pups where condition permitted
Postmortem examinations (parental animals):
SACRIFICE
All females and pups were sent to necropsy on or shortly after Day 4 post-partum, males were sent to necropsy after evaluation of the females, non pregnant females and females with total embryo foetal loss were sent to necropsy on Day 26 of the gestation period. All animals were given an intraperitoneal injection of sodium pentobarbitone. Once a suitable deep plane of anaesthesia had been established, the animal was exsanguinated by the severing of major blood vessels.
Where possible, the animals were killed in cage order.


GROSS NECROPSY
All parental animals were given a macroscopic examination under the general supervision of a pathologist for structural or pathological changes, with particular attention being paid to the reproductive organs. All offspring were given a macroscopic external and internal examination for signs of abnormality.
The uterus of each littering female and any apparently not pregnant female were immersed in 10% ammonium sulphide solution and implantation sites counted.Animals were weighed before being sent to necropsy. Testes and epididymides were dissected free from fat and other contiguous tissue and weighed together before fixation.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues from all terminal adult animals were retained, as appropriate:
cervix
kidney
ovaries
pituitary
prostate
coagulating gland
seminal vesicles
testes and epididymides
urinary bladder
uterus
vagina
gross lesions

All tissues were preserved in relevant preservatives.

Testes, epididymides and ovaries from all terminal adult animals, as appropriate, were embedded in paraffin wax and sectioned at a nominal thickness of 5 μm. Sections allocated for testicular staging were stained with periodic acid Schiff (PAS); all other sections were stained with haematoxylin and eosin.
The following tissues from Groups 1 and 4 only were microscopically examined by the study pathologist in the first instance:
Testes, epididymides and ovaries
Postmortem examinations (offspring):
SACRIFICE
On or shortly after Day 4 post partum, all surviving offspring were killed.

GROSS NECROPSY
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Data were processed, where appropriate, to give litter mean values, group mean values and standard deviations.
Reproductive indices:
A number of indices were used, where appropriate, to evaluate reproductive function.
Offspring viability indices:
No data available.
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
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female receiving 1000 mg/kg/day (number 73), was found dead on Day 11 with a slightly sore neck. There were no other findings at necropsy and therefore this death was not considered to be caused by treatment. There were no other unscheduled deaths during the study.
Clinical observations were unremarkable. There were no post-dosing observations recorded.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the pre-pairing period only, males receiving 500 and 1000 mg/kg/day gained slightly less weight than controls, however over the course of the study there was no significant adverse effect on mean body weight gain in males and females. Due to the reversibility of this finding, it was not considered to be an adverse effect of treatment.
Mean food intake in males receiving 1000 mg/kg/day was slightly reduced compared to controls during the pre-pairing period. This was considered likely to be a consequence of the large amounts of test material administered to this group rather than any systemic toxicity. There were no other effects on mean food intake.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on median pre-coital time, pregnancy rate, mating index, or fertility and fecundity indices
Group mean uterine/implantation data were unaffected by treatment. Live birth index, viability index and mean pup weight at Day 1 and Day 4, and pup necropsy data, all showed no adverse effect of treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There was no effect of treatment on mean testes/epididymides weight.

GROSS PATHOLOGY (PARENTAL ANIMALS)
From macroscopic examination, most tissues were unremarkable and the findings seen were generally consistent with the usual pattern of findings in animals of this strain and age. There were no macroscopic findings suggestive of effects of the test article.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic findings were generally infrequent, of a minor nature and consistent with the usual pattern of findings in animals of this strain and age. There were no microscopic findings in treated animals due to effects of the test article.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No signs of toxicity observed at any dose level tested.
Remarks on result:
other: Generation: all animals (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Group mean uterine/implantation data were unaffected by treatment. Live birth index, viability index and mean pup weight at Day 1 and Day 4, and pup necropsy data, all showed no adverse effect of treatment.
Reproductive effects observed:
not specified
Conclusions:
Administration of the test article by oral gavage to rats for at least 41 days at dose levels of 250, 500 and 1000 mg/kg/day elicited no signs of toxicity at any dose level tested.
The no observed adverse effect level (NOAEL) was at least 1000 mg/kg/day.
Executive summary:

In an Oral reproduction/developmental toxicity screening test in the rat (OECD 421, 1995) the test article was administered for at least 41 days to three groups each of ten male and ten female HsdHan:WIST strain rats by gavage at dose levels of 250, 500 and 1000 mg/kg/day. Administration of the test substance elicited no signs of toxicity at any dose level tested.The no observed adverse effect level (NOAEL) was at least 1000 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be good.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity of the test material to the rat was investigated in an oral reproduction/developmental toxicity screening test conducted in accordance with the standardised guideline OECD 421 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was administered in carboxymethyl cellulose to three groups each of ten male and ten female HsdHan:WIST strain rats by gavage at dose levels of 250, 500 and 1000 mg/kg/day. All animals were treated for the two weeks until pairing; males were treated throughout the pairing period until the day before necropsy. Females were treated throughout the pairing period and until Day 4 post-partum, inclusive. Dosing was occasionally deferred or omitted if the animal was in or near parturition. Males were treated for at least 8 weeks prior to necropsy.

Administration of the test material elicited no signs of toxicity at any dose level tested.

Under the conditions of this study, the no observed adverse effect level (NOAEL) was determined to be at least 1000 mg/kg/day.

In accordance with section 8.7.3 of Column 1 of Annex IX of Regulation (EC) No 1907/2006, it is considered justified to omit the extended one-generation reproductive toxicity study (EOGRTS) as it is not scientifically justified. Column 1 states that the EOGRTS should be proposed if the available repeated dose toxicity studies indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. The NOAEL established in the 90 day study was 207 mg/kg bw/day in males. The repeated dose result was selected as the key value in the CSA and used to derive the DNEL for long term effects. The registrant considers it unlikely that any subsequent reproductive toxicity studies would result in a lower DNEL and it is considered that the one calculated on the basis of the 90 day study will be suitably precautionary. Furthermore, an OECD 421 screening study showed no toxicity and no effects on reproductive parameters at the maximum dose level of 1000 mg/kg bw/day. It is therefore considered to be appropriate to waive the EOGRTS at this tonnage level as it is considered unlikely that further information would be yielded by a new study addressing this endpoint.


Short description of key information:
ORAL
NOAEL 1000 mg/kg/day (rat); OECD 421

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information
ORAL
NOAEL 1000 mg/kg/day (rat); OECD 421
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 2009 to 28 September 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 the relevant results.
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan (UK) Ltd
- Age at study initiation: 10 - 12 wks
- Weight at study initiation: Females: 175.6 - 229.8 g
- Fasting period before study: Not specified
- Housing: Single, exclusive room. Animals were housed in groups of up to five (pre pairing and post pairing), one female with one male (pairing) or individually (mated females) in cages that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 1989).
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester). Shortly before littering pregnant females were provided with wood shavings (Datesand Ltd, Manchester) for the duration of the lactation period.
The bedding was considered not to have contained any contaminants at a level which might have affected the integrity or outcome of the study.
- Diet: ad libitum
- Water: ad libitum
The diet and water were considered not to have contained any contaminants at a level which might have affected the integrity or outcome of the study.
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 40 to 70 %. On one occasion during the acclimatisation period the relative humidity was above the specified protocol range (72 %).
- Air changes: minimum of 15 air changes/hour
- Photoperiod: Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light (0600 to 1800 h) and 12 hours of darkness.


IN-LIFE DATES: From: 8 July 2009 To: 4 September 2009
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily.
The test article was formulated as a suspension in 0.5 % w/v aqueous carboxymethylcellulose following dispensary SOPs.
The formulations were prepared immediately before use, stirred on arrival at the animal room and were stirred continuously before and throughout dosing.

DIET PREPARATION
Not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data provided
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): No data supplied
- Purity: No data supplied
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A Charged Aerosol Detector (CAD) was used for analyses.

Formulations prepared at 1.0 and 200.0 mg/mL were analysed to determine homogeneity and stability. Formulations were to be considered homogeneous if the coefficient of variation (CV) of the results is ≤ 6.0 %. In addition all the homogeneity results should be within ± 10 % of the mean. Formulations are normally considered to be stable if the mean of the results at each time point are ± 10 % of the mean at 0 hour. Results were within these criteria over the 15 day room temperature storage period.

Homogeneity and Stability
Samples were removed in triplicate from the top and bottom of each formulation. These were analysed for test article concentration to determine homogeneity. The formulations were split into 2 aliquots. One aliquot was stored at room temperature (10 to 30 °C). After 1, 8 and 15 days the formulations were analysed to determine stability.
The other aliquot was stored refrigerated (1 to 10 °C) to be analysed if degradation was observed in the room temperature formulations.

Achieved concentration
Three samples were removed from the test article formulations. Two of the samples were analysed with the third to be analysed only if the samples results are outside the target range (90 to 110 % of nominal concentration). Two samples were taken from control formulations. One of these samples was analysed.

Formulations prepared for use at the start middle and end of dosing were analysed to determine achieved concentration. The target range for preparation of liquid formulations is 90 to 110 % of nominal. Results were within this range, with the exception of Groups 2 and 4 on Day 1 and Group 4 on Day 22. Test article was not detected in the Group 1 control samples.

Formulations prepared for use at the start middle and end of dosing were analysed to determine achieved concentration. The target range for preparation of liquid formulations is 90 to 110 % of nominal. Results were within the range of 91 to 109 %, with the exception of Groups 2 and 4 on Day 1 which were in the range of 63 to 116 % and Group 4 on Day 22 which was found to be in the range of 86 to 91 %. Repeat samples were taken from the formulations prepared on Day 6 and found to be in the range of 100 to 108 %. Test article was not detected in the Group 1 control samples.
Results for homogeneity and stability of the formulations at 1.00 and 200 mg/mL were all within the acceptance criteria of a CV ≤ 6 %, and concentrations were within 10 % of the mean over a period of 16 days.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until confirmation of mating
- Proof of pregnancy: vaginal plug or sperm in vaginal washing referred to as day 0 of pregnancy
- After - days of unsuccessful pairing replacement of first male by another male with proven fertility - no data provided
- Further matings after two unsuccessful attempts: no data provided
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: none specified
- Other: Daily vaginal washings were taken from each female and the stage of oestrus was recorded from the start of treatment until the confirmation of mating. On confirmation of mating vaginal washing was discontinued and the male was removed and rehoused. The day on which mating was confirmed was designated Day 0 of gestation.
Duration of treatment / exposure:
Two weeks until males paired with females. Females were treated throughout the pairing period and until Day 4 post partum, inclusive. Dosing was occasionally deferred or omitted if the animal was in or near parturition.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
200, 500 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level was based on a 90 day dietary rat toxicity study performed by the Sponsor where a dose level of 10 000 ppm (approximately equivalent to 830 mg/kg/day) elicited toxicity in the form of histopathological changes to the urinary bladder and kidney in both sexes. Therefore a high dose level of 1000 mg/kg/day was chosen for this study. The low dose level of 250 mg/kg/day was expected to be a no observed effect level (NOEL), and the intermediate dose level of 500 mg/kg/day was the geometric mean of the low and high dose levels.
Individual dose volumes were adjusted according to the latest recorded body weight.
- Rationale for animal assignment (if not random): The animals were assigned to treatment groups during the acclimatisation period using a total randomisation procedure. Treatment group positions in the room were assigned using a set of random number permutations.
Maternal examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATION: Yes
- Time schedule: All animals were examined at least once daily for signs of ill health or overt toxicity. All animals were examined twice daily to detect any which were dead or moribund.
- Cage side observations checked: Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded and a detailed individual record was maintained of the clinical condition of each animal on the days of body weight recording.
In addition, the animals were observed immediately after dosing and at 0.5, 1, 2 and 4 hours post dose for signs of reaction to treatment. From Day 23 of dosing animals were observed immediately after dosing, 0.5, and 1 hour post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Female body weights were recorded weekly prior to pairing and until confirmation of mating, on Days 0, 7, 14 and 20 of gestation and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes
The food consumed by each cage of animals was determined weekly during the pre-pairing periods.
Individual food intake of mated females was recorded for Days 0 to 6, 7 to 13 and 14 to 19 of gestation, and Days 1 to 3 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

SACRIFICE
Females were sent to necropsy on or shortly after Day 4 post-partum; non pregnant females and females with total embryo foetal loss were sent to necropsy on Day 26 of the gestation period. All animals were given an intraperitoneal injection of sodium pentobarbitone. Once a suitably deep plane of anaesthesia had been established, the animal was exsanguinated by the severing of major blood vessels.

GROSS NECROPSY
All parental animals were given a macroscopic examination for structural or pathological changes, with particular attention being paid to the reproductive organs.
The uterus of each littering female and any apparently not pregnant females were immersed in 10 % ammonium sulphide solution and implantation sites counted. Animals were weighed before being sent to necropsy.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues from all terminal adult animals were retained, as appropriate: cervix, kidney, ovaries, pituitary, urinary bladder, uterus, vagina and gross lesions.
All tissues were preserved in relevant preservatives.

Ovaries from all terminal adult animals, as appropriate, were embedded in paraffin wax and sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin. The ovaries from Groups 1 and 4 only were microscopically examined by the study pathologist in the first instance.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes. All parental animals were given a macroscopic examination with particular attention being paid to the reproductive organs.
Examinations included:
- Gravid uterus weight: Yes
- Uterine/implantation data: Yes
Fetal examinations:
The females were allowed to litter and the following data were recorded for each litter to Day 4 post-partum:
- number of pups born (live and dead)
- daily live litter size and sex (reported on Days 1, and 4)
- daily clinical observations
- individual pup weights on Days 1 and 4 post partum
- necropsy findings of dead pups where condition permitted

SACRIFICE
All pups were sent to necropsy on or shortly after Day 4 post-partum. All animals were given an intraperitoneal injection of sodium pentobarbitone. Once a suitable deep plane of anaesthesia had been established, the animal was exsanguinated by the severing of major blood vessels.

GROSS PATHOLOGY
All offspring were given a macroscopic external and internal examination for signs of abnormality.
Statistics:
Data were processed, where appropriate, to give litter mean values, group mean values and standard deviations.
Indices:
- Mating index: (Number of females with determined copulations / Number of oestrous cycles required for their insemination) x 100
- Female fecundity index: (Number of pregnant females / Number of females mated) x 100
- Female fertility index: (Number of pregnant females / Number of females paired) x 100
- Median pre-coital time: Time (day) by which half the females in the group had shown evidence of mating
- % Post-implantation loss: [(Number of implantations – number of live embryos) / Number of implantations] x 100
- Gestation index: (Number of females with live pups / Number of pregnant females) x 100
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female receiving 1000 mg/kg/day (number 73), was found dead on Day 11 with a slightly sore neck. There were no other findings at necropsy and therefore this death was not considered to be caused by treatment. There were no other unscheduled deaths during the study.
Clinical observations were unremarkable. There were no post-dosing observations recorded.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Over the course of the study there was no effect on mean body weight gain in females.
There were no effects on mean food intake in females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on median pre-coital time, pregnancy rate, mating index, or fertility and fecundity indices
Group mean uterine/implantation data were unaffected by treatment. Live birth index, viability index and mean pup weight at Day 1 and Day 4, and pup necropsy data, all showed no adverse effect of treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
From macroscopic examination, most tissues were unremarkable and the findings seen were generally consistent with the usual pattern of findings in animals of this strain and age. There were no macroscopic findings suggestive of effects of the test article.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic findings were generally infrequent, of a minor nature and consistent with the usual pattern of findings in animals of this strain and age. There were no microscopic findings in treated animals due to effects of the test article.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Group mean uterine/implantation data were unaffected by treatment. Live birth index, viability index and mean pup weight at Day 1 and Day 4, and pup necropsy data all showed no adverse effect of treatment.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Administration of the test article by oral gavage to rats for at least 41 days at dose levels of 250, 500 and 1000 mg/kg/day elicited no signs of toxicity at any dose level tested.
The no observed adverse effect level (NOAEL) was at least 1000 mg/kg/day.
Executive summary:

A study was carried out to investigate the potential effects of the test article on the reproductive/developmental toxicity in the rat when administered orally by gavage. The study was conducted in accordance with the standardised guideline OECD 421 under GLP conditions.

Male and female HsdHan:WIST rats were assigned to four groups (10 animals/sex/group). Each treated group received dose preparations containing the control article (0.5 % w/v aqueous carboxymethylcellulose) or 250, 500 or 1000 mg of test article/kg of body weight/day (mg/kg/day) at a dose volume of 5 mL/kg. Animals were treated for two weeks then paired for mating. Females were treated throughout the pairing period and until Day 4 post partum, inclusive. Dosing was occasionally deferred or omitted if the animal was in or near parturition.

Prepared formulations were considered to be acceptable for dosing to the study animals. There were no treatment-related unscheduled deaths during the study, and no treatment related clinical signs.

Overall, group mean food intake and group mean body weight gain showed no significant dose-related adverse effects of treatment.

Mating data were unaffected by treatment. There was no effect of treatment on mean uterine/implantation data or mean litter data.

At necropsy, macroscopic findings were unremarkable. Microscopic examination revealed no treatment-related findings.

Administration of the test article by oral gavage to rats for at least 41 days at dose levels of 250, 500 and 1000 mg/kg/day elicited no signs of toxicity at any dose level tested. The no observed adverse effect level (NOAEL) was at least 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available study was conducted in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be good.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity of the test material to the rat was investigated in an oral reproduction/developmental toxicity screening test conducted in accordance with the standardised guideline OECD 421 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was administered in carboxymethyl cellulose to three groups each of ten male and ten female HsdHan:WIST strain rats by gavage at dose levels of 250, 500 and 1000 mg/kg/day. All animals were treated for the two weeks until pairing; males were treated throughout the pairing period until the day before necropsy. Females were treated throughout the pairing period and until Day 4 post-partum, inclusive. Dosing was occasionally deferred or omitted if the animal was in or near parturition. Males were treated for at least 8 weeks prior to necropsy.

Administration of the test material elicited no signs of toxicity at any dose level tested.

Under the conditions of this study, the no observed adverse effect level (NOAEL) was determined to be at least 1000 mg/kg/day.

In accordance with section 1 of Annex XI of Regulation (EC) No 1907/2006, it is considered justified to omit the pre-natal developmental toxicity study required under point 8.7.2 of Annex XI as it is not scientifically justified. The NOAEL established in the 90 day study was 207 mg/kg bw/day in males. The repeated dose result was selected as the key value in the CSA and used to derive the DNEL for long term effects. The registrant considers it unlikely that any subsequent reproductive toxicity studies would result in a lower DNEL and it is considered that the one calculated on the basis of the 90 day study will be suitably precautionary. Furthermore, an OECD 421 screening study showed no toxicity and no effects on reproductive parameters at the maximum dose level of 1000 mg/kg bw/day. It is therefore considered to be appropriate to waive the developmental toxicity study at this tonnage level as it is considered unlikely that further information would be yielded by a new study addressing this endpoint.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproduction.