Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOEL (No Observed Effect Level) for reproductive toxicity was conisdered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 July 2016 and 25 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : FAT 92504/C TE
Physical State/Appearance: Blue paste
Purity: 86.4%
Batch Number: BOP 01-15
Label: FAT-Nr. 92504/C TE Lot. Nr.: BOP 01-15 bei
Raumtemperatur VD: 18.11.2020 (Retest Date)
Date Received: 14 March 2016
Storage Conditions: Room temperature, in the dark (14 March 2016 – 18 March 2016). Stored cold at
approximately 4°C in the
dark (18 March 2016 onwards)
Expiry Date: 18 November 2020
No correction for purity was made.
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on species / strain selection:
As recommended by the guideline
Sex:
male/female
Details on test animals and environmental conditions:
The animals were acclimatized for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 310 to 348 g, the females weighed 186 to 226g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided
in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding,
throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour
and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and
print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol
400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL)
Animal Numbers
Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 1000 4 250 12 (73-84) 12 (85-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group. The test item was administered daily by gavage using a stainless steel cannula attached to a disposable
plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. 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 estrus 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 (Day 0 of gestation) 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:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least nineteen days.
Formulations were initially prepared for one week and then fortnightly thereafter and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken and analyzed for concentration of FAT 92504/C TE at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.
Duration of treatment / exposure:
Up to eight weeks
Frequency of treatment:
Daily (except for females during parturition where applicable)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was
designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Days 43 and 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Parental animals: Observations and examinations:
None
Statistics:
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs for any of the animals considered to be related to the toxicity of the test item.
For all treatment groups, instances of blue fur staining by the test item were frequently observed in both
males and females from the first week of dosing at 1000 and 300 mg/kg bw/day and from the second
week of dosing at 100 mg/kg bw/day. Blue colored faeces and staining of the cage bedding were also
observed for animals of either sex from all treatment groups from the first week and second week of
dosing (respectively). These observations generally persisted throughout the remainder of the study.
One female treated with 100 mg/kg bw/day had increased salivation post dosing on Day 45 and one male
from this treatment group had pilo-erection between Days 29 and 37. In isolation and in the absence of
a similar effect at 300 or 1000 mg/kg bw/day, these observations were considered to be incidental and
unrelated to treatment. One control male, two males treated with 100 mg/kg bw/day and one male treate
d with 300 mg/kg bw/day showed incidences of noisy respiration. In the absence of a similar effect in
females or in either sex at 1000 mg/kg bw/day this finding was deemed likely to be due to the dosing
procedure.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on body weight development in
animals of either sex.
Males from all treatment groups showed a statistically significant increase in body weight gain (p<0.05)
during Week 4 of treatment. An isolated incidence of increased body weight gain is considered not to
represent an adverse effect of treatment
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption or food conversion efficiency (where calculated) in animals of either sex.
Statistical analysis of the data (where applicable) did not indicate any significant intergroup differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the hematological parameters examined. Statistical analysis of the data did not reveal any significant intergroup differences.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in chloride
concentration (p<0.05). Males treated with 1000 mg/kg bw/day also showed a statistically significant reduction in calcium concentration (p<0.05). All of the individual values were within background control
ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.
Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
During motor activity evaluations, males treated with 1000 mg/kg bw/day showed a statistically significant increase during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Other effects:
effects observed, non-treatment-related
Reproductive performance:
no effects observed
Mating
There was no effect of treatment on mating performance with all animals mating within four days after pairing.
Fertility
No treatment-related effects were detected in fertility.
One female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day were not
pregnant following positive evidence of mating. There was evidence of uterine dilation and inflammation
in the female treated with 100 mg/kg bw/day which may indicate a problem following mating and is most
likely to be the reason for the lack of pregnancy. No histopathological changes were evident in the female treated with 300 mg/kg bw/day however the male partner had tubular atrophy in the testes which was
the reason for the lack of pregnancy. In the absence of any fertility effects at 1000 mg/kg bw/day, these
intergroup differences were considered to be incidental.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
Statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.

Litter Responses
In total twelve females from the control group, eleven females each from the 100 and 300 mg/kg bw/day
dose groups and twelve females from the 1000 mg/kg bw/day dose group gave birth to a live litter and
successfully reared young to Day 5 of age. Female 22 was administered test item on Day 3 of lactation
due to a technical error. The data for this female has therefore been excluded from assessment from
Day 3 of lactation onwards. The following assessment of litter response is based on all remaining litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation
losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of
the data did not reveal any significant intergroup differences.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated
groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth and Development
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to
controls. The incidental clinical signs detected throughout the control and treated groups, consisting
of small size, pale, physical injury, missing or found dead were considered to be low incidence findings
observed in offspring in studies of this type and were considered unrelated to test item toxicity. On Day
5 of lactation, four litters from females treated with 1000 mg/kg bw/day were stained blue. This was
considered to be the result of the administration of a colored test item to the adult female and the transfer
of the colored material to the offspring following the dams normal grooming procedure.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Days 43 and 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

The oral administration of FAT 92504/C TE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day for either sex. The NOEL (No Observed Effect Level) for reproductive toxicity was determined to be 1000 mg/kg bw/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:
High quality GLP compliant guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Currently no study to assess reproductive and developmental toxicity of Disperse Blue 056 is available. However source substance Disperse Blue 054/077 was evaluated for potential to cause reproductive and/or developmental toxicity in a combined repeated dose toxicity study with reproductive and developmental screening.

In this study, the Disperse Blue 054/077 was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Adult males were terminated on Days 43 and 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. 

In this study, there was no effect of treatment on mating performance. All animals mated within four days of pairing. There were no treatment-related effects on conception rates and gestation lengths.Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum, sex ratio and offspring viability were comparable to controls. Offspring body weight gain and litter weights on Days 1 and 4 post partum were comparable to control litters. Surface righting reflex response was also comparable to controls. Gross pathological examination, histopathological examination and evaluation of organ weights also did not reveal any toxicologically significant adverse effects at any dose levels. Hence, the ‘No Observed Effect Level’ (NOEL) for reproductive as well as developmental toxicity for the source substance Disperse Blue 054/077 was considered to be 1000 mg/kg bw/day. Based on the principles of read across, Disperse Blue 056 is also expected to pose no hazards to reproductive as well as developmental systems/functions.

Effects on developmental toxicity

Description of key information

The NOEL (No Observed Effect Level) for developmental toxicity was considered to be 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 July 2016 and 25 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : FAT 92504/C TE
Physical State/Appearance: Blue paste
Purity: 86.4%
Batch Number: BOP 01-15
Label: FAT-Nr. 92504/C TE Lot. Nr.: BOP 01-15 bei
Raumtemperatur VD: 18.11.2020 (Retest Date)
Date Received: 14 March 2016
Storage Conditions: Room temperature, in the dark (14 March 2016 – 18 March 2016). Stored cold at
approximately 4°C in the
dark (18 March 2016 onwards)
Expiry Date: 18 November 2020
No correction for purity was made.
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on test animals and environmental conditions:
The animals were acclimatized for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 310 to 348 g, the females weighed 186 to 226g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol
400.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL)
Animal Numbers
Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 1000 4 250 12 (73-84) 12 (85-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group. T
he test item was administered daily by gavage using a stainless steel cannula attached to a disposable
plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol
400.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least nineteen days. Formulations were initially prepared for one week and then fortnightly thereafter and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken and analyzed for concentration of FAT 92504/C TE at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared
formulations were within ± 5% of the nominal concentration.
Details on mating procedure:
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. 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 estrus 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 (Day 0 of gestation) 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:
Up to eight weeks
Frequency of treatment:
Daily (except for females during parturition where applicable)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was
designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on
Days 43 and 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and
surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Maternal examinations:
Observations and examinations performed and frequency
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the prepairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity; see deviations from Study Plan. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions.
The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

3.4.5.4 Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were
carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).
Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anticoagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed.
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Sacrifice and pathology
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals Pituitary (post-fixation)
Brain Prostate and Seminal Vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Caecum Rectum
Coagulating gland Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes ♦
Jejunum Thyroid/Parathyroid
Kidneys Trachea
Liver Thymus
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina
* Eyes fixed in Davidson’s fluid
♦ preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were dispatched to the Test Site. The tissues from five selected control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted.

Other Information on methods:
Group mean values are generally calculated using non-rounded values therefore is it not always possible to calculate the exact group values from the individual values presented in the appendices.
For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring. For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.
Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea,
Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (nonparametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded.
Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum and at necropsy
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs for any of the animals considered to be related to the toxicity of the test item.
For all treatment groups, instances of blue fur staining by the test item were frequently observed in both
males and females from the first week of dosing at 1000 and 300 mg/kg bw/day and from the second
week of dosing at 100 mg/kg bw/day. Blue colored faeces and staining of the cage bedding were also
observed for animals of either sex from all treatment groups from the first week and second week of
dosing (respectively). These observations generally persisted throughout the remainder of the study.
One female treated with 100 mg/kg bw/day had increased salivation post dosing on Day 45 and one male
from this treatment group had pilo-erection between Days 29 and 37. In isolation and in the absence of
a similar effect at 300 or 1000 mg/kg bw/day, these observations were considered to be incidental and
unrelated to treatment. One control male, two males treated with 100 mg/kg bw/day and one male treate
d with 300 mg/kg bw/day showed incidences of noisy respiration. In the absence of a similar effect in
females or in either sex at 1000 mg/kg bw/day this finding was deemed likely to be due to the dosing
procedure.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on body weight development in animals of either sex.
Males from all treatment groups showed a statistically significant increase in body weight gain (p<0.05)
during Week 4 of treatment. An isolated incidence of increased body weight gain is considered not to
represent an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption or food conversion efficiency (where calculated) in animals of either sex.
Statistical analysis of the data (where applicable) did not indicate any significant intergroup differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the hematological parameters examined. Statistical analysis of the data did not reveal any significant intergroup differences.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in chloride
concentration (p<0.05). Males treated with 1000 mg/kg bw/day also showed a statistically significant red
uction in calcium concentration (p<0.05). All of the individual values were within background control
ranges and in the absence of a true dose related response or any associated histopathological correlates
the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.

Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
During motor activity evaluations, males treated with 1000 mg/kg bw/day showed a statistically significant increase during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured. Males treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in thymus
weight: both absolute and relative to terminal body weight, whilst females from this treatment group showed a statistically significant increase (p<0.05) in absolute and relative pituitary weight. All of the individual values for thymus weights and all but one absolute and relative value for the pituitary weights were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Adults
At necropsy, blue/green discoloration was observed for a number of tissues in animals of either sex from all treatment groups. These included gastrointestinal tract (stomach and small/large intestine), skin, mammary gland, mesenteric lymph nodes and adipose tissue. Green fluid in the urinary bladder was also evident in one 300 mg/kg bw/day male. These observations were deemed likely to be due to the uptake of the test item (blue paste) and/or its metabolite/s and in the absence of any adverse histopathological correlates were regarded as of no toxicological significance.
One male treated with 300 mg/kg bw/day had small testes and epididymides. Microscopic examination of these tissues revealed tubular atrophy in the testes. In the absence of a similar effect at 1000 mg/kg bw/day, the intergroup difference was considered to be incidental and unrelated to treatment. One male
and three females treated with 100 mg/kg bw/day, one female treated with 300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day had red discoloration of the lungs. Additionally, one control male, one male treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day showed increased pelvic space in one or both kidneys. Such observations are common in this type of study and these findings were deemed likely to be incidental. The remaining macroscopic findings (fluid filled uterus, mass on the ovaries, reddened caecum and a white patch in the right eye) were confined to either control animals or in animals treated with 100 mg/kg bw/day and in the absence of a similar effect seen at 300 or 1000 mg/kg bw/day, the intergroup differences were considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Mating
There was no effect of treatment on mating performance with all animals mating within four days after pairing.

Fertility
No treatment-related effects were detected in fertility.
One female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day were not pregnant following positive evidence of mating. There was evidence of uterine dilation and inflammation in the female treated with 100 mg/kg bw/day which may indicate a problem following mating and is most likely to be the reason for the lack of pregnancy. No histopathological changes were evident in the female treated with 300 mg/kg bw/day however the male partner had tubular atrophy in the testes which was the reason for the lack of pregnancy. In the absence of any fertility effects at 1000 mg/kg bw/day, these intergroup differences were considered to be incidental.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
Statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.

Litter Responses
In total twelve females from the control group, eleven females each from the 100 and 300 mg/kg bw/day dose groups and twelve females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. Female 22 was administered test item on Day 3 of lactation due to a technical error. The data for this female has therefore been excluded from assessment from Day 3 of lactation onwards. The following assessment of litter response is based on all remaining litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Necropsy
Necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 and 1000 mg/kg bw/day.
Six litters from females treated with 1000 mg/kg bw/day were stained blue or had blue colored contents in the stomach however this was considered to be the result of the administration of a colored test item to the adult female and the transfer of the colored material to the offspring.

Offspring Growth and Development
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting
of small size, pale, physical injury, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity. On Day 5 of lactation, four litters from females treated with 1000 mg/kg bw/day were stained blue. This was considered to be the result of the administration of a colored test item to the adult female and the transfer of the colored material to the offspring following the dams normal grooming procedure.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

Conclusions:
The 'No Observed Effect Level' for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Days 43 and 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

The oral administration of FAT 92504/C TE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day for either sex. While the 'No Observed Effect Level' for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/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:
High quality GLP compliant guideline 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
Additional information

As discussed in the section 'Effects on fertility' above, source FAT 92504 (Disperse Blue 054/077) did not have any adverse effects on development of the fetus/offsprings in the combined repeated dose toxicity study with reproductive and developmental screening. Hence, the NOEL for developmental toxicity was determined to be 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the above discussion, FAT 36002 does not warrant classification as per the CLP criteria (Regulation EC no. 1272/2008) criteria.