4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421 Screening for reproductive / developmental toxicity:

The oral administration of test substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not produce any adverse effects in adults or offspring. Based on the available results within the confines of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered 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 in-life phase: 08 August 2017 (first day of treatment) to 01 October 2017 (final day of necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

None that affected the purpose and scientific integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification : ST1031KH (MB002 Pigment#1)
Physical State/Appearance: Red powder
CAS Number : 56396-10-2
Purity : 100%
Batch Number : 20160212
Label : ST1031KH (MB002 Pigment#1) Batch:20160212
Date Received : 03/02/2017
Storage Conditions : Stored in darkness at ambient temperature and humidity; used/formulated in light.
Expiry Date : 01/02/2018

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for twenty days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. 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 291 to 359g, and were approximately eleven weeks old. The females weighed 191 to 240g, 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, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. 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 the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

The animals were housed in a single air-conditioned room within the 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; there were short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxy methylcellulose/0.5% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in 1% Carboxy methylcellulose/0.5% Tween 80. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least 13 Days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

DOSE ADMINISTRATION:
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 10 mL/kg of 1% Carboxy methylcellulose/0.5% Tween 80.
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:

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 estrous 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:

Samples of the test item formulation were taken on three occasions and analyzed for concentration of test substance. The results indicate that the prepared formulations were within ± 16% of the nominal concentration.

Duration of treatment / exposure:

Approximately six weeks (males) and eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

Daily.

Details on study schedule:

Chronological Sequence of Study:
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.

ii. 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. For the 14 days prior to pairing, pre-pairing vaginal smears were performed and assessed for females.

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. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) surface righting and clinical signs were also recorded during this period.

vi. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.

vii. The male dose groups were killed and examined macroscopically on 45.

viii. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.

ix.All females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels and dose volume were selected in collaboration with the Sponsor and are based on available toxicity data including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat. In the range finder investigation, at dose levels of 500 or 1000 mg/kg bw/day resulted in slightly lower overall body weight gains in males, which was not associated with an adverse effect on food intake in these males. The vehicle chosen for this study was Arachis oil at a dose volume of 4 mL/kg. A high dose level of 1000 mg/kg bw/day was therefore considered to be suitable for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. Further investigations with the test item revealed that Arachis oil was an unsuitable vehicle for chemical analysis purposes. Therefore, the proposed vehicle was changed to 1% Carboxymethyl cellulose/0.5% Tween 80 and the dose volume was increased to 10 mL/kg. It was considered unlikely that the change in vehicle would adversely affect the tolerance of the animals to the test item given the low toxicity observed in the range finding study.

Positive control:

None.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
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 the 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, 4 and 7 post partum. Body weights were also recorded for all animals at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. 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 for the periods covering post partum Days 1-4, 4-7, 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females 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.

REPRODUCTIVE PERFORMANCE:
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 estrous 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.

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

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight

Litter observations:

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, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

PHYSICAL DEVELOPMENT:
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Postmortem examinations (parental animals):

NECROPSY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum.
Any females which failed to achieve pregnancy or were killed around the same time as littering females.

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).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The epididymides, testes, seminal vesicles (with coagulating gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid were dissected free from fat for terminal kill animals from both sexes, and weighed post-fixation.

HISTOPATHOLOGY:
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:

Epididymides♦
Prostate
Glans penis
Seminal Vesicles (with coagulating gland)
Gross lesions
Testes♦
LABC (levator ani-bulbocavernous) muscle
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (with oviducts)
Ovaries
Vagina
Pituitary

Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered Formalin.

All tissues were dispatched to the histology processing Test Site for processing.
The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. 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.

Postmortem examinations (offspring):

NECROPSY:
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

Statistics:

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:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

Reproductive indices:

Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
ii. Fertility Indices: Mating Index (%) and Pregnancy Index (%)

Gestation and Parturition Data:
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation length
ii. Parturition Index

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

i. Implantation Losses (%)
ii. Live Birth and Viability Indices: The following indices were calculated for each litter;
- Live Birth Index (%)
- Viability Index 1 (%)
- Viability Index 2
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.

iii. Sex Ratio (% males):
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse clinical signs detected.

All 1000 mg/kg bw/day animals showed instances of fur stained by the test item between Days 2 to 54 and also three males and one female treated with 300 mg/kg bw/day showed fur stained by test material between Days 2 to 36. This finding is not unsurprising given the coloured nature of the test Item.

Incidental findings included one 1000 mg/kg bw/day female with mammary gland mass under the left hind limb. This was considered incidental and unrelated to treatment. At 100 mg/kg bw/day, one male exhibited noisy respiration between Days 23 and 32, this isolated finding was considered most likely to reflect difficulties dosing these particular animals rather than any treatment related effect.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were evident on body weights in treated males throughout the treatment period or in females during the maturation, gestation or lactation phases of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects were evident for food consumption or food conversion efficiency in treated males or in treated females during maturation, gestation or lactation.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

No findings were apparent which could be related to the administration of the test item in the tissues examined. There were no test item-related microscopic findings 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) or the evaluation of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle. All females showed regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrous on the day of necropsy.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating:
There was no effect of treatment on mating performance, all animals mated within four days of pairing.

Fertility:
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female treated with 300 mg/kg bw/day was non-pregnant. As lack of pregnancy only ocurred in one animal and no such findings were noted in animals treated with 1000 mg/kg bwday this finding was considered to be incidental and unrelated to treatment with the test item.

Gestation Length:
Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females were essentially similar to control.
One female treated with 300 mg/kg bw/day had a gestation length of 24½ days but subsequently suffered a total litter loss, as this was an isolated finding and no such effects were noted in animals treated with 1000 mg/kg bw/day this was considered to be un-related to treatment with the test item.

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At dose levels of 100, 300 or 1000 mg/kg bw/day there were no effects on reproductive performance

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Litter Responses:
In total twelve, twelve, ten, and twelve females from control, 100, 300, and 1000 mg/kg bw/day dose groups respectively gave birth to a live litter and successfully reared young to Day 13 of age. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:
There was no effect of treatment with the test item at any dose level on the mean number of implantations and post-implantation losses. Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.

Offspring Growth and Development:
There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 13 post partum. Litter weights for all treated dose groups were also comparable with controls.

When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.

Clinical signs detected in pups from treated dose groups included cold, small, weak, no milk in the stomach, thread like tail, tail missing, a physical injury, found dead and missing. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The oral administration of test substance to rats, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not produce any adverse effects in offspring.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The oral administration of test substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not produce any adverse effects in adults or offspring. Based on the available results within the confines of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction:

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

Methods:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and eight weeks (females) (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 (1% Carboxy methylcellulose/0.5% Tween 80) over the same period.

Clinical signs, body weight change, dietary intake 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 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results:

Adult Responses:

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

There were no adverse clinical signs detected.

Body Weight

No effects were evident on body weights in treated males throughout the treatment period or in females during the maturation, gestation or lactation phases of the study.

Food Consumption

No effects were evident for food consumption or food conversion efficiency in treated males throughout the treatment period or in treated females during maturation, gestation or lactation phases of the study.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance:

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle. All females showed regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrous on the day of necropsy.

Mating

There was no effect of treatment on mating performance.

Fertility

Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.

 

Gestation Length

Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females were essentially similar to control.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

There was no effect of treatment with the test item indicated by offspring body weight, body weight gain or litter weights, ano-genital distance on Day 1 post partum, visible nipple count in male offspring on Day 13 post partum or clinical signs up to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Pathology:

Necropsy

No macroscopic abnormalities were detected in adult animals which were considered to be related to an adverse effect of treatment with the test item.

Neither the type, incidence or distribution of macroscopic observations in offspring indicated any treatment-related effect up to a dose level of 1000 mg/kg bw/day.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Organ Weights

There were considered to be no treatment related effects detected in the organ weights measured.

Histopathology

No findings were apparent which could be related to the administration of the test item in the tissues examined. There were no test item-related microscopic findings 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) or the evaluation of follicles and corpora lutea in the ovaries.

Conclusion

The oral administration of test substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not produce any adverse effects in adults or offspring. Based on the available results within the confines of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered 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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Refer to effects on fertility section.

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the OECD 421 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level tested. The substance is therefore not classified for reproductive toxicity.