4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A reproduction/developmental screening study in rats (oral route) is available. Adverse effects on both parental and reproductive toxicity were observed. NOAEL for parental toxicity is 50 mg/kg bw; for reproductive toxicity is 50 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

other: OECD 421

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Full GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Raleigh, North Carolina)
Age at Study Start: Approximately eight weeks of age at initiation of treatment.
Physical and Acclimation
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed two-three per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study.
Housing
After assignment to study, animals were housed singly (two per cage for the Pig-a assay positive control group) in solid bottom stainless steel cages, except during the breeding phase of the study. The solid bottom cages contained corncob bedding. During breeding, one male (excluding the Pig-a assay positive control group) and one female were placed in stainless steel cages with wire mesh floors that are suspended above catch pans in order to better visualize copulation and plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material from approximately GD 0 until LD 4. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 12-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Temporary excursions from these ranges may occur on an infrequent basis; all observed ranges will be documented in the study file. The average number of air changes was 10.36 times/hour.
Enrichment
Enrichment for rats included the use of nylon bones during the pre-breeding phase, and paper nesting material during the gestation and lactation phase. Cages also had open areas on the sides for visualization of other rats.
Randomization and Identification
Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers. If a transponder stopped functioning or was lost, it was replaced with a new transponder that was correlated with the unique animal number.
Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

Once Daily via gavage

Details on mating procedure:

Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males were dosed daily for 14 days prior to mating and continuing throughout the mating for a total of at least 28 days. Female rats were dosed once daily for 14 days prior to breeding, and continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days) until necropsy.

Frequency of treatment:

Daily, via gavage

Details on study schedule:

Groups of 10 male and 10 female Crl:CD(SD) rats were administered MORTRACE™ SB Conc. daily, by gavage in Polyethylene glycol 400 (PEG 400) , at dose levels of 0, 10, 50, or 200 mg/kg/day. Male rats were dosed two weeks prior to breeding and continuing through breeding (up to two weeks) for a treatment period of at least 28 days. Female rats were dosed two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days) and until necropsy. Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition, litter size, pup survival, sex, pup body weight and the presence of gross external morphological alterations were assessed. In addition, hematology was evaluated in females and a gross necropsy and histopathology of the adults was conducted with an emphasis on organs of the reproductive system. An integrated in vivo gene mutation Pig-a assay was also conducted on six males/group to evaluate the mutagenic potential of the test material. The Pig-a assay included a positive control group (6 males) that was dosed with 20 mg/kg N-Nitroso-N-ethylurea on test days (TD) 1-3. Test material administration for both males and females began on April 15, 2015. The adult males were necropsied on May 14 and 15, 2015. The adult females were necropsied on May 26, 2015 to June 8, 2015. Pups were euthanized on postnatal day 4.

Remarks:

Doses / Concentrations:
0, 10, 50, 200 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that are clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals will be observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Clinical examinations were conducted on all animals pre-exposure and at least once daily throughout the study. During the exposure period, these examinations were conducted approximately one hour after dosing. Females were observed for signs of parturition beginning on or about GD 20 (see litter data). Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Thereafter, male body weights were recorded weekly throughout the study (excluding the Pig-a assay positive control group). Females were weighed weekly during the pre-breeding and breeding periods. During gestation, females were weighed on gestation days (GD) 0, 7, 14, 17 and 20. Females that delivered litters were weighed on lactation days (LD) 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly until termination. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20 and LD 1-4.
Feed consumption was determined weekly during the two week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption for males was not measured. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not measured for females that failed to mate or failed to deliver a litter.

Sperm parameters (parental animals):

The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 µm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Litter observations:

Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Pup body weights were inadvertently not recorded for animal #1194 on LD 1. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects and discarded. Any pups found dead on PND 0 underwent a lung float test to determine whether they were born dead or alive.

Postmortem examinations (parental animals):

Adult Necropsy
Adult males (fasted) were submitted for necropsy after at least four weeks of exposure. Adult females (fasted) were terminated on LD 5-8, or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, fasted rats were weighed in the animal room (excluding the Pig-a assay positive control group) and submitted alive for necropsy. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen. Animals utilized in the PigA assay (see genetox section) had blood collected from the orbital sinus, following this, they were placed in a CO2 chamber to continue anesthesia. Under a deep plane of anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised.
The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al., (1964) for approximately one minute and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
The hindlimbs and sternum from all females were collected and preserved at necropsy in 10 percent formalin for histopathological examination of bone marrow.
Weights of the epididymides, kidneys, liver, spleen, testes were recorded and organ:body weight ratios calculated.
Representative samples of tissues listed in teh OECD guideline were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s. Transponders were removed and placed in jars with the tissues.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and a blood sample was not obtained.
Histopathology:
Histologic examination of the tissues was conducted on all control and high-dose adult rats including one female in the high-dose group which was removed from study in a moribund condition on test day 42. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue was life threatening.

Hematology:
To further evaluate potential treatment-related effects following exposure to the test material, blood samples for hematology were collected from all adult females. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Blood was not obtained from animals that died or were euthanized in a moribund condition prior to their scheduled necropsy.
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.
Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Neutrophils (NEUT)
Lymphocytes (LYMP)
Monocytes (MONO)
Eosinophils (EOS)
Basophils (BASO)
Large Unstained Cells (LUC) which include, atypical lymphocytes,
large lymphocytes, plasma cells, and blasts
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)

Note: A manual differential WBC count was performed as appropriate per department Standard Operating Procedures.

Postmortem examinations (offspring):

All pups surviving to LD 4 were examined for gross external alterations, euthanized with sodium pentobarbital solution followed by decapitation, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Gestation index = (No. females delivering a viable litter/No. females with evidence of mating) x 100
Gestation survival index = percentage of delivered pups alive at birth
Postimplantation loss = (No. implants – No. viable offspring)/(No. implants) x 100

Offspring viability indices:

Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease in high dose males; high dose females decreased BW during gestation (GD14 - 20) and lactation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decrease in high dose males; high dose females decreased BW during gestation (GD14 - 20) and lactation

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hypertrophy (cetrilobular/midzonal) of hepatocytes in high dose males and females

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see below

All animals survived until scheduled termination with the exception of one female given 200 mg/kg/day which was removed from study in a moribund condition due to dystocia on GD 25.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity

Dose descriptor:

NOAEL

Remarks:

Parental general toxicity

Effect level:

ca. 50 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on liver (increased relative and absolute weight) females and males, increased spleen weights (males only), hematolgical changes, body weight decreases (males and females), Liver histopathology.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Treatment related reduction in litter size in the high dose group (200 mg/kg bw).

Dose descriptor:

NOEL

Remarks on result:

other: A NOEL for this generation was not reported in this study.

Reproductive effects observed:

not specified

Hematology

Females given 200 mg/kg/day had a treatment-related decrease in percent neutrophils and a treatment-related increase in percent lymphocytes as compared to controls. There were no treatment related alterations in total white blood cell counts, and no treatment-related effects in any of the erythrocytic parameters, in females at any dose level.

There were no treatment-related effects at any dose level on mating indices, gestation length, pup survival, post-implantation loss, or sex ratio.

In the 200 mg/kg/day dose group there were treatment-related decreases in conception, fertility, gestation and gestation survival indices . There was also an increase in the time to mating and an increased incidence of dystocia. There were no effects on conception, fertility, or gestation indices in the 10 or 50 mg/kg/day dose groups. Additionally, there were no effects on time to mating or the incidence of dams with dystocia in the 10 or 50 mg/kg/day dose groups.

	Dose Level (mg/kg/day)
Parameter (mean %)	0	10	50	200
Male conception index (%)	100.0 (10/10)	80.0 (8/10)	90.0 (9/10)	70.0 (7/10)
Female conception index (%)	100.0 (10/10)	80.0 (8/10)	90.0 (9/10)	70.0 (7/10)
Male fertility index (%)	100.0 (10/10)	80.0 (8/10)	90.0 (9/10)	70.0 (7/10)
Female fertility index (%)	100.0 (10/10)	80.0 (8/10)	90.0 (9/10)	70.0 (7/10)
Gestation index (%)	90.0 (9/10)	100.0 (8/8)	100.0 (9/9)	71.4 (5/7)
Gestation survival index (%)	100.0 (99/99)	98.8 (83/84)	100.0 (101/101)	88.2 (45/51)
Time to mating (days)	2.4	2.1	2.1	3.9

In the 200 mg/kg/day dose group, there was a treatment-related increase in the number of litters (4/5) with one or more dead pups. Treatment-related clinical observations associated with pup mortality (pale and yellow skin) were also observed in the high-dose litters. No treatment-related observations were observed in the 10 or 50 mg/kg/day litters. Additionally, no malformed pups were observed at any dose level tested.

Consistent with effects on offspring survival (see previous section), there was a treatment-related reduction in mean litter size in the 200 mg/kg/day group. There were no effects on pup body weights in any treated group and no effects on litter size in the 10 or 50 mg/kg/day dose groups.

	Mean litter size
Dose Level (mkd):	0	10	50	200
Born Live	11.0	10.4	11.2	9.0
Born Dead	0.0	0.1	0.0	1.2
Day 1	11.0	10.3	11.2	8.8
Day 4	10.9	10.1	10.9	8.6

Conclusions:

Administration of Mortrace SB at 200 mg/kg bw produce clear evidence of parental toxicity with effects noted on the liver, spleen, hematopoetic system. In addition to this, there was a clear decrease in reproductive performance at this dose level with effects on conception, fertiloty, gestation and gestation survival indices. There were also clear effects on the offspring survival with a decreased litter size in the top dose level. There were no effects on any parameter observed in the 50 mg/kg bw dose group or the 10 mg/kg bw dose group. Consequently the NOEL for reproductive and parental toxicty is 50 mg/kg bw/day

Executive summary:

The purpose of this study was to evaluate the potential effects of MORTRACE™ SB Conc on reproductive function, and prenatal/early neonatal growth and survival of the offspring. Furthermore, the integrated in vivo gene mutation Pig-aassay evaluated the mutagenic potential of the test material. 

Groups of 10 male and 10 female Crl:CD(SD) rats were administered MORTRACE™ SB Conc. daily, by gavage in Polyethylene glycol 400 (PEG 400), at dose levels of 0, 10, 50, or 200 mg/kg/day. Male rats were dosed two weeks prior to breeding and continuing through breeding (up to two weeks) for a treatment period of at least 28 days. Female rats were dosed two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), lactation (four days) and until necropsy. Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition, litter size, pup survival, pup sex, pup body weight and the presence of gross external morphological alterations were assessed. In addition, hematology was evaluated on females and a gross necropsy and histopathology of the adults was conducted with an emphasis on organs of the reproductive system. An integrated in vivo gene mutation Pig-a assay was also conducted on six males/group to evaluate the mutagenic potential of the test material. The Pig-a assay included a positive control group (6 males) that was dosed with 20 mg/kg N-Nitroso-N-ethylurea on test days (TD) 1-3. Mutation frequency in reticulocytes (RET) and red blood cells (RBC) of each designated male was determined by flow cytometry quantification of CD59-negative RET and RBC (RET^CD59-and RBC^CD59-).

Males and females given 200 mg/kg/day had treatment-related decreases in body weights and body weight gains compared to controls throughout most of the study, with no corresponding effects on feed consumption. 

Females given 200 mg/kg/day had a treatment-related decrease in percent neutrophils and an increase in percent lymphocytes as compared to controls.

The majority of males and females given 50 or 200 mg/kg/day had treatment-related gross pathological observations of yellow discoloration of the adipose tissue throughout the body which was interpreted to be caused by the dye nature of the test material. Additional treatment-related gross pathologic observations were limited to males given 200 mg/kg/day and consisted of dark livers and spleens, and perineal soiling. Treatment-related organ weight effects were limited to males and females given 200 mg/kg/day and consisted of increased absolute (10.5 and 9.5%, respectively) and relative (24.8 and 15.6%, respectively) liver weights and increased absolute (18.3%) and relative (33.2%) spleen weights (males only) compared to controls.

Histopathology in livers of males and females given 50 or 200 mg/kg/day demonstrated treatment-related very slight or slight centrilobular/midzonal hypertrophy of hepatocytes, with increased cytoplasmic eosinophilia. Males given 200 mg/kg/day also had treatment-related congestion of the red pulp of the spleen and very slight or slight increased extramedullary hematopoiesis (erythroid cell) of the spleen.

Two females given 200 mg/kg/day had complications of parturition (dystocia), manifest as protracted delivery.  These females also exhibited clinical signs of vulvar discharge, and one had pale skin/mucous membranes. One of these females was euthanized in a moribund condition as a secondary consequence of dystocia. Other treatment-related reproductive effects occurred in the 200 mg/kg/day group and consisted of decreases in conception, fertility, gestation and gestation survival indices, and increased time to mating. There was a treatment-related increase in the number of litters (4/5) with one or more dead pups and clinical observations associated with pup mortality (pale and yellow skin) in the high-dose litters. Consistent with effects on offspring survival, there was a treatment-related reduction in mean litter size in the 200 mg/kg/day group.

No significant change was observed in the frequency of RET^CD59-or RBC^CD59-in animals treated with the test material. Therefore, MORTRACE™ SB Conc Fuel Marking System was negative in this in vivo gene mutation Pig-a assay under the experimental conditions used.

Based on the non-adverse histopathological changes in the liver, the no-observed-adverse-effect level (NOAEL) for general male and female toxicity was 50 mg/kg/day. The no-observed-effect level (NOEL) for reproductive toxicity was 50 mg/kg/day, based on the reproductive effects observed in the high-dose group.

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Acceptable - a screening study for reproductive toxicity is available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Available toxicity data for Reproductive/developmental toxicity endpoint:

A standard guideline OECD 421 reproductive/developmental screening study is available for this substance. This study also included an assessment of in vivo genotoxicity (Pig-a assay) which is discussed in section 7.6.

Groups of 10 male and 10 female Crl:CD(SD) rats were administered MORTRACE™ SB Conc. daily, by gavage in Polyethylene glycol 400 (PEG 400), at dose levels of 0, 10, 50, or 200 mg/kg/day. Male rats were dosed two weeks prior to breeding and continuing through breeding (up to two weeks) for a treatment period of at least 28 days. Female rats were dosed two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), lactation (four days) and until necropsy. Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition, litter size, pup survival, pup sex, pup body weight and the presence of gross external morphological alterations were assessed. In addition, hematology was evaluated on females and a gross necropsy and histopathology of the adults was conducted with an emphasis on organs of the reproductive system.

General toxicological findings:

Males and females given 200 mg/kg/day had treatment-related decreases in body weights and body weight gains compared to controls throughout most of the study, with no corresponding effects on feed consumption. Females given 200 mg/kg/day had a treatment-related decrease in percent neutrophils and an increase in percent lymphocytes as compared to controls.

The majority of males and females given 50 or 200 mg/kg/day had treatment-related gross pathological observations of yellow discoloration of the adipose tissue throughout the body which was interpreted to be caused by the dye nature of the test material. Additional treatment-related gross pathologic observations were limited to males given 200 mg/kg/day and consisted of dark livers and spleens, and perineal soiling. Treatment-related organ weight effects were limited to males and females given 200 mg/kg/day and consisted of increased absolute (10.5 and 9.5%, respectively) and relative (24.8 and 15.6%, respectively) liver weights and increased absolute (18.3%) and relative (33.2%) spleen weights (males only) compared to controls.

Histopathology in livers of males and females given 50 or 200 mg/kg/day demonstrated treatment-related very slight or slight centrilobular/midzonal hypertrophy of hepatocytes, with increased cytoplasmic eosinophilia. Males given 200 mg/kg/day also had treatment-related congestion of the red pulp of the spleen and very slight or slight increased extramedullary hematopoiesis (erythroid cell) of the spleen.

The very slight hypertrophy observed in the liver at the 50 mg/kg bw/day dose level was considered to be non-adverse due to the absence of any other correlated findings. Consequently the No observed Adverse effect level for general parental toxicity was 50 mg/kg bw/day.

 

Reproductive findings

In the 200 mg/kg/day group there were treatment-related decreases in conception and fertility indices. 3/10 dams with evidence of mating had no litter or evidence of pregnancy. Furthermore, animals in the 200 mg/kg/day group had increased time to mating and incidence of dystocia (with 2/10 dams displaying complications of parturition).  Together, these effects resulted in a treatment-related decrease in gestation index, with 5/7 pregnant dams delivering a live litter.  The two females given 200 mg/kg/day that had complications of parturition (dystocia - manifesting as protracted delivery) also exhibited clinical signs of vulvar discharge, and one had pale skin/mucous membranes. One of these females was euthanized in a moribund condition as a secondary consequence of dystocia. There was a treatment-related increase in the number of litters (4/5) with one or more dead pups and clinical observations associated with pup mortality (pale and yellow skin) in the high-dose litters. Consistent with effects on offspring survival, there was a treatment-related reduction in mean litter size in the 200 mg/kg/day group.

 

There were no effects on conception, fertility, or gestation indices in the 10 or 50 mg/kg/day dose groups.  Additionally, there were no effects on time to mating or the incidence of dams with dystocia in the 10 or 50 mg/kg/day dose groups.

 

Based on the non-adverse histopathological changes in the liver, the no-observed-adverse-effect level (NOAEL) for general male and female toxicity was 50 mg/kg/day. The no-observed-effect level (NOEL) for reproductive toxicity was 50 mg/kg/day, based on the reproductive effects observed in the high-dose group.

Although these findings do indicate the potential for this substance to produce reproductive toxicity, no further testing is planned at this time. The registration level for substance is currently <100t and more extensive testing at this tonnage band is not typically required. This substance is also used as a fuel marler and as such is formulated into petroleum which significantly limits the potentail for oral and inhalation exposure. Dermal exposure route is not considered to be of significant concern (refer to Toxicokinetics section and the low dermal bioavailability prediction).

Short description of key information:
A reproduction/developmental screening study in rats (oral route) is available. Adverse effects on both parental and reproductive toxicity were observed. NOAEL for parental toxicity is 50 mg/kg bw; for reproductive toxicity is 50 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
This is the appropriate OECD guideline screening study for this endpoint at this registration level.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The available data on this substance do not allow for a definitive assessment of the mode of action for reproductive toxicity. However there are some pertinent observations that do provide context for the nature of the toxicity observed.

The substance is an azo compound with very low water solubility. The predicted oral bioavailability is very low (refer to TK section). Therefore it is unlikely that the parent compound is directly responsible for the observed systemic findings.

The azo bond is vulnerable to metabolism by gut microflora and to a limited extent, the liver. Once the azo bond is reduced the subsequent metabolites include 4 -nitroaniline.

There is a high degree of concordance between the observer toxicity with this substance and that observed with 4 -nitroaniline. It is therefore likely that the ultimate toxicant is this metabolite and not ht eparent compound. It is unclear what the other metabolite 4-amino-2,6-di-sec-butylphenol

contributes to to the toxicity profile.

4 -nitroaniline is not classified for reproductive and developmental toxicity, however this appears due to the low dose levels used in the available studies; top dose is limited by the methemoglobinemia and anemia produced by 4 -nitroaniline.

Anemia and methemoglobinemia are known to produce adverse effects during pregnancy due to the additional stress placed on the pregnant rats.

Considering that it is likely the systemic toxicity results from exposure to 4 -nitroaniline, it is probable that the observed reproductive findings are secondary to more genral toxicity. However without further data it is not possible to further support this conclusion.

An important consideration relating to the systemic toxicity is the potential for this to occurr via different dose routes. Reductive metabolism does not occur in the lung and due to very low water solubility this substance will not be well absorbed. Therefore via inhalation the conversion to 4 -nitroaniline will not occurr and thus the observed effects on reproduction (and genreral systemic toxicity) will also not occurr.

Via the dermal route, there is evidence that azo bond reduction by skin bacteria can occur and foollowing metabolism 4 -nitroaniline can penetrate the skin. However it is likely that the kinetics of the metabolism and subsequent skin penetration would result in a lower C-max and longer Tmax, potnetially lessening the potency of the dermal route versus the oral route. However additional research is needed before the relevance of the findings to the dermal route can be clarified.

Justification for classification or non-classification

In the OECD 421 at the top dose level (200 mg/kg bw) there were treatment-related effects on time to mating, conception, fertility and gestation indices. At this dose level there were also clear effects on parental toxicity (effects on the liver, bodyweights and hematological parameters). There were no findings relating to the testes, epididymis, ovaries, oviducts, uterus, or sperm parameters that would provide any explanation for the cause of the effects on mating, conception, fertility and gestation indices. In the available repeated dose toxicity study the systemic toxicity was limited to hematological parameters, bodyweight and organ weight changes (including spleen and liver). No effects were observed on sex organs.

Based on the findings from the key study, it is considered appropriate to classify Mortrace SB for reproductive toxicity (fertility). With respect to the classification category, Category 2 is proposed for the following reason:

The key study is a screening study and while there is evidence that the substance produced effects on fertility parameters, these effects were concurrent with parental toxicity (including evidence of anemia in the males and neutropenia in the females (effects also observed in the other repeated dose toxicity studies – refer to repeated dose toxicity section) which could have contributed to the observed reproductive toxicity findings (also refer to th Mode of action analysis).