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REACH

N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

EC number: 212-344-0 | CAS number: 793-24-8

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

The fertility effects are based on an OECD 443 extended 1 -generation reproductive study and its range-finder, which was based on an OECD 421.

The key effect is dystocia, which was found in multiple treatment groups in both the main study and the DRF.

In the DRF, rats were dosed at 100, 75 or 50 mg/kg/day 6PPD with a 2 week pre-breed and 15 rats/group. Five rats were either sacrificed in extremis or with total litter loss around the time of parturition at 100 mg/kg/day. There were also 2 rats at 75 mg/kg/day and 1 rat at 50 mg/kg/day with similar findings.

In the main OECD 443, rats were dosed at 60, 20 and 7 mg/kg/day, with a 10 week prebreed and 25 rats per group. A6 60 mg/kg/day there were 4 rats either found dead or sacrificed with dystocia around parturition and also 3 total litter losses. There were 2 found dead and 1 total litter loss at 20 mg/kg/day and none at 7 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018 - December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate accordig to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for 6PPD.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patterns of the registered substance in coatings, exposure-based waiving is not possible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes

Limit test:

Justification for study design:

The study design was based on the OECD 443 guideline. The developmental neurotoxicity cohort was conducted based on an order from the European Chemicals Agency

Specific details on test material used for the study:

Identification: N-1,3-Dimethylbutyl-N′-Phenyl-p-Phenylenediamine (also referred to as 6PPD; CAS No. 793-24-8)
Lot No.: W7F25PN001
Receipt Date: 23 Oct 2017
Physical Description: Dark brown pellets
Purity: 96.9%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light, under nitrogen
Supplier: Eastman Chemical Company

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, Jun 2018, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to meet guideline recommendations. Five extra females (and corresponding males) were included in the high-dose group on the current study to accommodate potential higher losses in this group, based on observations of dystocia, moribundity, mortality, and total litter losses in the previous dose range-finding study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Identification
Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning
Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 9999 01).

Environmental Acclimation
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.

Selection, Assignment, and Disposition of Animals
F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups.
To reduce variability among the F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 4 F1 pups/sex/litter from all available litters (≥ 20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts. Cohort 1 (A and B) was designated for reproductive/developmental toxicity assessment. Animals assigned to Cohort 1B were maintained on study for possible breeding when the animals were between 90 and 120 days of age to generate an F2 generation; however, additional breeding was not required on this study. Cohort 2 (A and B) was designated for developmental neurotoxicity assessment. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill a designated cohort, the following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B, Cohort 2A, and Cohort 2B

Housing
On arrival, the F0 animals were group housed (2 to 3 animals of the same sex). During cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until euthanasia
Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF was provided ad libitum throughout the study, except during designated procedures .
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director. The following treatments (excluding any dietary supplementations or addition of water bottles) were administered.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Animals were treated once daily, 7 days a week for the specific number number of days for thier sex and cohort .

Details on mating procedure:

Males and females were cohabitated at the appropriate times specified by their groups (presented below) 1:1 in the same cage. Animals were separated with a sign of copulation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation and Analysis

Preparation of Vehicle
The vehicle, corn oil, was dispensed approximately weekly for administration to Group 1 control animals and preparation of the test substance formulations. For administration to Group 1 control animals, an adequate amount of the vehicle was dispensed into daily aliquots, which were stored refrigerated (target of 5°C), protected from light, until use. The vehicle was also stirred continuously during dosing. Details of the dispensing of the vehicle have been retained in the Study Records.

Preparation of Test Substance
Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared every 3 days for the first 3 dosing preparations then approximately weekly thereafter, and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5°C), protected from light, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study

Records.Analytical Method
Analyses were performed by high performance liquid chromatography using ultraviolet absorbance detection using a validated analytical procedure.1,

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (1.0 mL) from the 1 mg/mL nondosing formulation and from the first dosing formulations were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean concentration was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Test substance formulations have been previously shown to be stable at concentrations of 10 to 200 mg/mL for at least 10 days at refrigerated temperature.3 Therefore, stability of test substance formulations at concentrations above 10 mg/mL will not be assessed on this study.
During the course of this study, stability of the prepared formulation was determined at room temperature for 24 hours and at refrigerated storage (target of 5°C) for 3 days at a concentration of 1 mg/mL. Duplicate sets of each sample (1.0 mL) were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Stability results were considered acceptable if the sample analysis results were not < 90% of the concentration determined by the initial analysis of each formulation. After acceptance of the analytical results, backup samples were discarded.

Duration of treatment / exposure:

F0 males were dosed orally by gavage for 70 consecutive days prior to mating and continuing through the day prior to euthanasia (for a minimum of 10 weeks). F0 females were dosed orally by gavage for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia (following completion of weaning for all F1 litters). The offspring selected for the F1 generation began dosing on the day of weaning until the day prior to euthanasia (PND 21 [Cohort 2B], PND 91 [Cohort 1A], PND 78 [Cohort 2A], and PND 98 [Cohort 1B]). All animals were dosed at approximately the same time each day .

Frequency of treatment:

Treatment was daily, 7 days a week for the appropriate date range for each sex/group

Details on study schedule:

See "other methods" section

Dose / conc.:

7 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

No. of animals per sex per dose:

25/sex/group except in the 60 mg/kg/day groups which had 30/ex

Control animals:

yes, concurrent vehicle

Details on study design:

Parental animals (the F0 as identified in the study)were pretreated for 70 days, followed by up to 2 weeks of mating, through gestation and lactation. F0 animals were sacrificed at weaning, which is when the F1 animals began treatment (F1 was the term used in the study report). The F1 animals were treated for the periods specified by their group assignments.

Positive control:

Parental animals: Observations and examinations:

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily, and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

Sperm parameters (parental animals):

Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer
The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (IDENT Stain, based on Hoechst bisbenzimide, stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.

Litter observations:

Litter weights were recorded on PND 1. Pups were sexed and AGD was measured.

Postmortem examinations (parental animals):

All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

Postmortem examinations (offspring):

Scheduled Euthanasia
On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital.
On PND 21, nonselected pups were euthanized by carbon dioxide inhalation.

Necropsy
On PND 4, all culled pups were subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.
On PND 21, nonselected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

Organ Weights
The organs identified were weighed at necropsy from up to 10 nonselected F1 pups/sex/group on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Tissue Collection and Preservation
Representative specimens with malformations or gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin.
Representative samples of the tissues were collected from PND 4 culled pups and preserved in 10% neutral buffered formalin.
Representative samples of the tissues identified were collected from PND 21 nonselected pups and preserved in 10% neutral buffered formalin.

Statistics:

See "other information" as there is not enough space in this box

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Mortality:

mortality observed, treatment-related

Description (incidence):

*Terminology used in the study report is F0 and F1, and that is retained here*

One F0 male each in the 7 and 60 mg/kg/day groups and 3 and 5 F0 females in the 20 and 60 mg/kg/day groups, respectively, were found dead or euthanized in extremis during the study. Of these rats, 2 and 5 of the females in the 20 and 60 mg/kg/day groups, respectively, were found dead or euthanized in extremis at the time of parturition or early lactation (Gestation Day 21 through Lactation Day 2); moribundity and mortality were considered test substance-related and adverse.
Male No. 9639 in the 7 mg/kg/day group was found dead on Study Day 26. This male had no remarkable clinical or macroscopic observations or effects on body weight or food consumption, and microscopically was noted with congestion of the liver, lungs and kidneys. A cause of death was not determined; however, this death was not considered test substance-related because there were no other indications of toxicity at this dosage level and no test substance-related mortality in males at 20 or 60 mg/kg/day. Male No. 9647 in the 60 mg/kg/day group was euthanized in extremis on Study Day 94 after swallowing the dosing cannula; the cause of death was accidental and not considered to be test substance-related. Female No. 9557 in the 20 mg/kg/day group was found dead on Study Day 2 due to a gavage error; a perforated esophagus and dark red contents in the thoracic cavity were noted at necropsy. All other F0 parental animals in the control, 7, 20, and 60 mg/kg/day groups survived to the scheduled necropsy.
No significant test substance related clinical observations were noted during the generation at the daily examinations or approximately 3 hours postdosing at any dosage level. Findings noted in the test substance treated groups, including hair loss or scabbing on various body surfaces, and red material around the nose and/or mouth, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Weekly
No test substance related effects on mean body weights or body weight gains were noted in the 7, 20, and 60 mg/kg/day groups. The values in the test substance treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences for mean body weight gains compared to the control group occurred infrequently in all groups; however, the changes were transient, not of sufficient magnitude to result in statistically significant differences in mean absolute body weights or mean body weight gains for the premating period (Study Days 0–69, males and females) or entire generation (Study Days 0–128, males), and/or did not occur in a dose-related manner. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Gestation
Mean maternal body weights and body weight gains were unaffected by test substance administration during gestation. Differences between the control, 7, 20, and 60 mg/kg/day groups were slight and not statistically significant.

Lactation
Mean maternal body weights and body weight gains were unaffected by test substance administration during lactation. Differences between the control, 7, 20, and 60 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Weekly
Mean food consumption, evaluated as g/animal/day, and food efficiency in the 7, 20, and 60 mg/kg/day groups were unaffected by test substance administration. The values in the test substance treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences in food consumption and/or food efficiency compared to the control group occurred infrequently in all groups; however, the changes were transient and did not occur in a dose-related manner. Other differences from the control group were slight, did not occur in a dose related manner, and/or were not statistically significant.

Gestation
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during gestation. Differences between the control, 7, 20, and 60 mg/kg/day groups were slight and not statistically significant.

Lactation
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance administration during lactation. Differences between the control, 7, 20, and 60 mg/kg/day groups were slight and not statistically significant.

Food efficiency:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

There were no test substance-related effects on hematology parameters. Statistically significantly lower mean corpuscular hemoglobin concentration and statistically significantly higher reticulocyte counts were noted in the 60 mg/kg/day group males compared to the control group. Higher mean red blood cell distribution width was noted in 60 mg/kg/day females compared to concurrent controls. These findings were not considered test substance related due to the low magnitude of the changes, the absence of other related hematologic changes, and/or any correlating microscopic changes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Mean T4 levels in the 20 and 60 mg/kg/day group males and females were statistically significantly higher than the control group. In the absence of effects on mean thyroid weights and microscopic findings, given that a correlating change in TSH levels was not observed, and due to the values being within the Charles River Ashland historical control data range (version 2019.02), the higher mean T4 levels in these groups were not considered test substance related. Mean T4 levels in the 7 mg/kg/day group males and females and mean TSH levels in the 7, 20, and 60 mg/kg/day group males and females were comparable to the control group.

Higher mean albumin, total protein, and globulin concentrations were noted in the 60 mg/kg/day group males and females; the differences from the control group were statistically significant. Mean cholesterol levels in the 20 and 60 mg/kg/day group males and the 60 mg/kg/day group females were higher than the control group; the differences were statistically significant. Other statistically significant differences in serum chemistry values in the test substance-treated groups (higher mean calcium levels and lower mean chloride levels in the 7 and/or 60 mg/kg/day group females) compared to the control group were not considered test substance-related due to the small magnitude of change and/or lack of a dose response.

Urinalysis findings:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

There were no test substance-related effects on urinalysis parameters. The only statistically significant difference from the control group was a higher mean urobilinogen level in the 60 mg/kg/day group females. This finding was not considered test substance-related because there were no other effects on urinalysis parameters or correlating microscopic changes and no corresponding changes in the males.

Behaviour (functional findings):

not examined

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Immunological findings:

not examined

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

There were treatment-related findings in the liver and the kidneys of 7, 20, and 60 mg/kg/day males and 60 mg/kg/day females.
In 7, 20, and 60 mg/kg/day males, there was dose-related increase in incidence and severity of vacuolation of the liver. Vacuolation in the liver in females was limited to the 60 mg/kg/day group. Vacuolation was characterized by multiple clear vacuoles expanding the cytoplasm of the hepatocytes especially in the midzonal region of the hepatic lobules. When minimal, a few widely scattered hepatocytes had distended cytoplasm filled with these clear vacuoles and there was no lobular distribution. This macrovesicular vacuolation caused no displacement of the nucleus. The vacuolation may have contributed in part to higher liver weights but was not the primary cause for elevation in liver weights. When marked (Animal No. 9685, 60 mg/kg/day), the vacuolation correlated to gross observation of pale liver.
In 7, 20, and 60 mg/kg/day males, there was dose-related increase in incidence and severity of pigment deposition in the kidneys. Pigment deposition in females was limited to 60 mg/kg/day group. Pigment deposition was characterized as yellow to brown pigment in the cytoplasm of proximal convoluted tubular epithelium and within the lumen of these proximal convoluted tubules. This pigment did not distort the tubular epithelial cells or result in any parenchymal damage to the tubule. Pigment was present in higher incidence and severity in males than in females. The pigment was not the cause of treatment-related higher kidney weights. The pigment had no correlation to any treatment-related clinical pathology findings or urinalysis changes and was nonadverse.
The Hall’s special stain and the Prussian Blue special stains on selected male kidneys revealed that the pigment was generally negative for bile and/or iron. One selected kidney (Animal No. 9712) had a minimal amount of positive iron staining in the kidney, but this staining did not account for the full amount of golden brown pigment observed in the H&E stained tissue. Therefore, the pigment visualized in the H&E stained section is of unknown origin but may be related to the 6PPD or its metabolite(s).
There were no other 6PPD-related histologic changes. Remaining histologic changes were considered to be incidental findings. One 60 mg/kg/day female (Animal No. 9624) had moderate dilatation of all chambers of the heart; however, this isolated finding was considered unrelated to 6PPD since there was no microscopic findings in the cardiac myofibers of this animal. One 60 mg/kg/day male (Animal No. 9744) had focal minimal necrosis of skeletal muscle, and this isolated finding was also considered unrelated to 6PPD.
In all groups of females, including the controls, there was minimal to moderate mineralization of the outer stripe of the kidney medulla. This mineralization was considered secondary to the 5K96 diet, and there was no difference in incidence or severity between control and treated groups. Any variation in the incidence of chronic progressive nephropathy and/or basophilic tubules in controls vs treated males or females was considered toxicologically insignificant and related to the 5K96 diet.
The ovaries of F0 animals frequently had reduced numbers of corpora lutea and retained antral follicles. The incidence of these changes showed no dose response profile, and was 6/25, 6/25, 4/22, and 5/25 in the 0, 7, 20 and 60 mg/kg/day groups, respectively. Many of these animals were identified as animals with reduced fertility. The incidence of follicular cysts (focal or multifocal) was 2/25, 0/25, 1/22, and 4/25, respectively. The cysts were slightly larger than a tertiary ovulatory follicle but were not noted on gross examination. There was no difference in the appearance of the cysts between the various dose groups. The cysts were considered incidental, within an expected incidence rate for adult fertile rats, and unrelated to 6PPD administration.
There were no microscopic findings to correlate to treatment-related changes in the clinical pathology, such as regenerative red cell changes (lower MCHC, higher reticulocyte counts and higher red cell distribution width) in 60 mg/kg/day females, higher urobilinogen in 60 mg/kg/day females, or with the higher albumin, total protein and globulin in 60 mg/kg/day males and females.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Other effects:

not specified

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

No test substance related effects were observed on F0 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

*Terminology used in the study report is F0 and F1, and that is retained here*

No test substance related effects on F0 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. Three, 3, and 2 males in the control, 7 and 20 mg/kg/day groups, respectively, did not sire a litter. Three, 4, and 2 females in these same respective groups were determined to be nongravid.
The mean numbers of days between pairing and coitus in the test substance treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Two and 5 females in the 20 and 60 mg/kg/day groups were found dead or euthanized in extremis at the time of parturition or early lactation (Gestation Day 21 through Lactation Day 2). Moribundity and mortality noted during the time of parturition and early lactation was attributed to test substance-related dystocia and/or prolonged labor in the 20 and 60 mg/kg/day groups, although mean gestation lengths for the surviving females in these 2 groups and the 7 mg/kg/day group were between 22.0 to 22.2 days and were comparable to the control group mean (22.0 days) and the Charles River Ashland historical control data (21.8 days). No signs of dystocia were noted at 7 mg/kg/day.

*Terminology used in the study report is F0 and F1, and that is retained here*

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

7 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

7 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

other: Dystocia

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Critical effects observed:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

There were no test substance-related effects on F1 survival at any dosage level. Three males in the control group and 1 male and 1 female in the 7 mg/kg/day group were found dead. One male and 1 female in each of the control and 60 mg/kg/day groups were euthanized in extremis. Male No. 9552-07 and Female No. 9552 12 in the 60 mg/kg/day group were euthanized in extremis on PND 27 and 26, respectively, following clinical observations of cool, pale body and/or extremities beginning up to 2 days prior to euthanasia; these animals weighed only 27 g and 25 g, respectively, on PND 21 compared to respective group means of 46 g and 44 g. A cause of moribundity could not be determined for these animals, but the moribund condition was likely a continuation of the effects noted on body weights for this litter during the preweaning period. In the 7 mg/kg/day group, Male No. 9606-04 and Female No. 9606-10 were found dead on PND 57 and 91, respectively. Male No. 9606-04 had dark red discoloration in the lungs at necropsy; pulmonary hemorrhage was determined to be the cause of death microscopically. A cause of death could not be determined for Female No. 9606-10. In the absence of any unscheduled deaths for F1 males and females in the 20 mg/kg/day group, the mortality in the single litter at 7 mg/kg/day was not considered test substance-related.
In the control group, Male Nos. 9528-01 and 9556 05 in the control group were found dead on PND 22 and 24, respectively, as a result of intubation errors; esophageal perforation was noted at necropsy for both males. Male No. 9556 07 in the control group was found dead on PND 35. There were no clinical or macroscopic observations for this male; the cause was death was determined to be pulmonary hemorrhage microscopically. Male No. 9594-01 in the control group was euthanized in extremis due to a swollen forelimb; at necropsy, the radius and ulna were noted as bent and the ulna was thickened. Female No. 9539-08 in the control group was euthanized in extremis on PND 25 after clinical observations of cool body and pale extremities; this female weighed only 26 g on PND 21 compared to a group mean of 48 g. This death was likely a continuation of the low body weights noted for this animal during the preweaning period.
All other F1 animals in the control, 7, 20, and 60 mg/kg/day groups survived to the scheduled necropsy.
No test substance related clinical observations were noted during the F1 generation at the daily examinations or approximately 3 hours postdosing at any dosage level. Findings noted in the test substance treated groups, including red, yellow, or clear material, hair loss, and/or scabbing on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Mean absolute body weights in the 60 mg/kg/day group males and females were 6.1% and 8.3% lower, respectively, than the control group on PND 21 due to the test substance-related decrements in mean body weight gain at the end of the preweaning period. Mean body weight gain in the 60 mg/kg/day group females was slightly lower than the control group during PND 21–28, resulting in a mean absolute body weight that was 8.6% lower than the control group on PND 28. Mean body weight in the 60 mg/kg/day males also remained slightly lower than controls (5.9%) on PND 28. None of the differences were statistically significant. These effects were considered test substance related but not adverse due to the transient nature and because the differences in body weights were a continuation of the effects noted during the preweaning period. No other effects on mean body weights or body weight gains were noted in the 60 mg/kg/day group F1 males and females following weaning. The only statistically significant difference from the control group was a transient, lower mean body weight gain in the males during PND 90–98.
No test substance related effects on mean body weights and body weight gains were noted in the 7 and 20 mg/kg/day groups. The values in these groups were generally similar to the control group values throughout the generation. None of the differences from the control group were statistically significant

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Food consumption, evaluated as g/animal/day, and food efficiency in the 7, 20, and 60 mg/kg/day groups were unaffected by test substance administration. The values in the test substance treated groups were generally similar to the control group values. The only statistically significant differences from the control group were higher mean food consumption in the 60 mg/kg/day group males during PND 42–49 and 56–63 and in the 20 mg/kg/day group females during PND 56–63 and lower mean food efficiency in the 7 and 60 mg/kg/day group males during PND 63–70. These differences were transient and/or not dose-responsive.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

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

not examined

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Dose-related increases in the mean absolute numbers of white blood cells and lymphocytes were noted in the 7, 20, and 60 mg/kg/day group males; the differences from the control group were statistically significant for white blood cells at 20 and 60 mg/kg/day and for lymphocytes at 60 mg/kg/day. These findings were considered test substance-related but not adverse because there were no other adverse effects on hematology parameters or correlating microscopic findings. Higher reticulocyte counts were noted in the 20 and 60 mg/kg/day group males compared to the control group; differences were generally statistically significant. These findings were not considered test substance related due to the lack of a dose response. No other test substance-related findings were noted in hematology parameters; other statistically significant differences from the control group (lower mean corpuscular hemoglobin and higher red blood cell distribution width in the 60 mg/kg/day group males) were not considered test substance-related due to the small magnitude of change.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Thyroid Hormone
There were no test substance-related effects on thyroid hormones for F1 males and females at any dosage level prior to necropsy on PND 91. None of the differences were statistically significant and were observed in a non-dose-responsive fashion.

Higher mean albumin, total protein, and globulin concentrations were noted in the 60 mg/kg/day group males and females; the differences from the control group were statistically significant. Mean cholesterol levels in the 20 and 60 mg/kg/day group males and the 60 mg/kg/day group females were higher than the control group; the differences were statistically significant. These findings were considered test substance-related but not adverse because there were no other adverse effects on serum chemistry parameters or correlating adverse microscopic findings. Other statistically significant differences in serum chemistry values in the test substance-treated groups (lower mean chloride levels in the 60 mg/kg/day group males) compared to the control group were not considered test substance-related due to the small magnitude of change.

Urinalysis findings:

no effects observed

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

There were no test substance-related effects on urinalysis parameters. The only statistically significant difference from the control group was a higher mean pH value in the 60 mg/kg/day group females.

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Cohort 1A
There was treatment-related higher absolute adrenal gland weight in the 20 (14.8%, statistically significant) mg/kg/day females; higher absolute adrenal gland weights in the 60 (21.9%, statistically significant) mg/kg/day females; higher adrenal gland weight relative to final body weight and brain weight in the 60 mg/kg/day females (statistically significant); and higher adrenal gland weight relative to final body weight and relative to brain weight (both statistically significant) in the 60 mg/kg/day males. These adrenal gland weights had no microscopic correlation and were within the Charles River Ashland historical control range. The changes in adrenal weights were considered nonadverse and of little to no toxicologic significance.
There were higher absolute (14.9%) kidney weight and higher kidney weights relative to final body weight and brain weight in the 60 mg/kg/day females (all statistically significant); and higher kidney weights relative to final body weight and brain weight (both statistically significant) in the 60 mg/kg/day males. These higher kidney weights were within the Charles River Ashland historical control range, and any variation in the kidney weights between groups was of no or little toxicological significance.
There were higher absolute (15.3%) liver weights and higher liver weights relative to final body weight and brain weight (all statistically significant) in the 20 mg/kg/day females; higher absolute (30.7%) liver weights and higher liver weights relative to final body weight and brain weight (all statistically significant) in the 60 mg/kg/day females; and higher absolute (15.3%) liver weights and higher liver weights relative to final body weight and brain weight (all statistically significant) in the 60 mg/kg/day males. These higher liver weights had no microscopic correlation and were considered to be a nonadverse adaptive change.
Other statistically significant weight changes were considered incidental, because they were in a direction opposite to that normally expected for a toxicologic lesion, only affected 1 relative weight, and/or showed no dose response profile. Included in these changes were: higher left cauda epididymis weights (relative to body weight and relative to brain weight; statistically significant) in the 60 mg/kg/day males; higher (statistically significant) LABC muscle weight relative to final body weight in the 7 mg/kg/day males; and higher (statistically significant) right testis weight relative to brain weight in the 60 mg/kg/day males.

Cohort 1B
There were no treatment-related effects on organ weights in the F1 Cohort 1B animals. Only limited tissues were weighed in Cohort 1B, including male reproductive tissues, female reproductive tissues, and the pituitary gland.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of 6PPD. There were dark red area(s) in the lungs and dark discoloration of the lungs limited to the treated males and females. In males, dark red area(s) in the lungs were present in 0/18, 3/19, 2/18 and 3/19 males and dark red discoloration of the lungs in 0/18, 0/19, 1/18 and 1/19 males in the control, 7, 20, and 60 mg/kg/day groups, respectively. In females, dark red area(s) in the lungs were reported in one 20 mg/kg/day female. The gross findings corresponded to hemorrhage and/or congestion, but there was no dose-response profile for incidence or severity of these microscopic changes in the lungs.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Neuropathology
Macroscopic Examination (Cohorts 2A and 2B)
At the scheduled F1 male and female necropsies, no test substance related findings were observed for the brain and spinal cord at any dosage concentration.

Brain Weights and Measurements (Cohorts 2A and 2B)

There were no treatment-related effects on brain weights or gross brain measurements. In the Cohort 2B animals, there was lower group mean brain weight in the 60 mg/kg/day males, but this change was considered a direct consequence of the 6PPD-related statistically significant lower final body weight in that group. The group mean brain weight in the 60 mg/kg/day group was within the neurotoxic historical control range for PND 21 male Sprague Dawley rats (mean ± 2 standard deviations). Because a lower group mean brain weight in the 60 mg/kg/day male was attributed to a 6PPD-related effect on body weight, it did not reflect a developmental effect.

Microscopic Examination (Cohorts 2A and 2B)
There were no microscopic findings in the brains of males or females from Cohorts 2B or 2A.

Morphometric Analysis (Cohort 2A)
There was statistically significant lower width of the caudate-putamen (S3) and lower thickness of the hippocampus (S5) in the 60 mg/kg/day males at PND 78. There were no 6PPD-related changes in the female microscopic measurements. These lower measurements had no correlation to microscopic findings or correlation to brain weights or gross brain measurements at PND 78. These 2 findings without correlating changes in other brain sites of the same dose group, between sexes, or between cohorts, suggest that the changes were unrelated to treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

There were no treatment-related microscopic findings in the tissues of F1 males or females in any treatment group. In the control and high-dose group females, there was minimal to marked mineralization of the outer strip of the medulla, and variable degrees of basophilic tubules in the outer cortex. As with the F0 cohorts, these changes were considered secondary to the 5K96 diet. There was no difference in incidence or severity between control and treated groups.
There were no treatment-related effects on ovarian follicle counts in F1 females from Cohort 1A. Any variation in counts between those groups was due to normal biological variability.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Other effects:

not specified

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Details on results:

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

The mean lengths of estrous cycles for F1 females in the test substance-treated groups during PND 75–91 were similar to the control group value. In addition, the percentage of females cycling in the test substance-treated groups were similar to the control group; there was no test substance-related effect on the percentage of females cycling regularly. None of the differences were statistically significant.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

No test substance related effects were observed on F1 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

not examined

Description (incidence and severity):

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Not a valid metric as the F2 was not bred

*Data in "P1" boxes is cohort 1A and 1B after weaning. Data prior to weaning is under F1*

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Neurotoxicity cohort with no adverse findings

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance administration. Forty-two (13), 19(6), 45(14), and 111(20) pups (litters) in the control, 7, 20, and 60 mg/kg/day groups, respectively, were found dead or euthanized in extremis. Eighteen (8), 9(5), 17(10), and 29(13) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Body weight and weight changes:

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Mean male and female pup birth weights (PND 1) were comparable across all groups. Mean pup body weight gains in the 60 mg/kg/day group were slightly lower than the control group throughout the preweaning period (PND 1–21), resulting in mean body weights that were 8.1% and 7.4% lower for males and females, respectively, compared to the control group on PND 21. The differences were not statistically significant but were considered test substance-related and adverse based on the magnitude of the difference from the control group.
Mean male and female pup body weights and body weight changes in the 20 mg/kg/day group were slightly lower than the control group however, the differences in mean body weights remained within 5% of controls and were hence considered test substance-related but nonadverse. Mean male and female pup body weights and body weight changes in the 7 mg/kg/day group were unaffected by parental test substance administration throughout the preweaning period. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Value was not examined as measurements are birth to weaning

Food efficiency:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Value was not examined as measurements are birth to weaning

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

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Ophthalmological findings:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Haematological findings:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Thyroid Hormone Analysis
PND 4 Culled Pups

There were no test substance-related effects on mean T4 levels at any dosage level in the PND 4 culled pups. Differences were observed in a non-dose responsive manner.

PND 21 Pups Selected for Hormone Analysis

There were no test substance-related effects on mean T4 and TSH levels at any dosage level in male and female pups on PND 21. Mean T4 levels in the 7, 20, and 60 mg/kg/day group male and female pups were slightly lower than the control group. However, the changes were not dose-responsive in males and there were no test substance related effects on mean thyroid weights in males or females. Therefore, the lower mean T4 levels were not considered test substance-related.

Urinalysis findings:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Sexual maturation:

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Balanopreputial Separation

Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of balanopreputial separation were 45.7, 47.0, and 47.1 days in the 7, 20, and 60 mg/kg/day groups, respectively, when compared to 47.7 days in the control group. Mean body weights at the age of attainment were 232.0, 240.9, and 238.1 g in the same respective groups compared to 245.3 g in the control group. None of the differences from the control group were statistically significant.

Vaginal Patency

Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of vaginal patency were 35.0, 35.9, and 36.4 days in the 7, 20, and 60 mg/kg/day groups, respectively, when compared to 36.3 in the control group. Mean body weights at the age of attainment were 123.5, 123.3, and 124.3 g in the same respective groups compared to 129.7 g in the control group. None of the differences from the control group were statistically significant.

The mean ages at the first occurrence of estrus in the 7, 20, and 60 mg/kg/day groups (37.3, 37.8, and 39.5 days, respectively) were comparable to the control group (38.3 days). In addition, the duration from vaginal opening to first estrus in these same respective groups (3.2, 2.4, and 2.8 days) was also comparable to the control group (2.7 days). None of the differences were statistically significant.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 7, 20, and 60 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

There were no retained areolae/nipple anlagen in the F1 male pups following parental exposure to the test substance, when evaluated on PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

No test substance related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for F1 pups on PND 21 at any dosage level when the test substance treated groups were compared to the control group. None of the differences from the control group were statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Forty-two (13), 19(6), 45(14), and 111(20) pups (litters) in the control, 7, 20, and 60 mg/kg/day groups, respectively, were found dead or euthanized in extremis from PND 0 through the selection of the F1 generation. Aside from the absence or presence of milk in the stomach, internal findings included the following malformations: Pup No. 9556-01 in the control group had craniorachischisis (cranial vault and spinal column opened through the sacral region), consisting skeletally of absent parietal, frontal, interparietal, and supraoccipital bones; fused, malformed, misshapen, malproportioned arches and/or centra, dorsal aspects of arches reflected more laterally than normal, unco-ossified centra, and extra site of ossification. Pup No. 9605 17 in the 20 mg/kg/day group had a cleft palate (palatine plates not joined, entire length) and fused sternebrae; this pup was also noted with variations of 25 presacral vertebrae and reduced ossification of the 13th ribs. There were no malformations noted in the 60 mg/kg/day group; therefore, the malformations and variations noted in a single pup in the 20 mg/kg/day group were not considered test substance related. Other findings noted in the test substance-treated groups, including hemorrhagic ring around the iris, renal papillae not developed or not fully developed, and/or distended ureters, were noted infrequently, similarly in the control group, or not in a dose related manner. No other internal findings were noted.

PND 4 Culled Pups
No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of culled pups euthanized on PND 4. Findings, including hemorrhagic iris or hemorrhagic ring around iris, renal papillae not fully developed, and accessory spleen were noted infrequently, similarly in the control group, or not in a dose-related manner. No other internal findings were noted.

PND 21 Nonselected Pups and Pups Euthanized Due to Death of Dam

At the scheduled necropsy on PND 21 (nonselected pups), no internal findings were noted in pups at any dosage level.
At the necropsy of pups euthanized due to the death of dam, internal findings, including milk not present in the stomach, renal papillae not developed and distended ureter and distended urinary bladder, were noted for the 5 pups in 2 litters (Nos. 9548 and 9555) in the 60 mg/kg/day group. No other internal findings were noted.

Histopathological findings:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Other effects:

not specified

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Auditory Startle Response (Cohort 2A)
The auditory startle response habituation paradigm was conducted as a longitudinal assessment with selected F1 animals evaluated on PND 23. Administration of 7, 20, and 60 mg/kg/day had no significant effect on auditory startle responsiveness. PEAK and Tpeak values for all trials combined were similar in the F1 males and females among the test substance-treated and the control groups. No statistically significant differences from the control group were noted when analyzed by a repeated measures analysis, and the values were within the Charles River Ashland historical control data ranges (version 2018.02). No effects were noted in the pattern of the habituation response over the entire 50-block test session in adult animals.

Functional Observational Battery (Cohort 2A)
Home Cage Observations
Home cage parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Handling Observations
Handling parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Open Field Observations
Open field parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Sensory Observations
Sensory parameters were unaffected by test substance administration. The only statistically significant difference from the control group was fewer females in the 7 mg/kg/day group that turned slowly and walked away from the tail pinch stimulus. There was no dose-response relationship.

Neuromuscular Observations
Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Physiological Observations
Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated males and females when compared to the control group on PND 65.

Locomotor Activity (Cohort 2A)
Locomotor activity patterns (total activity as well as ambulatory activity counts) in F1 males and females were unaffected by parental test substance administration at all dosage levels when evaluated on PND 65. Values obtained from the 6 epochs evaluated (0–10, 11–20, 21–30,
31–40, 41–50, and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data (version 2018.01). Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the Charles River Ashland historical control data ranges, and/or did not occur in a dose related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance treated groups when the F1 animals were evaluated at PND 65.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

*Data in the F1 boxes are for the F1 pups from birth through weaning, at which point data is in the P2 boxes*

Dose descriptor:

NOAEL

Generation:

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

Clinical signs:

not specified

Description (incidence and severity):

The F2 generation was not bred

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

The F2 generation was not bred

Mortality / viability:

not specified

Description (incidence and severity):

The F2 generation was not bred

Body weight and weight changes:

not specified

Description (incidence and severity):

The F2 generation was not bred

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

The F2 generation was not bred

Food efficiency:

not specified

Description (incidence and severity):

The F2 generation was not bred

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

not specified

Description (incidence and severity):

The F2 generation was not bred

Ophthalmological findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Haematological findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Clinical biochemistry findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Urinalysis findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Sexual maturation:

not specified

Description (incidence and severity):

The F2 generation was not bred

Anogenital distance (AGD):

not specified

Description (incidence and severity):

The F2 generation was not bred

Nipple retention in male pups:

not specified

Description (incidence and severity):

The F2 generation was not bred

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

The F2 generation was not bred

Gross pathological findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Histopathological findings:

not specified

Description (incidence and severity):

The F2 generation was not bred

Other effects:

not specified

Description (incidence and severity):

The F2 generation was not bred

Behaviour (functional findings):

not specified

Description (incidence and severity):

The F2 generation was not bred

Developmental immunotoxicity:

not specified

Description (incidence and severity):

The F2 generation was not bred

Critical effects observed:

Reproductive effects observed:

yes

Lowest effective dose / conc.:

20 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Text Table 37
Results of Homogeneity Analyses

	1 mg/mL	Group 2 (1.4 mg/mL)	Group 4 (12 mg/mL)
Homogeneity Assessment of the 06 Aug 2018 Formulations
Mean Concentration (mg/mL)	0.976	N/A	N/A
RSD (%)	0.76	N/A	N/A
Mean Concentration % of Target	97.6	N/A	N/A
Homogeneity Assessment of the 09 Aug 2018 Formulations
Mean Concentration (mg/mL)	N/A	1.37	12.0
RSD (%)	N/A	1.5	0.39
Mean Concentration % of Target	N/A	97.7	99.8
3-Day Refrigerated Resuspension Homogeneity Assessment of the 06 Aug 2018 Formulations
Mean Concentration (mg/mL)	0.967	N/A	N/A
RSD (%)	0.80	N/A	N/A
Mean Concentration % of Target	96.7	N/A	N/A
8-Day Refrigerated Resuspension Homogeneity Assessment of the 06 Aug 2018 Formulations
Mean Concentration (mg/mL)	0.956	N/A	N/A
RSD (%)	0.56	N/A	N/A
Mean Concentration % of Target	95.6	N/A	N/A
N/A = Not applicable.

Text Table 38
Results of Stability Analyses

	Mean Concentration, mg/mL (% of Time Zero)
	1 mg/mL^a
Minimum 24-Hour Room Temperature Storage	0.983 (101)
3-Day Refrigerated Storage	0.970 (99.4)
^aStability of test substance formulations between concentrations of 10 and 200 mg/mL following at least 10 days of refrigerated storage was previously established.³

Text Table 39
Results of Concentration Analyses

	Mean Concentration, mg/mL (% of Target)
Date of Preparation	Group 2 (1.4 mg/mL)	Group 3 (4 mg/mL)	Group 4 (12 mg/mL)
09 Aug 2018	1.37 (97.6)	4.04 (101)	12.0 (99.7)
15 Aug 2018	1.40 (100)	3.96 (99.0)	11.8 (97.9)
22 Aug 2018	1.42 (101)	3.93 (98.4)	11.6 (96.4)
29 Aug 2018	1.38 (98.3)	4.02 (100)	12.0 (99.8)
26 Sep 2018	1.35 (96.2)	3.67 (91.6)	11.2 (93.5)
31 Oct 2018	1.39 (99.1)	4.06 (101)	11.5 (96.1)
29 Nov 2018 (Batch A)	1.40 (100)	4.27 (107)	12.6 (105)
29 Nov 2018 (Batch B)	1.40 (99.8)	4.24 (106)	12.1 (101)
03 Jan 2019	1.36 (97.2)	3.88 (97.1)	11.7 (97.6)
07 Feb 2019	1.35 (96.4)	3.85 (96.2)	11.7 (97.8)
14 Feb 2019	1.38 (98.8)	3.74 (93.5)	11.3 (94.5)

Text Table 40
Test Substance-Related Unscheduled Deaths (F₀Females)

Female No.	Type of Death/Day	Clinical Observations	Relevant Macroscopic and Microscopic^ Findings
20 mg/kg/day
9532	FD/GD 24	GD 23: decreased activity, pale/cool body GD 24: yellow diarrhea around rectum, delay in skin turgor, unstable to walk, dilated pupils*	Enlarged adrenal glands (adrenal cortical hypertrophy^), suppurative inflammation of lungs and uterus^, myeloid hyperplasia of bone marrow^, 14 dead fetuses in utero cause of death: dystocia, septicemia
9538	FD/GD 21	GD 20: pale body, pale ears and extremities, hunched in corner at rest	11 dead fetuses and 1 early resorption in utero cause of death: dystocia
60 mg/kg/day
9548	EE/LD 0	Postcohabitation Day 24 to LD 0: partially closed eyes Postcohabitation Day 25: hunched posture, piloerection, dermal atonia, yellowish green diarrhea, LD 0: unstable gait* Delivered 13 dead pups and 4 live pups	Dark red areas in stomach (erosion^), small thymus (lymphoid depletion^) cause of moribundity: dystocia^
9550	EE/GD 22	GD 22: decreased activity, severe delay in skin turgor/dehydration, eyes partially closed, yellow diarrhea, pale, cool to touch	Enlarged adrenal glands (adrenal cortical hypertrophy^), pale kidneys (mineralization/cytoplasmic vacuolation^), white areas on the liver (vacuolation^), dark red areas in the stomach, small and pale spleen (red and white pulp atrophy^), small thymus (lymphoid depletion^), 16 live fetuses and 2 early resorptions in utero cause of moribundity: dystocia^
9555	EE/LD 0	LD 0: cool body, eyes partially closed, red vaginal discharge, pale, cool, piloerection*, Delivered 1 dead pup and 2 live pups	Pale kidneys (mineralization/cytoplasmic vacuolation^), dark red areas in the stomach (erosion^), dark red uterine contents (dilation^), lymphoid depletion of thymus^, white pulp atrophy of spleen, 11 dead fetuses and 1 early resorption in utero cause of moribundity: dystocia^
FD= Found dead. EE= Euthanized in extremis. GD= Gestation day. LD= Lactation day. ^= Microscopic findings. * Observations recorded by Veterinary Staff.

Text Table 40 (continued)
Test Substance-Related Unscheduled Deaths (F₀Females)

Female No.	Type of Death/Day	Clinical Observations	Relevant Macroscopic and Microscopic^ Findings
60 mg/kg/day
9603	EE/LD 2	LD 1: piloerection, severe delay in skin turgor*, Delivered 5 dead pups and 12 live pups (that subsequently died or were missing)	Pale and enlarged adrenal glands (adrenal cortical hypertrophy^), pale liver (vacuolation), small thymus (lymphoid depletion^), white pulp atrophy of spleen^, erosion of stomach^ cause of moribundity: dystocia^
9624	EE/GD 24	GD 23 and/or 24: cool/pale body, piloerection, partially closed eyes, yellowish diarrhea, delay in skin turgor, hunched, pale, cool to touch	Enlarged adrenal glands (adrenal cortical hypertrophy^), pale kidneys (cytoplasmic vacuolation^), kidneys- rough surface (mineralization^), dark red contents in stomach, pale spleen (red and white pulp atrophy^), 14 live fetuses and 2 late resorptions in utero cause of moribundity: dystocia^
FD= Found dead. EE= Euthanized in extremis. GD= Gestation day. LD= Lactation day. ^= Microscopic findings. * Observations recorded by Veterinary Staff.

Text Table 41
Results of F₀Reproductive Performance

Parameter	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
Parameter	0	7	20	60	CRL HC^a Mean (Range)
Male Mating Index (%)	96.0	100.0	91.7	100.0	98.0 (83.3–100.0)
Female Mating Index (%)	96.0	100.0	91.7	100.0	98.0 (83.3–100.0)
Male Fertility Index (%)	88.0	87.5	91.7	100.0	93.9 (80.0–100.0)
Female Fertility Index (%)	88.0	84.0	91.7	100.0	93.9 (80.0–100.0)
Male Copulation Index (%)	91.7	87.5	100.0	100.0	95.7 (80.0–100.0)
Female Conception Index (%)	91.7	84.0	100.0	100.0	95.7 (80.0–100.0)
Estrous Cycle Length (days)	4.2	4.3	4.4	4.3	4.2 (3.9–5.2)
Pre-Coital Interval (days)	2.4	3.1	2.5	3.2	2.7 (1.4–4.5)
^a Charles River Ashland historical control data (version 2019.02).

Text Table 42
Adverse Parturition Findings (F₀Females)

Female No.	Type of Death/Day	Clinical and Veterinary Observations
20 mg/kg/day
9532	FD/GD 24	GD 23: decreased activity, pale/cool body; GD 24: yellow diarrhea around rectum, delay in skin turgor, unstable to walk, dilated pupils*
9538	FD/GD 21	GD 20: pale body, pale ears and extremities, hunched in corner at rest
60 mg/kg/day
9548	EE/LD 0	Postcohabitation Day 24 to LD 0: partially closed eyes; Postcohabitation Day 25: hunched posture, piloerection, dermal atonia, yellowish green diarrhea; LD 0: unstable gait*
9550	EE/GD 22	GD 22: decreased activity, severe delay in skin turgor/dehydration, eyes partially closed, yellow diarrhea, pale, cool to touch
9555	EE/LD 0	LD 0: cool body, eyes partially closed, red vaginal discharge, pale, cool, piloerection*
9603	EE/LD 2	LD 1: piloerection, severe delay in skin turgor*
9624	EE/GD 24	GD 23 and/or 24: cool/pale body, piloerection, partially closed eyes, yellowish diarrhea, delay in skin turgor, hunched, pale, cool to touch
FD= Found dead. EE= Euthanized in extremis. GD= Gestation day. LD= Lactation day. * Observations recorded by Veterinary Staff.

Text Table 43
Incidence of Selected Histopathologic Findings, F₀Males and Females

Dosage (mg/kg/day):	Males				Females
Dosage (mg/kg/day):	0	7	20	60	0	7	22	60
Liver^a	25	24	25	29	25	25	22	25
Vacuolation	0	12	16	25	0	0	0	3
Minimal	-	11	15	18	-	-	-	3
Mild	-	1	1	3	-	-	-	0
Moderate	-	0	0	3	-	-	-	0
Marked	-	0	0	1	-	-	-	0
Kidney^a	25	23	25	29	25	25	22	25
Pigment	0	7	12	22	0	0	0	15
Minimal	-	7	12	17	-	-	-	15
Mild	-	0	0	5	-	-	-	0
a - Number of tissues examined from each group.

Text Table 44
F₁Postnatal Survival

Day	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
Day	0	7	20	60	CRL HC^a Mean (Range)
PND 0–1	97.3	98.6	93.9	90.1	98.3 (88.1–100.0)
PND 1–4	91.1	94.3	89.9	79.2	98.3 (87.5–100.0)
PND 4–7	91.3	94.9	93.4	83.1	99.5 (96.7–100.0)
Birth to PND 4	84.2	92.6	85.2	76.0	95.7 (84.5–100.0)
PND 4–21	86.9	94.2	87.5	77.9	99.6 (95.8–100.0)
^a Charles River Ashland historical control data (version 2019.02).

Text Table 45
F₀Total Litter Losses

Female No.	Dosage Level (mg/kg/day)	Day of Litter Loss
9530	0	Lactation Day 0
9533	0	Lactation Day 2
9576	0	Lactation Day 9
9634	20	Lactation Day 2
9536	60	Lactation Day 2
9549	60	Lactation Day 7
9592	60	Lactation Day 9
9603	60	Lactation Day 2
9614	60	Lactation Day 4

Conclusions:

Based on the lack of adverse systemic effects in the F0 males and females at dosages up to 60 mg/kg/day, a dosage level of 60 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic toxicity of 6PPD when administered orally to Crl:CD(SD) rats. Based on the lack of F0 male reproductive effects at any dosage level, 60 mg/kg/day was considered to be the NOAEL for F0 male reproductive toxicity. Based on dystocia in the F0 20 and 60 mg/kg/day groups, 7 mg/kg/day was considered to be the NOAEL for F0 female reproductive toxicity. Based on effects of F1 pup survival and body weight gains at 60 mg/kg/day, a dosage level of 20 mg/kg/day was considered be the NOAEL for F1 neonatal toxicity. Based on the lack of adverse effects on the F1 adult males and females or on neurobehavioral parameters at any dosage level, 60 mg/kg/day was considered to be the NOAEL for F1 adult systemic and neurotoxicity.
Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117,2 a second generation was not assessed on this study.

Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance, N‑1,3-Dimethylbutyl-Nˈ-Phenyl-p-Phenylenediamine (hereafter referred to as 6PPD) on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and testing for effects that may occur as a result of pre- and postnatal chemical exposure.

Animals were dosed via oral gavage daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The offspring selected to become the F₁generation were dosed beginning at weaning through the day prior to euthanasia. Direct offspring exposure during the pre-weaning period was demonstrated on a previous dose range-finding study[i]where quantifiable levels of 6PPD were measured in maternal milk obtained from dams exposed to 6PPD at dosage levels of 50, 75, and 100 mg/kg/day, on Lactation Day 13.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, thyroid hormones, clinical pathology, gross necropsy findings, sperm parameters, organ weights and brain measurements, and histopathologic, neuropathologic and brain morphometric examinations.

F₁animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 for reproductive/developmental toxicity assessments and Cohort 2 for developmental neurotoxicity assessments.

Two and 5 of the F₀females in the 20 and 60 mg/kg/day groups were found dead or euthanized in extremis during the time of parturition or early lactation (Gestation Day 21 through Lactation Day 2) as a result of dystocia and/or prolonged labor; moribundity and mortality of these 2 early death animals were considered test substance-related and adverse. There were 3 other early deaths that were not considered test article related: One F₀male in the 7 mg/kg/day group and 1 female in the 20 mg/kg/day group were found dead and 1 male in the 60 mg/kg/day group was euthanized in extremis. These 3 unscheduled deaths were not considered test substance-related due to the lack of a dose response or an undetermined cause of death (7 mg/kg/day) or an accidental cause of moribundity (swallowing the dosing cannula or esophageal perforation). All other F₀animals survived to the scheduled necropsies.

In addition, 3, 0, 1, and 5 females, respectively, in the control, 7, 20, and 60 mg/kg/day groups were noted with total litter losses between Lactation Days 0 and 9. Total litter losses noted in the 60 mg/kg/day group correlated with lower mean F₁pup postnatal survival and were considered test substance-related and adverse. For surviving F₀animals, there were no significant test substance-related clinical findings at the daily examinations or approximately 3 hours postdosing at any dosage level.

For surviving F₀animals, there were no significant test substance‑related clinical findings at the daily examinations or approximately 3 hours postdosing at any dosage level.

Mean F₀body weights, body weight gains, food consumption, and food efficiency throughout the treatment period, including during gestation and lactation for females, were unaffected by test substance administration at all dosage levels.

F₀male and female mating and fertility, male copulation, and female conception indices, mean estrous cycle lengths, mean precoital intervals, and sperm parameters in the test substance‑‑treated groups were comparable to the control group. Test substance-related dystocia and/or prolonged labor was noted for 2 and 5 females in the 20 and 60 mg/kg/day groups, respectively, although mean gestation lengths for the surviving females in these 2 groups and the 7 mg/kg/day group were between 22.0 to 22.2 days and were comparable to the control group mean (22 days).

No test substance-related effects on mean T₄and TSH levels or on hematology, coagulation, and urinalysis parameters were noted in the F₀males and females at any dosage level. Test substance‑related higher mean albumin, total protein, and globulin concentrations were noted in the 60 mg/kg/day group males and females, and higher mean cholesterol levels were noted in the 20 and 60 mg/kg/day group males and the 60 mg/kg/day group females.

A test substance-related gross observation of pale liver was noted in 1 male each in the 20 and 60 mg/kg/day groups. Administration of 6PPD to F₀males and females resulted in higher liver weights in the 20 and 60 mg/kg/day males and 60 mg/kg/day females; higher kidney weights in the 60 mg/kg/day males and females; and nonadverse microscopic findings of vacuolation in the liver and pigment in the proximal convoluted tubules of the kidney in 7, 20 and 60 mg/kg/day males and 60 mg/kg/day females. The higher kidney and liver weights were considered nonadverse.

Lower mean F₁postnatal survival (76.0% from birth to PND 4) was noted in the 60 mg/kg/day groupbeginning on PND 1 compared to the control group which was considered test substance‑relatedand adverse. The mean number of pups born, live litter size, and percentage of males per litter atbirth in the 7, 20, and 60 mg/kg/day groups and postnatal survival in the 7 and 20 mg/kg/day groups were unaffected by the test substance.

There were no test substance-related effects on F₁pup clinical condition, anogenital distance, or areolae/nipple anlagen noted at any dosage level. Mean male and female pup birth weights (PND 1) were comparable across all groups. Lower mean male and female pup body weight gains in the 60 mg/kg/day during PND 1–21 resulted in mean body weights that were 8.1% and 7.4% lower for males and females, respectively, on PND 21 (not statistically significant) compared to the control group. No test substance-related effects on F₁pup preweaning body weights or body weight gains were noted at 7 and 20 mg/kg/day.

There were no test substance-related effects on mean F₁T₄levels noted at any dosage level on PND 4 or T₄and TSH levels on PND 21.

No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of F₁pups that were found dead, culled pups on PND 4, or at the scheduled necropsy on PND 21. No test substance‑related effects on organ weights were observed for F₁pups on PND 21 at any dosage level.

The mean ages and body weights at attainment of balanopreputial separation and vaginal patency and mean ages at the first occurrence of estrus at 7, 20, and 60 mg/kg/day were unaffected by test substance administration.

There were no test substance-related effects on F₁survival or clinical signs following weaning at any dosage level. Lower mean F₁body weights were noted in the 60 mg/kg/day group males and females through PND 28 due to the lower mean body weight gains noted during the last week of the preweaning period. These findings were considered test substance-related but not adverse because of the transient nature and because these effects were a continuation of the test substance related effects on mean pup body weight gain during the preweaning period.

There were no test substance-related effects on mean F₁estrous cycle lengths or spermatogenic parameters noted at any dosage level.

No test substance-related effects on mean T₄and TSH levels, or on hematology, coagulation and urinalysis parameters were noted in the F₁males and females on PND 91 at any dosage level. Higher mean albumin, total protein, and globulin concentrations were noted in the 60 mg/kg/day group males and females, and higher mean cholesterol level was noted in the 20 and 60 mg/kg/day group males and the 60 mg/kg/day group females. These findings were considered test substance-related but not adverse because there were no other adverse effects on serum chemistry parameters or correlating adverse microscopic findings.

No test substance-related macroscopic findings were noted at any dosage level at the PND 91 necropsy.

Administration of 6PPD to F₁males and females was associated with higher adrenal gland weights in the 60 mg/kg/day males and in the 20 and 60 mg/kg/day females; higher kidney weights in the 60 mg/kg/day males and females; and higher liver weights in the 60 mg/kg/day males and 20 and 60 mg/kg/day females (Cohort 1A, PND 91). There were no 6PPD-related microscopic findings, and the higher liver and kidney weights were considered nonadverse.

Administration of 6PPD to F₁males and females was not associated with any evidence of developmental neurotoxicity. There were no test substance-related effects on F₁startle response, functional observational battery parameters, or motor activity noted at any dosage level. There were no test substance-related macroscopic findings for the brain and spinal cord and no effects on brain weights or measurements at any dosage level. Furthermore, there were no test substance-related microscopic findings in the neurological tissues or effects on brain morphometry.

Based on the lack of adverse systemic effects in the F₀males and females at dosages up to 60 mg/kg/day, a dosage level of 60 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F₀systemic toxicity of 6PPD when administered orally to Crl:CD(SD) rats. Based on the lack of F₀male reproductive effects at any dosage level, 60 mg/kg/day was considered to be the NOAEL for F₀male reproductive toxicity. Based on dystocia in the F₀20 and 60 mg/kg/day groups, 7 mg/kg/day was considered to be the NOAEL for F₀female reproductive toxicity. Based on effects of F₁pup survival and body weight gains at 60 mg/kg/day, a dosage level of 20 mg/kg/day was considered be the NOAEL for F₁neonatal toxicity. Based on the lack of adverse effects on the F₁adult males and females or on neurobehavioral parameters at any dosage level, 60 mg/kg/day was considered to be the NOAEL for F₁adult systemic and neurotoxicity.

Based on the lack of any equivocal effects on the general categories of triggers for the production of a second-generation, as described in the OECD Guidance Document 117, a second generation was not assessed on this study.

[i]

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 7 mg/kg bw/day
Study duration:: subchronic
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

IUCLID won't allow our text to be pasted here for some reason, so see the "additional information" section below.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: GLP study, comparable to guideline study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Route of administration:: oral: gavage
Vehicle:: corn oil
Analytical verification of doses or concentrations:: yes
Details on mating procedure:: positve mating was confirmed by the presence of a copulatory plug
Duration of treatment / exposure:: GD 6 - 15
Frequency of treatment:: once daily
Duration of test:: study termination on gestation day 20
No. of animals per sex per dose:: 25
Control animals:: yes, concurrent vehicle
Details on study design:: Sex: female
Duration of test: sacrifice on GD 20
Dose descriptor:: NOAEL
Effect level:: 50 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Dose descriptor:: LOAEL
Effect level:: 100 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day
Basis for effect level:: other: teratogenicity
Abnormalities:: not specified
Developmental effects observed:: not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Identification: N-1,3-Dimethylbutyl-N′-Phenyl-p-Phenylenediamine (also referred to as 6PPD)
Lot No.: W7F25PN001
Receipt Date: 23 Oct 2017
Expiration Date: None
Physical Description: Dark brown pellets
Purity: 96.9%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light, under nitrogen
Supplier: Eastman Chemical Company

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System
Receipt
On 29 Dec 2017, time-mated female New Zealand White rabbits were received from Covance Research Products, Inc., Denver, PA on Gestation Day 1, 2, or 3. The animals were 6 to 7 months old at receipt and weighed between 2910 and 3869 g on Gestation Day 0.

Justification for Test System and Number of Animals
The New Zealand White rabbit is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the New Zealand White rabbit. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The number of animals selected for this study was based on the United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommend evaluation of approximately 20 females/group with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 females/group at termination.

Animal Identification
Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).

Quarantine
After receipt at the Testing Facility, the New Zealand White rabbits were acclimated prior to the initiation of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.
Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the Study Records.

Husbandry
Housing
Animals were individually housed in stainless steel perforated floor cages suspended above appropriate bedding and equipped with an automatic watering valve.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 61°F to 71°F (16°C to 22°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322 was provided ad libitum throughout the study, except during the acclimation period when food was provided according to Testing Facility SOPs.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment or to aid in maintaining the animals’ oral or gastrointestinal health.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Route of administration:

oral: gavage

Vehicle:

other: methyl cellulose 400cP

Details on exposure:

The vehicle, 1% Methylcellulose (MC) (400 cP) in deionized water, was prepared approximately weekly and stored refrigerated (2°C to 8°C). For administration to Group 1 control animals, an adequate amount of the vehicle was dispensed into daily aliquots, which were stored refrigerated (2°C to 8°C), protected from light, until use. The vehicle was stirred continuously during dosing. Details of the preparation and dispensing of the vehicle have been retained in the Study Records. Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (2°C to 8°C), protected from light, until use. The dosing formulations were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by high performance liquid chromatography using ultraviolet absorbance detection using a validated analytical procedure.

Concentration Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were sent to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were sent to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% at a concentration within the acceptable limits (85% to 115% of the target concentration). After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with 003871142 demonstrated that the test substance is stable in the vehicle when prepared and stored refrigerated (2°C to 8°C) for up to 12 days at concentrations of 1 to 100 mg/mL. Stability data have been retained in the study records for 00387114.2

Details on mating procedure:

Rabbits were received time-mated from the supplier.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose from Gestation Day 7 through 28, inclusively. All animals were dosed at approximately the same time each day.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose from Gestation Day 7 through 28, inclusively. All animals were dosed at approximately the same time each day.

Duration of test:

28 Days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

24, with 28 at the high dose only

Control animals:

yes, concurrent vehicle

Maternal examinations:

Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage and a detailed clinical observation was performed once daily, beginning on the day of receipt and lasting through Gestation Day 29. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded 3 hours postdose.

Body Weights
Animals were weighed individually on Gestation Days 0 (by supplier), 4, and 7–29.
Gravid uterine weight was collected and net body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated for each gravid female at the scheduled laparohysterectomy (see Appendix 1 - Study Protocol and Deviations).

Food Consumption
Food consumption was quantitatively measured on Gestation Days 4–29.

Ovaries and uterine content:

The uterus was weighed, and the ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. The placentae were also examined. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as developmental variations or malformations. Representative photographs of all malformations, as appropriate, were included in the Study Records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the Study Records as needed and as appropriate for comparison, when possible.
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis

External
Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Nonviable fetuses (the degree of autolysis is minimal or absent) were examined, crown-rump length measured, weighed, sexed and tagged individually. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Visceral (Internal)
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads from all fetuses were examined by a mid coronal slice.
All carcasses were eviscerated, skinned, and fixed in 100% ethyl alcohol for subsequent examination of skeletons.

Skeletal
Each eviscerated fetus, following fixation in alcohol, was stained with Alizarin Red S and Alcian Blue The skeletal examination was made following this procedure.

Statistics:

See below as character limits prevent entry here.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related increased incidences of decreased defecation and brown material on the facial area were noted sporadically throughout the study at the daily examinations in the 50 and 100 mg/kg/day groups compared to control group. The decreased defecation correlated with reduced food consumption noted in these group throughout the treatment period (see Section 8.4.). In addition, an increased occurrence of mucoid feces was noted in the 100 mg/kg/day group compared to the control group between Gestation Days 21 and 23 and was considered test substance-related. Other clinical observations noted at the daily examinations in the test substance-treated groups, including rales, soft feces, green staining of the fur on the dorsal trunk, and brown material, scabbing, or hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, were not toxicologically relevant, and/or occurred in a manner that was not dose related.
No test substance related clinical observations were noted at approximately 3 hours following dose administration at any dosage level.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females in the 100 mg/kg/day group aborted during the study.

Female No. 4169 in this group aborted 1 dead fetus on Gestation Day 22 following marked body weight losses (13.4%) and limited food consumption (0–32 g/day) from Gestation Days 7–22. Clinical observations noted during the treatment period at the daily examinations for this female included a thin body during Gestation Days 18 22 and decreased defecation sporadically primarily during Gestation Days 14 22; red material on the cage floor was noted on the day of abortion. At necropsy, Female No. 4169 was internally normal and had 5 dead fetuses and 2 late resorptions with no apparent malformation in utero.

Female No. 4195 in the 100 mg/kg/day group aborted 1 dead fetus on Gestation Day 24 following body weight losses (18.3%) from Gestation Days 7–24 and reduced food consumption (1 20 g/day) from Gestation Day 14–24. Decreased defecation and brown material on various body surfaces were noted sporadically at the daily examinations for this female during Gestation Days 16–24. At necropsy, Female No. 4195 was internally normal and had 4 viable fetuses with no apparent malformations in utero.

Female No. 4249 in the 100 mg/kg/day group aborted 2 dead fetuses on Gestation Day 24 following body weight losses (12.7%) and reduced food consumption (0 47 g/day) from Gestation Days 7–24. Clinical observations noted for this female at the daily examinations included sporadic decreased defecation from Gestation Days 12–23, mucoid feces on Gestation Day 21, and brown material on the facial area on Gestation Day 22. At necropsy, Female No. 4249 was internally normal and had 13 viable fetuses with no apparent malformations in utero. The aforementioned abortions at 100 mg/kg/day were considered test substance-related.
In the 25 mg/kg/day group, Female No. 4225 was found dead on Gestation Day 28. This animal was noted with gasping and brown material around the nose and mouth on the day of death, but there were no effects on body weight gain and food consumption noted for this female prior to death. At necropsy, Female No. 4225 was noted with an accessory spleen, brown matting of the skin, lungs that were not fully collapsed, and dark red areas in the lungs and had 8 dead fetuses with no apparent malformations in utero. In the absence of other signs of toxicity at this dosage level, this mortality was not considered test substance-related. All other females survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight losses and lower mean body weight gains were noted in the 50 and 100 mg/kg/day groups during Gestation Days 7–10, 10–13, and 13–20; differences from the control group were generally significant (p < 0.01). Mean body weight gains in these groups were comparable to or slightly higher than the control group during Gestation Days 20–29. The decrements in body weight gain in the 50 and 100 mg/kg/day groups resulted in significantly (p < 0.01) lower mean body weight gains when the entire treatment period (Gestation Days 7 29) was evaluated. Mean body weights were lower in the 50 mg/kg/day group (5.0% to 5.5%) and 100 mg/kg/day group (5.1% to 10.0%) compared to the control group during Gestation Days 20 27 and 10–29, respectively; differences were generally significant (p < 0.05 or p < 0.01). Significantly (p < 0.05 or p < 0.01) lower mean gravid uterine weight at 100 mg/kg/day and net body weight change at 50 and 100 mg/kg/day were noted compared to the control group. The aforementioned effects on mean body weight, body weight gains, and gravid uterine weight were considered test substance-related and adverse. Mean net body weights at 50 and 100 mg/kg/day and gravid uterine weight at 50 mg/kg/day were comparable to the control group.
Mean body weights, body weight gains, net body weight, net body weight change, and gravid uterine weight in the 25 mg/kg/day group were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 100 mg/kg/day groups was generally lower than the control group beginning on Gestation Day 7, and continuing through Gestation Day 24 or 25. Thereafter, mean food consumption in these groups were comparable to the control group. Mean food consumption in the 50 and 100 mg/kg/day groups was lower than the control group when the cumulative intervals of Gestation Days 7–10, 10–13, 13–20, and 7–29 were evaluated; differences were generally significant (p < 0.05 or p < 0.01). The reductions in mean food consumption noted in the 50 and 100 mg/kg/day groups were generally dose-responsive and corresponded to the reduced mean body weight gains or body weight losses (see Section 8.3.) and were considered test substance related and adverse due to the effect on mean body weights. However, when interpreting the effect of test substance administration on food consumption, individual food consumption of rabbits in the 50 and 100 mg/kg/day dose groups should be taken into account due to the variability noted in food consumption patterns for these females. Several females in the 50 and 100 mg/kg/day groups experienced periods of 3–14 days of markedly reduced food consumption during the implantation period (Gestation Days 7–20). These females tended to have increased food consumption during the fetal period (Gestation Days 21–29; with the exception of the 3 females that aborted on either Gestation Day 22 or 24 at 100 mg/kg/day), although their food consumption levels did not recover completely.
Food consumption in the 25 mg/kg/day group was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food efficiency:

not examined

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

not examined

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, treatment-related

Description (incidence and severity):

Test substance-related higher mean absolute and relative (to net body weight) liver weights were noted in the 50 and 100 mg/kg/day groups; differences from the control group were generally significant (p < 0.01).
No other test substance related effects on organ weights (absolute and relative to net body weight) were observed at any dosage level when the test substance treated groups were compared to the control group. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were observed at dosage levels of 25, 50, and 100 mg/kg/day. However, increased incidences of green discoloration of the fur were noted in the 50 and 100 mg/kg/day groups compared to the control group and was not considered toxicologically relevant. Other macroscopic findings observed in the test substance treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related. With the exception of 1 female each in the control and 100 mg/kg/day group, all females were determined to be gravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Three females in the 100 mg/kg/day group aborted during the study.

Female No. 4169 in this group aborted 1 dead fetus on Gestation Day 22 following marked body weight losses (13.4%) and limited food consumption (0–32 g/day) from Gestation Days 7–22. Clinical observations noted during the treatment period at the daily examinations for this female included a thin body during Gestation Days 18 22 and decreased defecation sporadically primarily during Gestation Days 14 22; red material on the cage floor was noted on the day of abortion. At necropsy, Female No. 4169 was internally normal and had 5 dead fetuses and 2 late resorptions with no apparent malformation in utero.
Female No. 4195 in the 100 mg/kg/day group aborted 1 dead fetus on Gestation Day 24 following body weight losses (18.3%) from Gestation Days 7–24 and reduced food consumption (1 20 g/day) from Gestation Day 14–24. Decreased defecation and brown material on various body surfaces were noted sporadically at the daily examinations for this female during Gestation Days 16–24. At necropsy, Female No. 4195 was internally normal and had 4 viable fetuses with no apparent malformations in utero.

Female No. 4249 in the 100 mg/kg/day group aborted 2 dead fetuses on Gestation Day 24 following body weight losses (12.7%) and reduced food consumption (0 47 g/day) from Gestation Days 7–24. Clinical observations noted for this female at the daily examinations included sporadic decreased defecation from Gestation Days 12–23, mucoid feces on Gestation Day 21, and brown material on the facial area on Gestation Day 22. At necropsy, Female No. 4249 was internally normal and had 13 viable fetuses with no apparent malformations in utero. The aforementioned abortions at 100 mg/kg/day were considered test substance-related.

In the 25 mg/kg/day group, Female No. 4225 was found dead on Gestation Day 28. This animal was noted with gasping and brown material around the nose and mouth on the day of death, but there were no effects on body weight gain and food consumption noted for this female prior to death. At necropsy, Female No. 4225 was noted with an accessory spleen, brown matting of the skin, lungs that were not fully collapsed, and dark red areas in the lungs and had 8 dead fetuses with no apparent malformations in utero. In the absence of other signs of toxicity at this dosage level, this mortality was not considered test substance-related. All other females survived to the scheduled necropsy.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean litter proportion of postimplantation loss in the 100 mg/kg/day group, primarily consisting of early resorptions (11.6 % per litter) was higher when compared to the concurrent control group (3.5% per litter). The difference was not statistically significant when compared to the concurrent control group; however, the value exceeded the maximum mean value in the Charles River Ashland historical control data version 2018.01 (10.15% per litter). A corresponding lower (not statistically significant) mean litter proportion of viable fetuses was noted in this group (88.4% per litter) when compared to the concurrent control group (96.5% per litter) and the minimum mean value in the Charles River Ashland historical control data (89.85% per litter).

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were comparable across all groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Dead fetuses:

effects observed, treatment-related

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean fetal body weights (male, female, and combined) were noted in the 50 (39.3 g, 38.6 g, and 39.0 g, respectively) and 100 (33.9 g, 34.6 g, and 35.0 g, respectively) mg/kg/day groups when compared to the concurrent control group (43.6 g, 41.8 g, and 42.7 g, respectively) and the mean values in the Charles River Ashland historical control data (42.370 g, 41.124 g, and 41.792 g, respectively). Differences from the concurrent control were generally significant (p < 0.05 or p < 0.01). These changes were considered adverse. The effect on mean fetal weights at the 50 and 100 mg/kg/day dosage levels generally corresponded with the lower mean maternal food consumption observed during the period of fetal growth in these groups.
Intrauterine growth at 25 mg/kg/day and survival at 25 and 50 mg/kg/day were unaffected by test substance administration; differences from the control group were slight, not statistically significant, and/or not dose-responsive. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were comparable across all groups.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were noted in 1 fetus each in the control and 25 mg/kg/day groups. Fetus No. 4187-01 in the 25 mg/kg/day group was noted with polydactyly consisting 1 extra digit on a hindpaw. This finding was noted in a single fetus in the low-dose group, and therefore was not considered test substance-related. In the control group, Fetus No. 4257-02 had anophthalmia (unilateral), exencephaly with an open eyelid (unilateral), cheilognathoschisis (involving right naris), and microtia (unilateral; bent).
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were observed in 5, 2, 4, and 1 fetuses in the control, 25, 50, and 100 mg/kg/day groups, respectively. Fetus No. 4219-05 in the 100 mg/kg/day group and Fetus Nos. 4237-01, 4220-02, and 4213-08 in the 50 mg/kg/day group were noted with vertebral anomalies with or without rib anomalies (consisting of fused cervical centrum, extra arches, ribs, centrum; malpositioned arches and centrum; absent centrum; malpositioned centrum; and/or a small centrum or cervical arch). Four, 1, and 2 fetuses in the control, 25, and 50 mg/kg/day groups, respectively, including Fetus No. 4237-01 that was noted with a vertebral anomaly were noted with costal cartilage anomalies consisting of malpositioned, fused, or bifurcated costal cartilage and a malaligned sternebra. In the 25 mg/kg/day group, Fetus No. 4177-01 was noted with a skull anomaly (small interparietal bone). These malformations were noted infrequently and/or did not occur in a dose-related manner, and therefore were not considered to be test substance related. In the control group, Fetus No. 4206 01 was noted with severely malaligned sternebra(e).
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue developmental malformations were noted in 5(4), 8(5), 11(5), and 5(5) fetuses (litters) in the control, 25, 50, and 100 mg/kg/day groups, respectively. Lobular agenesis of the lungs (right accessory lobe absent) was noted in 5(4), 7(4), 10(4), and 4(4) fetuses (litters) in the same respective groups. In the 100 mg/kg/day group, Fetus No. 4172-05 was noted with hydrocephaly (both lateral ventricles). Fetus No. 4213-04 in the 50 mg/kg/day group was noted with tetralogy of Fallot. In the 25 mg/kg/day group, Fetus No. 4187-01 was noted with an interrupted aortic arch (brachiocephalic trunk and left carotid and subclavian arteries arose from the ascending aorta; ductus arteriosus communicated with the descending aorta); this fetus was also noted with an external malformation (see Section 8.8.1.). The visceral malformations noted in the test substance-treated groups were not attributed to the test substance because they occurred in single fetuses or infrequently, were not observed in a dose-related manner, and/or the differences in the mean litter proportions were not statistically significant when compared to the concurrent control group.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Cystic oviducts were observed in Fetus No. 4242-02 in the 25 mg/kg/day group and Fetus Nos. 4174-03 and 4174-09 in the 100 mg/kg/day group. Fetus Nos. 4198-02 and 4230-06 in the 25 and 50 mg/kg/day groups, respectively, had a distended stomach Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for Fetus No. 4251-07 in the 50 mg/kg/day group. Fetus No. 4263-08 in the 100 mg/kg/day group was observed with a cystic liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Text Table 7
Results of Homogeneity Analyses

	Group 2 (5 mg/mL)	Group 4 (20 mg/mL)
Homogeneity Assessment of the 29 Dec 2017 Formulations
Mean Concentration (mg/mL)	5.24	20.5
RSD (%)	2.7	3.1
Mean % of Target	105	103
10-Day Refrigerated Resuspension Homogeneity Assessment of the 29 Dec 2018 Formulations
Mean Concentration (mg/mL)	4.79	19.7
RSD (%)	1.7	1.6
Mean % of Target	95.8	98.5

Text Table 8
Results of Concentration Analyses

	Mean Concentration, mg/mL (% of Target)
Date of Preparation	Group 2 (5 mg/mL)	Group 3 (10 mg/mL)	Group 4 (20 mg/mL)
29 Dec 2017	5.38 (108)	9.71 (97.1)	20.2 (101)
19 Jan 2018	5.50 (110)	10.5 (105)	20.1 (100)

Conclusions:

Abortions at 100 mg/kg/day were noted in the presence of mean body weight losses, lower mean body weight gains, and corresponding reduced food consumption for females at this dosage level. Mean body weight losses, lower mean body weight gains, and lower food consumption were also noted at 50 mg/kg/day. Individual variability in maternal food consumption was noted for females in the 50 and 100 mg/kg/day groups and therefore, the individual data should be taken into account while interpreting summary results. In addition, higher maternal liver weights were noted at 50 and 100 mg/kg/day. Based on these results, a dosage level of 25 mg/kg/day was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity. A higher mean litter proportion of postimplantation loss and corresponding lower mean litter proportion of viable fetuses were noted at 100 mg/kg/day and lower mean fetal weights were noted at 50 and 100 mg/kg/day. Given the relationship between reduced maternal food consumption and increase in post-implantation losses and decrease in fetal weights, and given the severity and time period of the reduced food consumption in individual animals in this study, as well as the postimplantation loss during the implantation period (Gestation Days 7–20), it is not possible to assign the increase in mean litter proportion of postimplantation loss (and lower mean litter proportion of viable fetuses) in the 100 mg/kg/day group to either the test substance or the treatment-related decreases in food consumption.
However, based on the aforementioned developmental effects at 50 and 100 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the NOAEL for embryo/fetal development when 6PPD was administered orally by gavage to time-mated New Zealand White rabbits.

Executive summary:

The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no‑observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. The study was conducted according to OECD Test Guideline 414 and 6PPD was administered to pregnant rabbits at dosage levels of 25, 50, and 100 mg/kg/day (vehicle 1% Methylcellulose in deionized water) from Gestation Day 7 through 28, inclusively.

Animals were dosed via oral gavage once daily during Gestation Days 7–28.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, intrauterine growth and survival, gross necropsy findings, organ weights, and fetal examinations.

Test substance-related abortions were noted in the 100 mg/kg/day group. Three females in this group aborted on Gestation Day 22 or 24 following marked body weight losses (12.7% to 18.3% from Gestation Day 7 to the day of abortion) and reduced food consumption (≤ 47 g/day). Clinical observations noted at the daily examinations for these females included a thin body, decreased defecation, and/or brown material on various body surfaces. One female in the 25 mg/kg/day group was found dead on Gestation Day 28. This animal was noted with gasping and brown material around the nose and mouth on the day of death, but were no effects on body weight gain and food consumption noted for this female prior to death. Notable macroscopic findings for this female at necropsy included lungs that were not fully collapsed and dark red areas in the lungs. In the absence of other signs of toxicity at this dosage level, the mortality at 25 mg/kg/day was not considered test substance-related. All other females survived to the scheduled necropsy.

Many of the test substance-related effects were related to marked to severe decrements in food consumption in individual animals in the 50 and 100 mg/kg/day groups. Test substance-related increased incidences of decreased defecation and brown material on the facial area at 50 and 100 mg/kg/day and mucoid feces at 100 mg/kg/day were noted sporadically at the daily examinations compared to the control group. No test substance‑related clinical observations were noted at approximately 3 hours following dose administration at any dosage level.

Test substance-related mean body weight losses and lower mean body weight gains with corresponding reduced food consumption were noted in the 50 and 100 mg/kg/day groups during Gestation Days 7–10, 10‑13, and 13–20. During Gestation Days 21–29, mean body weight gains in these groups were comparable to or higher than the control group, while mean food consumption generally remained lower until Gestation Day 24 or 25; thereafter mean food consumption was comparable to the control group. However, when interpreting the effect of test substance administration on food consumption, individual food consumption of rabbits in the 50 and 100 mg/kg/day dose groups should be taken into account due to the variability noted in food consumption patterns for these females. Several females in the 50 and 100 mg/kg/day groups experienced periods of 3–14 days of marked to severe reductions in food consumption during the implantation period (period of major organogenesis; Gestation Days 7–20). These females generally had increased food consumption during the fetal period (Gestation Days 21‑29; with the exception of the 3 females that aborted in the 100 mg/kg/day group), although their food consumption levels did not recover completely. The decrements in body weight gains in the 50 and 100 mg/kg/day groups resulted in lower mean body weight gains when the entire treatment period (Gestation Days 7–29) was evaluated. As a result, mean body weights in the 50 and 100 mg/kg/day groups were lower (5.0% to 5.5% and 5.1% to 10.0%, respectively) than the control group during Gestation Days 20–27 and 10–29, respectively. Test substance‑related lower mean gravid uterine weight was noted at 100 mg/kg/day and lower net body weight changes were noted at 50 and 100 mg/kg/day. The mean net body weights in these groups were similar to the control group.

There were no test substance-related internal findings noted for females that survived to the scheduled necropsy on Gestation Day 29. However, increased incidences of green discoloration of the fur were noted at necropsy in the 50 and 100 mg/kg/day groups compared to the control group but was not considered toxicologically relevant.

Test substance-related higher mean absolute and relative (to net body weight) liver weights were noted in the 50 and 100 mg/kg/day groups.

A higher mean litter proportion of postimplantation loss was noted in the 100 mg/kg/day group, resulting in a lower mean litter proportion of viable fetuses. In the 50 and 100 mg/kg/day groups, test substance-related lower (up to 9.9% and 22.2%, respectively) mean fetal body weights were noted compared to the control group. Increased in early resorptions, spontaneous abortions, and decreased fetal weights have been previously demonstrated[i]in rabbits upon feed restriction, and thereby reduced food consumption, during the implantation period; the magnitude of these findings corresponded with the degree of the effects on food consumption. However, in this study it was not possible to assign the increase in mean litter proportion of postimplantation loss (and lower mean litter proportion of viable fetuses) in the 100 mg/kg/day group to either the test substance or the treatment-related decreases in food consumption, due the severity and time period of the reduced food consumption in individual animals and the postimplantation loss during the implantation period (Gestation Days 7–20).

Intrauterine survival at 25 and 50 mg/kg/day, intrauterine growth at 25 mg/kg/day, and fetal morphology (external, visceral, and skeletal) at 25, 50, and 100 mg/kg/day were unaffected by test substance administration.

Abortions at 100 mg/kg/day were noted in the presence of mean body weight losses, lower mean body weight gains, and corresponding reduced food consumption for females at this dosage level. Mean body weight losses, lower mean body weight gains, and lower food consumption were also noted at 50 mg/kg/day. Individual variability in maternal food consumption was noted for females in the 50 and 100 mg/kg/day groups and therefore, the individual data should be taken into account while interpreting summary results. In addition, higher maternal liver weights were noted at 50 and 100 mg/kg/day. Based on these results, a dosage level of 25 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity. A higher mean litter proportion of postimplantation loss and corresponding lower mean litter proportion of viable fetuses were noted at 100 mg/kg/day and lower mean fetal weights were noted at 50 and 100 mg/kg/day. Given the relationship between reduced maternal food consumption and increase in post-implantation losses and decrease in fetal weights, and given the severity and time period of the reduced food consumption in individual animals in this study, as well as the postimplantation loss during the implantation period (Gestation Days 7–20), it is not possible to assign the increase in mean litter proportion of postimplantation loss (and lower mean litter proportion of viable fetuses) in the 100 mg/kg/day group to either the test substance or the treatment-related decreases in food consumption.

However, based on the aforementioned developmental effects at 50 and 100 mg/kg/day, a dosage level of 25 mg/kg/day was considered to be the NOAEL for embryo/fetal development when 6PPD was administered orally by gavage to time-mated New Zealand White rabbits.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 25 mg/kg bw/day
Species:: rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

The potential maternal, embryotoxic and teratogenic effects of 6PPD were evaluated in a teratology study with Sprague-Dawley rats (Monsanto 1987d). 6PPD was administered orally by gavage to three groups of 25 bred Sprague-Dawley female rats. The rats were treated once daily from gestation day 6 through 15. Dose levels of 50, 100 and 250 mg/kg bw/day were selected. For comparative purpose, a concurrent control group composed of 25 bred females was dosed with corn oil, the vehicle control material. Throughout gestation, all females were observed twice daily for appearance and behaviour, and body weights and food consumption were recorded at appropriate intervals. On day 20 of gestation, all females were sacrificed for Cesarean section. Fetuses were weighed, sexed and examined for external, skeletal and soft tissue anomalies and developmental variations. Survival was 100 % in all study groups. The pregnancy rate in this study was 80% in the control group and 92 % in each of the treated groups. Clinical signs which could be attributed to the administration of 6PPD occurred in a dose-related trend in the mid and high dose groups. Salivation prior to dosing, soft stool, diarrhea, decreased defecation and green fecal discoloration were the most remarkable clinical signs. Single observations of soft stool, salivation prior to dosing and clear wet material around the mouth in the 50 mg/kg/day group cannot be directly attributed to test material administration. Maternal body weights and body weight gain were not adversely affected by the administration of 6PPD. Food consumption was slightly reduced in the 250 mg/kg/day group during the first three days of treatment. Food intake among maternal animals in this group was slightly increased for the remainder of gestation with statistical significance occurring from days 16-20 of gestation. No pathological changes occurred in the dams which could be considered treatment-induced in this study. Intrauterine survival as well as the foetal weights was comparable between the control and all 6PPD treated groups. The type and frequency of foetal malformations and developmental variations observed in this study were not indicative of a teratogenic response. One malformation occurred in the control group and two foetuses with malformations were found in the mid dose group. More importantly, no malformations were observed in the 250 mg/kg/day group. Developmental variations occurred with similar frequency between the control and the treated groups.

In conclusion, 6PPD administered to pregnant rats during the period of major organogenesis, was neither teratogenic nor embryo/fetotoxic at dose levels of 250 mg/kg/day and less. An increase in clinical signs occurred at dose levels of 100 and 250 mg/kg/day in a dose-dependent manner. The 50 mg/kg/day dosage was considered a NOAEL for any maternal effect.

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In a rabbit developmental study the objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no‑observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. The study was conducted according to OECD Test Guideline 414 and 6PPD was administered to pregnant rabbits at dosage levels of 25, 50, and 100 mg/kg/day (vehicle 1% Methylcellulose in deionized water) from Gestation Day 7 through 28, inclusively.

Test substance-related higher mean absolute and relative (to net body weight) liver weights were noted in the 50 and 100 mg/kg/day groups.

Abortions at 100 mg/kg/day were noted in the presence of mean body weight losses, lower mean body weight gains, and corresponding reduced food consumption for females at this dosage level. Mean body weight losses, lower mean body weight gains, and lower food consumption were also noted at 50 mg/kg/day. Individual variability in maternal food consumption was noted for females in the 50 and 100 mg/kg/day groups and therefore, the individual data should be taken into account while interpreting summary results. In addition, higher maternal liver weights were noted at 50 and 100 mg/kg/day. Based on these results, a dosage level of 25 mg/kg/day was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity. A higher mean litter proportion of postimplantation loss and corresponding lower mean litter proportion of viable fetuses were noted at 100 mg/kg/day and lower mean fetal weights were noted at 50 and 100 mg/kg/day. Given the relationship between reduced maternal food consumption and increase in post-implantation losses and decrease in fetal weights, and given the severity and time period of the reduced food consumption in individual animals in this study, as well as the postimplantation loss during the implantation period (Gestation Days 7–20), it is not possible to assign the increase in mean litter proportion of postimplantation loss (and lower mean litter proportion of viable fetuses) in the 100 mg/kg/day group to either the test substance or the treatment-related decreases in food consumption.

Justification for classification or non-classification

6PPD is being classified as a category 1B reproductive toxicant based on an OECD 443 extended 1-generation reproductive toxicity study. Classification is justified as in both the main 443 study and in the dose range-finder, dystocia was observed amongst dams at the top 2 doses. A lower dosing regimen was used in the main study, but dystocia was still found.

Additional information

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Registration Dossier

Registration Dossier

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Diss Factsheets

N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Effects on developmental toxicity

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

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