N,N'-di-sec-butyl-p-phenylenediamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There were no effects of fertility based on an OECD 421 study

Link to relevant study records

Reference

Endpoint:

screening for reproductive / 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

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

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 developmental toxicity endpoint (GLP or non-GLP). 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.

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

- Weight of evidence
There are no data on the registered substance itself.

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

- Substance-tailored exposure driven testing
To date, exposure-based waiving of this endpoint has not been accepted by the Agency. It is almost impossible to formulate a sufficiently robust case for exposure based waiving due to the need to demonstrate 'No' exposure

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The arguments presented in this proposal demonstrate that there are inadequate alternative approaches and waving justifications to address the information requirement.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: • EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: N,N’-Di-sec-butyl-p-phenylenediamine
Appearance: Red/Brown oily liquid
Batch: W 8A18GN001
Purity/Composition: See Certificate of Analysis
Test item storage: I n refrigerator (2-8°C) protected from light container flushed with nitrogen
Stable under storage conditions until: 22 January 2019 (expiry date)

Species:

rat

Strain:

other: Wister Han

Details on species / strain selection:

The species/strain was selected as they are considered appropriate for this study design.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Receipt
On 11 Apr 2018, female Crl: WI(Han) rats were received and on 25 Apr 2018, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 11 weeks old and weighed between 262 and 329 g and females were 14 weeks old and weighed between 201 and 245 g.
A health inspection was performed before the initiation of dosing.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
Prior to start of the pretest period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pretest period, reserve females were numbered R1 through R7 at random by indelible marker.
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 29 days prior to start of the pretest period (females) or 15 days before the commencement of dosing (males).

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-54 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 32-54 days.

The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced). Female nos. 41 and 76 (Group 1 and 4, respectively), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on the results of a dose range finder with oral administration of N,N’-Di-sec-butyl-p-phenylenediamine in rats (Test Facility Study No. 20140514), and in an attempt to produce graded responses to the test item.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle was continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20140513).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ~ 10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20140513) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20140513.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-54 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 32-54 days. Pups were not administered the test material and any exposure was through milk.

Frequency of treatment:

Daily for the duration of the treatment period

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder (Test Facility Study No. 20140514).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Litter observations:

Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Live pups were weighed individually on PND 1, 4, 7 and 13.

Sex was externally determined for all pups on PND 1 and 4.

Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

All male pups in each litter were examined for the number of areola/nipples on PND 13.

Postmortem examinations (parental animals):

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

The organs identified for examination were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Tissues were processed at Schaijk. Tissue processing and trimming was performed at the Test Facility. All other histology procedures were performed at Schaijk.
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:

All animals: Gross lesions/masses

All animals of Groups 1-4: Epididymis, thyroid gland, liver, ovaries and testes.

Unscheduled deaths (sacrificed in extremis found dead): Tissues identified in Text Table 7 (except animal identification, mammary gland, parathyroid gland and pituitary gland).


All tissues as defined under Histology – F0-Generation were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
For the testes of all males of Groups 1 and 4, and all males that failed to sire before mating , a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist


Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the animal facility. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Postmortem examinations (offspring):

Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. For details see also section 4.11.1.
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.
In addition, blood was collected from two pups per litter (see also section 4.11.1), and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for blood sampling were the same pups as selected for thyroid preservation.

Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room .
On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure

Statistics:

STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Offspring viability indices:

From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical signs were noted at 10, 30 and 60 mg/kg.
Green discolouration of the urine was noted at dose group 4 (60 mg/kg) in both males and females from Day 5 of treatment onwards. From Day 6 of treatment, green discoloration was also noted in females at dose group 3 (30 mg/kg) and from Day 12 of treatment also in males at dose group 3 (30 mg/kg).
Salivation was seen after dosing among male and female animals of the 30 and 60 mg/kg dose group from Day 2 of treatment onwards, among female animals of the 10 mg/kg dose group from Day 15 of treatment onwards and among male animals of the 10 mg/kg dose group Day 8 to 21 of treatment. This sign was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Salivation was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.
No additional findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male (no. 34) at 60 mg/kg was sacrificed in extremis on Day 21 of treatment. Prior to early sacrifice this animal was noted with hunched posture, labored respiration, rales, diarrhea, a lean posture and piloerection, and had a remarkable body weight loss (about 16% between Day 21 and Day 15). At necropsy soft yellowish nodules on the right side of the epididymides were noted as well as a pale discoloration of the liver. Moderate hepatocellular necrosis of the periportal area was considered to be the main cause of moribundity for this animal. This death was considered to be related to the treatment with the test item as the liver was identified as a target organ for this test item (see 9.2.7 and 9.2.8).
There were three unscheduled deaths during the study that were not considered test item related.
One female (no. 75) at 60 mg/kg died shortly after dosing on Day 32 of treatment (Day 14 of Post Coitum). During the macroscopic examination, the contents of the lungs and trachea were foamy, swollen lungs and a perforation was found in the trachea. These findings indicate that this death was related to the gavage procedure. This animal was pregnant and in the uterus a total of 10 fetuses and one resorption was found.
One control female (no. 45) and one female (no. 66) at 30 mg/kg were found dead in their cages on Day 5 and 35 of treatment, respectively. No clinical signs were present (except for salivation and green discolouration of the urine in female no. 66) and food consumption were considered within normal range on the previous days of treatment. For female no. 45 a body weight loss of 4% was observed from Day 3 to 4 of treatment. For female no. 66 no body weight loss was noted at the last recorded body weight. Female no. 66 was pregnant (Post coitum Day 17) and in the uterus, 13 fetuses and two resorptions were noted. No macroscopic findings were noted in these animals other than autolysis and at microscopic examination no cause of death could be determined. One of these deaths occurred in the control group and was therefore unrelated to the test item. For the death of the Group 4 animal, there were no findings that could relate her death to the treatment with the test item

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg, body weight and body weight gain were reduced from Day 5 (body weight) or Day 4 (body weight gain) of treatment onwards in males, resulting in a 8% lower mean body weight at Day 15 of the mating period compared to the control.
Body weight gain and mean body weights of males treated up to 30 mg/kg and females treated up to 60 mg/kg were considered not to be affected by treatment. Any statistically significant changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (before and after correction for body weight) was slightly reduced in both males and females at 60 mg/kg during the first 6 Days of the treatment period. From Day 6 of treatment onwards, food consumption levels were considered within normal range.
No treatment-related changes in food consumption before or after correction for body weight were observed in males and females treated up to 30 mg/kg.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analyses:
A decrease in total T4 was observed for males at 60 mg/kg (relative difference from controls: 28%) which exceeded the lower limit of the historical control range. Although not statistically significant, total T4 was slightly decreased at 10 and 30 mg/kg, but remained within the historical control range . In females, serum levels of T4 were considered not to be affected by treatment.
An increase in TSH (not dose-dependent) was observed for males at 30 and 60 mg/kg (relative difference from controls: 4.20 and 3.86 x, respectively), but values remained within the historical control range . In females, serum levels of TSH were considered not to be affected by treatment.

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A test item-related macroscopic finding was noted in the liver of females:
Enlarged was recorded in 8/10 females at 60 mg/kg/day and 6/10 females at 30 mg/kg/day, compared to 2/10 and 3/10 in respectively the control and 10 mg/kg/day treated females.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
In males periportal hepatocellular hypertrophy was recorded in 2/10 males at 30 mg/kg/day (minimal) and in all males at 60 mg/kg/day (minimal – moderate).
In one male at 60 mg/kg/day moderate hepatocellular necrosis was recorded.
In females diffuse hepatocellular hypertrophy (minimal-slight) with increased incidence and/or severity of cytoplasmic rarefaction (minimal-slight) was recorded at 60 mg/kg/day. The cytoplasmic rarefaction recorded for females of the remaining dose groups was considered to be within background for non-fasted lactating females.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for female no. 66 at 30 mg/kg (spontaneous death at Day 35 of treatment, Day 17 Post coitum). For two females (no. 45 in the control group and no. 69 at 30 mg/kg) estrous cycle could not be determined. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were 4/10 couples, one in each group, without offspring. For couple 61/21 (30 mg/kg/day) massive tubular atrophy of the testes and massive reduced sperm in the epididymides was the cause of infertility. The incidence of this finding was within the normal range of background pathology encountered in rats of this age and strain and was considered to be unrelated to treatment. For the remaining couples (control couple 48/08; 10 mg/kg/day treated couple 59/19 and 60 mg/kg/ day treated couple 73/33) histopathologic evaluation did not reveal any changes in the reproductive organs that could explain the lack of offspring
In addition three couples were evaluated because of spontaneous death before delivery: one control couple (female 45 died before mating/male 05), one couple at 30 mg/kg/day (pregnant female 66/male 26) and one couple at 60 mg/kg/day (pregnant female 75/male 35). There were no findings in the reproductive organs of these couples suggesting infertility.

Reproduction Data
Estrous Cycle

Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for female no. 66 at 30 mg/kg (spontaneous death at Day 35 of treatment, Day 17 Post coitum). For two females (no. 45 in the control group and no. 69 at 30 mg/kg) estrous cycle could not be determined. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Mating Index

Mating index was not considered to be affected by treatment. Except for one female (no. 61) at 30 mg/kg, all females showed evidence of mating.
The mating indices were 90% for the 30 mg/kg group and 100% for the other groups.
The unsuccessful mating of female no. 61 (Group 3) was due to infertility of the male she was paired with (no. 21 (Group 3); see section 9.2.8 for histopathologic changes in the testes and epididymides of this male). This unsuccessful mating was considered to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Precoital Time

Precoital time was not considered to be affected by treatment. Except for female no. 61 (Group 3), all females showed evidence of mating within four days.

Number of Implantation Sites

Number of implantation sites was not considered to be affected by treatment. All values were within normal limits .

Fertility Index

Fertility index was not considered to be affected by treatment. The fertility indices were 89, 90, 100 and 90% for the control, 10, 30 and 60 mg/kg groups, respectively.
There was one female not pregnant in both the control, 10 mg/kg and 60 mg/kg groups. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Developmental Data

Gestation Index and Duration

Gestation index and duration of gestation were not considered to be affected by treatment.
The gestation indices were 100% for all groups.

Parturition/Maternal Care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

ost-Implantation Survival Index

Post-implantation survival index (the total number of offspring born compared to the total number of uterine implantations) was not considered to be affected by treatment.
Post-implantation survival index was 85, 88, 98 and 89% for the control, 10, 30 and 60 mg/kg groups, respectively. All values remained within the historical control range .

Litter Size

Litter size was not considered affected by treatment.
Live litter sizes were 9.9; 12.3; 11.9 and 11.8 living pups/litter for the control, 10, 30 and 60 mg/kg groups, respectively. For controls, live litter size was somewhat low compared to historical control data, live litter sizes for the other groups were within the normal range of the historical control data .

Live Birth Index

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment.
The live birth indices were 100, 98, 100 and 100% for the control, 10, 30 and 60 mg/kg groups, respectively.
Two pups at 10 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
 
Viability Index

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 99, 100 and 100% for the control, 10, 30 and 60 mg/kg groups, respectively.
One pup at 10 mg/kg was found missing on PND 2, which was most likely cannibalised. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Lactation Index

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
The lactation indices were 100, 99, 100 and 100% for the control, 10, 30 and 60 mg/kg groups, respectively. One pup at 10 mg/kg (litter no. 54) was overlooked at culling on PND 4 and was therefore culled at PND 9.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other: No feproductive findings

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

A P1 mating was not conducted

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

A P1 mating was not conducted

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For the two pups of female no. 54 (10 mg/kg) who were found dead at first litter check, absence of milk in the stomach was noted on Day 1.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 99, 100 and 100% for the control, 10, 30 and 60 mg/kg groups, respectively.
One pup at 10 mg/kg was found missing on PND 2, which was most likely cannibalised. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of male and female pups were reduced for dose Groups 2 and 4 from postnatal Day 1 onwards. For dose Group 3, body weights were reduced on postnatal Day 1 and 4. These statistically significant changes were considered to be unrelated to treatment as they occurred in the absence of a dose-response relationship and remained within the historical control range, and were considered to have arisen as a result of slightly high control values when compared to historical controls

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
Mean serum T4 in male PND 14-16 pups at 30 mg/kg was statistically significantly lower than that in control pups (20%). This change was considered to be unrelated to treatment as it occurred in the absence of a dose-response relationship and values remain within the range of the historical control data .
Additional measurements of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 60 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

other: No findings

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

There was no F2 generation

Key result

Reproductive effects observed:

no

Mean Percent Liver Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg/day):	10		30		60	10	30	60
LIVER
Absolute	0		16**		25**	9	19	29**
Relative to body weight	5		24**		37**	7	15*	27**
*: P<0.05, **: P<0.01

Summary Test Item-Related Microscopic Liver Findings (including premature decendents)

	Males				Females
Dose level (mg/kg/day):	0	10	30	60	0	10	30	60
LIVER^a	10	10	10	10	10	10	10	10
Hepatocelular hypertrophy,                                     periportal
Minimal	-	-	2	5	-	-	-	-
Slight	-	-	-	4	-	-	-	-
Moderate	-	-	-	1	-	-	-	-
Hepatocelular hypertrophy,                                     diffuse
Minimal	-	-	-	-	-	-	-	3
Slight	-	-	-	-	-	-	-	7
Necrosis, hepatocelular
Moderate	-	-	-	1	-	-	-	-
Cytoplasmic rarefaction
Minimal	-	-	-	-	5	4	4	1
Slight	-	-	-	-	-	1	3	9

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of N,N’-Di-sec-butyl-p-phenylenediamine were established:
Parental NOAEL: 30 mg/kg/day (based on mortality observed at 60 mg/kg/day).

Note: In this study, a marked reduction of total T4 was observed at 60 mg/kg/day (in males) which was considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.
Reproduction NOAEL: 60 mg/kg/day
Developmental NOAEL: 60 mg/kg/day

Executive summary:

The objectives of this study were to determine thepotential toxic effects of N,N’-Di-sec-butyl-p-phenylenediamine when given orally by gavage to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Males were treated for 29 days and females that delivered were treated for 50-54 days. Females which failed to deliver were treated for 32-54 days.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 10, 30, 60 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20140514).

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity.

The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle determination,measurement of thyroid hormone T4 (F₀-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Formulation analysis confirmed that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously. For the formulation of Group 4, the mean accuracy was slightly below the target concentration (i.e. 89% of target). As the deviation from the target concentration was very small, accuracy of Group 4 formulations was considered acceptable. 

Parental results

Parental toxicity was observed at 60 mg/kg.

At 60 mg/kg bw/day there was one treatment-related death: One male had to be sacrificed in extremis on Day 21 of treatment. This animal was noted with hunched posture, labored respiration, rales, diarrhea, a lean posture and piloerection, and had a remarkable body weight loss (about 16% over one week). Moderate hepatocellular necrosis of the periportal area was considered to be the main cause of moribundity for this animal and as the liver is identified asa[GS1] target organ for the test item, this death was considered to be related to the treatment with the test item.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No developmental effects were found in an OECD 414 prenatal developmental toxicity study

Link to relevant study records

Reference

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

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

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 developmental toxicity endpoint (GLP or non-GLP). 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.

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

- Weight of evidence
There are no data on the registered substance itself.

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

- Substance-tailored exposure driven testing
To date, exposure-based waiving of this endpoint has not been accepted by the Agency. It is almost impossible to formulate a sufficiently robust case for exposure based waiving due to the need to demonstrate 'No' exposure

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The arguments presented in this proposal demonstrate that there are inadequate alternative approaches and waving justifications to address the information requirement.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: N,N’-Di-sec-butyl-p-phenylenediamine
Appearance: Red/Brown oily liquid
Batch: W8A18GN001
Purity/Composition: See certificate of analysis (see Appendix 4)
Test item storage: In refrigerator (2-8°C) protected from light container flushed with nitrogen
Stable under storage conditions until: 22 January 2019 (expiry date)

Species:

rat

Strain:

other: Wister Han

Details on test animals or test system and environmental conditions:

On 05 Jun 2018 (first 11 animals: Subgroups 1 and 2) and 22 Jun 2018 (second 11 animals: Subgroups 3 and 4), time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). Animals weighed between 184 and 256 g at the initiation of dosing. A health inspection was performed upon receipt of the animals.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified by indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
One day after receipt (Subgroups 1 and 2) or on the day of receipt (Subgroups 3 and 4), animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of the mean for each set of animals.

Housing
On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The daily average temperature during the study period was between 20 to 21°C with an actual daily mean relative humidity of 40 to 77% (see deviations in Appendix 7). 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
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) 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 were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 24 hours after adding the vehicle to the test item. Details of the preparation and dispensing of the test item have been retained in the Study Records. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Administration of Test Materials
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20140513 ).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory . Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ~ 10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20140513) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20140513.

Details on mating procedure:

Time-mated females were used on the study, so this does not apply.

Duration of treatment / exposure:

Gestation day 6-20

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

Dose / conc.:

0 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were treated with the test article from gestation days 6-20, at which time a caesarian section was performed. Dams were given a gross necropsy, and the fetuses were given a full gross examination, fresh visceral examination and skeletal exam for variations and malformations

Maternal examinations:

Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F0-Generation
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. For animals from Subgroup 3, clinical observations started on Day 3 post-coitum

The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect premature birth.

Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. For animal no. 88, the first body weight value was recorded on Day 3 post-coitum. In order to monitor the health status, animal no. 88 was also weighed on Day 14 post-coitum.

Food Consumption – F0-Generation Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

Water Consumption – F0-Generation Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Ovaries and uterine content:

All animals (including females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals that delivered early).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Fetal examinations:

Fetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. A visceral and skeletal examination was also performed for the nonviable fetuses (see deviations in Appendix 7).

Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) 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 ref 1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar ref 2.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique ref 3. After examination, the tissues without variation or malformations were discarded .
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson ref 4.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads ).
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

on-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical signs were observed in all treated groups:
Persistent green discoloration of the urine was observed among all treatment groups starting at 10 mg/kg. This clinical sign was noted in all females at 30 and 60 mg/kg, after 1-2 days of treatment (from Day 7-8 post-coitum onwards) and in 15 out of 22 animals at 10 mg/kg, on average starting during the second week of treatment (for animals nos. 23, 26, 27, 28, 29, 30, 32, 33, 40, 41, 43 and 44 starting on Days 13-19 post-coitum and for animals nos. 24, 31 and 42 starting on Days 7-11 post-coitum).

Piloerection was observed for several consecutive days in 4 out of 22 animals at 60 mg/kg (nos. 80, 81, 82 and 88), with two of them (no. 80 and 88) presenting with hunched posture for 2 and 10 consecutive days, respectively. Animal no. 88 was also noted with reflux on one single day (Day 13 post-coitum), flat gait for 8 consecutive days, slight and transient laboured respiration and rales for 1-3 days together with slight pale ears and ptosis for 1-2 days.

Salivation noted in one control, 2 animals at 10 mg/kg and among 11 and 10 animals at 30 and 60 mg/kg, respectively was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Other clinical signs observed during the treatment period included slight rales in one mid-dose animal on one single day and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and were not considered to be toxicologically relevant.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg, body weight gain was statistically significantly lower than control mean on Day 9 post-coitum. This decrease in body weight gain was mainly attributed to 6 high-dose females (nos. 68, 72, 78, 80, 83 and 88) that showed a body weight loss of 1-9% or absent body weight gain over Days 6-9 post-coitum, followed by a recovery afterwards (except for female no. 80 that was noted with low body weight gain during the entire study period as presented with only early resorptions). From Day 12 post-coitum until the end of the treatment period, mean body weight gain of animals at 60 mg/kg returned back to normal values. Mean body weights at 60 mg/kg remained in the same range as controls over the study period.
Body weight and body weight gain at 10 and 30 mg/kg was considered to be unaffected by treatment.
No toxicologically relevant changes in body weight gain corrected for gravid uterus were noted by treatment up to 60 mg/kg. The statistically significant increase in body weight corrected for gravid uterus at 30 mg/kg was considered to be unrelated to treatment since no trend was apparent regarding dose and since mean values were within the historical control range .

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg, body weight gain was statistically significantly lower than control mean on Day 9 post-coitum. This decrease in body weight gain was mainly attributed to 6 high-dose females (nos. 68, 72, 78, 80, 83 and 88) that showed a body weight loss of 1-9% or absent body weight gain over Days 6-9 post-coitum, followed by a recovery afterwards (except for female no. 80 that was noted with low body weight gain during the entire study period as presented with only early resorptions). From Day 12 post-coitum until the end of the treatment period, mean body weight gain of animals at 60 mg/kg returned back to normal values. Mean body weights at 60 mg/kg remained in the same range as controls over the study period.

Body weight and body weight gain at 10 and 30 mg/kg was considered to be unaffected by treatment.

No toxicologically relevant changes in body weight gain corrected for gravid uterus were noted by treatment up to 60 mg/kg. The statistically significant increase in body weight corrected for gravid uterus at 30 mg/kg was considered to be unrelated to treatment since no trend was apparent regarding dose and since mean values were within the historical control range .

Food efficiency:

not examined

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

not specified

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):

A test item-related increase in mean liver weight and liver/body weight ratio was observed at a dose-related severity . Relative mean liver weights were around 23% and 37% higher than concurrent control mean at 30 and 60 mg/kg, respectively. Higher liver weights were also observed in a previously performed Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Han rats with N,N’-Di-sec-butyl-p-phenylenediamine (Test Facility Study no. 20140515) in which non-adverse test item-related microscopic alterations were recorded in the liver of females treated at 60 mg/kg for 28 days, consisting of hepatocellular hypertrophy and/or increased cytoplasmic rarefaction, with correlating increased relative liver weight (27%).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment up to 60 mg/kg .

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3 vs 11.4). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. The higher number of early resorptions observed in these two high-dose females could be regarded as secondary to maternal toxicity (body weight loss over Days 6-9 post-coitum) and as such not considered a direct effect of the test item on fetal development

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3 vs 11.4). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. The higher number of early resorptions observed in these two high-dose females could be regarded as secondary to maternal toxicity (body weight loss over Days 6-9 post-coitum) and as such not considered a direct effect of the test item on fetal development

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal body weights were higher in both sexes at 30 and 60 mg/kg when compared to concurrent controls. Mean combined weights were 8% and 6% significantly higher than control mean, respectively. Mean values were also higher than the 95th percentile of the historical control data (combined weight P95=5.4 gram).
No statistically significant changes in fetal body weight (both sexes) were noted at 10 mg/kg.
Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.6 and 5.5 gram for the control, 10, 30 and 60 mg/kg groups, respectively.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to controls (9.3 vs 11.4). This was considered to be related to a higher incidence of early resorptions (caused by litter A078 and A080; each consisting of 9 and 12 early resorptions). The higher number of early resorptions observed in these two high-dose females could be related to their body weight loss over Days 6-9 post-coitum and, therefore, was regarded as secondary to maternal toxicity and as such not considered a direct effect of the test item on fetal development.
Mean litter sizes were 11.4, 10.2, 10.6 and 9.3 fetuses/litter for the control, 10, 30 and 60 mg/kg groups, respectively.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to controls (9.3 vs 11.4). This was considered to be related to a higher incidence of early resorptions (caused by litter A078 and A080; each consisting of 9 and 12 early resorptions). The higher number of early resorptions observed in these two high-dose females could be related to their body weight loss over Days 6-9 post-coitum and, therefore, was regarded as secondary to maternal toxicity and as such not considered a direct effect of the test item on fetal development.
Mean litter sizes were 11.4, 10.2, 10.6 and 9.3 fetuses/litter for the control, 10, 30 and 60 mg/kg groups, respectively.


Mean fetal body weights were higher in both sexes at 30 and 60 mg/kg when compared to concurrent controls. Mean combined weights were 8% and 6% significantly higher than control mean, respectively. Mean values were also higher than the 95th percentile of the historical control data (combined weight P95=5.4 gram).
No statistically significant changes in fetal body weight (both sexes) were noted at 10 mg/kg.
Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.6 and 5.5 gram for the control, 10, 30 and 60 mg/kg groups, respectively.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment up to 60 mg/kg.
Two externally malformed fetuses were observed in this study: snout proboscis was noted in one control fetus (A002-03) and one fetus at 10 mg/kg (A037-03) was noted with a small lower jaw. Both findings were skeletally confirmed. Besides, during evisceration of this low-dose fetus (A037-03) prior to skeletal staining, the visceral malformation absent eye was also noted. At the incidence observed (occurrence in a single fetus), presence only in the control group and absence of a dose-response relationship, these malformations were considered to be of spontaneous origin and, therefore, unrelated to treatment with the test item.
No external variations were noted in any of the groups

Skeletal malformations:

no effects observed

Description (incidence and severity):

No skeletal malformations were noted by treatment up to 60 mg/kg.
With regard to the observed skeletal variations, there was a treatment-related decrease in bent ribs at 30 and 60 mg/kg. Incidences were 22.5%, 21.7%, 11.4% and 5.9% in the control, 10, 30 and 60 mg/kg groups, respectively (reaching statistical significance in the high-dose group). The decreased incidence of bent ribs could be attributed to the higher mean fetal body weight observed in the mid- and high-dose groups. This finding was not considered to be toxicologically relevant as incidences were within the historical control range (mean=14.0%, P5-P95=1.8-26.1%) and since a lower incidence for bent ribs is not considered to have any detrimental effect.
Any other skeletal variations occurred in the absence of a dose-related trend, low occurrence (i.e. in one single fetus) and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered to be treatment related.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 60 mg.
Two viscerally malformed fetuses were observed in the current study: one control fetus was noted with a small eye (A012-08) and one fetus at 10 mg/kg (A037-03) presented with an absent eye. Both findings were skeletally confirmed and were considered to have occurred by chance, at the incidence observed and in the absence of a dose-response relationship.
Only one visceral variation (small supernumerary liver lobes) was observed in the control and all treated groups. At the low incidence observed (well within the historical control range) and in the absence of a dose-response relationship, this finding was not considered to be related to treatment. Other observed visceral variations (discoloured liver and convoluted ureter) were only present in the control group and, therefore, considered not to be test item-related.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Early resorptions

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg .
The developmental NOAEL for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg.

Executive summary:

The objectives of this study were to determine the potential of N,N’-Di-sec-butyl-p-phenylenediamine to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 10, 30, 60 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20140512)

 

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F₀-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights (liver), number of corpora lutea, (gravid) uterine weight and uterine contents.

In addition, the following parameters were determined for the F₁-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

No mortality occurred during the study period.

Test item-related clinical signs were observed in all treated groups:

Persistent green discoloration of the urine was observed among all treatment groups starting at 10 mg/kg. This clinical sign was noted in all treated animals at 30 and 60 mg/kg, after 1-2 days of treatment (from Day 7-8 post-coitum onwards) and in 15 out of 22 animals at 10 mg/kg, on average starting during the second week of treatment. Although no clear cause could be attributed to this finding, it was considered not to be toxicologically relevant since clear signs of systemic toxicity would have been observed in case of renal toxicity among animals presenting with this abnormal colour in urine (no relevant clinical signs were observed in any of the Group 2 and 3 animals and in 18 out of 22 animals in Group 4 presenting with green urine).

At 60 mg/kg, 4 out of 22 animals were noted with hunched posture and/or piloerection for several consecutive days, with one of them additionally presenting with reflux on one single day (Day 13 post-coitum), flat gait for 8 consecutive days, slight and transient laboured respiration and rales for 1-3 days together with slight pale ears and ptosis for 1-2 days.

At 60 mg/kg, mean body weight gain and food consumption were statistically significantly lower than control mean during the first days of treatment (over Days 6-9 post-coitum), followed by a recovery afterwards. As complete recovery was noted, the transient decrease in mean body weight gain and food consumption was not considered adverse. From Day 12-15 post-coitum onwards a significant increase in food consumption was observed at 60 mg/kg (15-16%vscontrol), that might have occurred to compensate for the initial decrease in food intake observed during the first days of the treatment period.

No toxicologically relevant changes in mean body weight, body weight gain and food consumption were observed at 10 and 30 mg/kg over the entire study period. The slight increase in food consumption (<10%vscontrol) observed at 30 mg/kg over Days 12-21 post-coitum was considered non-adverse.

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment up to, and including, 60 mg/kg.

A test item-related increase inmean liver weight and liver/body weight ratiowas observed in a dose-response manner. Relative mean liver weights were 23% and 37% higher than concurrent control mean at 30 and 60 mg/kg, respectively. In a previously performed Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Han rats with N,N’-Di-sec-butyl-p-phenylenediamine (Test Facility Study no. 20140515) a test item-related increase in mean relative liver weight was also observed in females treated at 30 and 60 mg/kg for at least 28 days (15% and 27%vscontrol, respectively), together with incidental/background hepatic microscopic findings at 30 mg/kg and non-adverse test item-related microscopic alterations at 60 mg/kg (hepatocellular hypertrophy with increased incidence and/or severity of cytoplasmic rarefaction). However, thepossible adversity of thedose-dependent moderate increase in liver weightobserved in the current study was not assessed histopathologically and was, therefore, not taken into account when determining the maternal NOAEL.

The number of pregnant females, corpora lutea, implantation sites, and pre-implantation loss in the control and treatment groups was in the range of normal biological variation.

There were 2 females, one each at 30 and 60 mg/kg that had an early delivery on the day of scheduled necropsy (1 viable pup + 11 viable fetuses and 4 viable pups + 10 dead fetuses, respectively). Although this number of fetal deaths was noticed in a female that delivered early on the day of schedule necropsy (Day 21 post-coitum), a relationship to treatment could not be completely discarded as it occurred in the high dose group.

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3vs11.4). The % per litter of viable fetuses in this high dose group was also lower when compared with concurrent control mean (89.1%vs97.5%) and the 5^thpercentile of the historical control data (90.8%). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. While maternal toxicity may have played a role in the increase in early resorptions in the two affected dams in the 60 mg/kg group, it was not possible to assign a cause and effect relationship nor was it possible to rule out a direct effect of the test item on the survival of the embryo.

Mean combined fetal body weights were 8% and 6% highervscontrol mean at 30 and 60 mg/kg, respectively, and values were outside the historical control range. The increase in mean fetal body weights in both sexes could partly be explained by the slightly higher mean food consumption (absolute and relative) noted in females at 30 and 60 mg/kg from Day 12-15 post-coitum onwards. This observation in fetal body weight was considered not to be of toxicologically relevance due to the slight magnitude of the change (less than 10%), absence of test item-related malformations or detrimental developmental variations and as the opposite effect (i.e. a decrease) would be expected in case of intrauterine/fetal growth retardation.

No statistically significant changes in fetal body weights (both sexes) were noted at 10 mg/kg.

The treatment-related decrease in the skeletal variation bent ribs observed at 30 and 60 mg/kg could be related by the higherfetal body weightobserved in both groups. This variation was considered not to be toxicologically relevant as incidences were within the historical control range and since a lower incidence for bent ribs is not considered to have any detrimental effect.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. sex ratio, external and visceral malformations and developmental variations, and skeletal malformations).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg (based on clinical signs at 60 mg/kg).

The developmental NOAEL for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg (due to the higher incidence of early resorptions in 2 females at 60 mg/kg).

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification for reproductive/developmental effects is required as the only effects seen were a minor chnage in the number of early resorptions in 2/25 high dose dams.