2,5-bis-isocyanatomethyl-bicyclo[2.2.1]heptane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03 February 2021 - 30 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The objective of this study was to select dose levels for the extended one generation reproductive toxicity study in Wistar Han rats (Test Facility Study No. 20263211).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Principles of method if other than guideline:

The potential toxic effects of NBDI when given orally by gavage for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated.
The dose levels in this study were selected to be 200 and 500 mg/kg/day, based on information by the Sponsor (28-day repeated dose toxicity study with oral administration of NBDI in rats, reference ECHA dossier). From Day 7 onwards the high dose level was lowered to 400 mg/kg/day. Ten animals per sex per dose were exposed to the test substance.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane

Cas Number:

74091-64-8

IUPAC Name:

Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane

Test material form:

liquid

Specific details on test material used for the study:

Physical description: Clear colourless liquid.
Storage conditions: In refrigerator (2-8°C) protected from light container flushed with nitrogen.
Composition correction factor: No correction factor required.
Test item handling: Handle in glove box or AtmosBag (nitrogen environment).
Chemical name: Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane
CAS number: 74091-64-8
Specific gravity/density: 1.14 at 20°C

Test animals

Species:

rat

Strain:

other: Crl:WI(Han) Outbred (SPF)

Details on species / strain selection:

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 to be used in this study is 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: (P) 10-11 weeks (males); 13-14 weeks (females).
- Weight at study initiation: (P) Males: 261-309 g; Females: 205-232 g.
- Fasting period before study: Not reported.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in
polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages. During the lactation phase, females were housed in Makrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding and were equipped with water bottles. Group-housed animals were separated during designated procedures/activities.
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum, municipal tap water.
- Contaminants: It is considered that there are no known contaminants in the feed and water that would interfere with the objectives of the study.
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C (actual temperature; target: 18-24).
- Humidity (%): 29 to 52% (actual humidity; target 40-70).
- Air changes (per hr): at least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark.

IN-LIFE DATES: From: 03 February 2021 To: 30 April 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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 twice weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations 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.

VEHICLE
- Justification for use and choice of vehicle: Commonly used vehicle.
- Concentration in vehicle: Not reported.
- Amount of vehicle (if gavage): 4 mL/kg (total, including test substance).
- Specific gravity: 0.92.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 consecutive days.
- Proof of pregnancy: sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. This was not necessary in this study.
- Further matings after two unsuccessful attempts: no.
- After successful mating each pregnant female was caged: once mating has occurred the males and females will be separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis as indicated below.

Dose Formulation Sample Collection Schedule:
- Week 1 of treatment: 26 Feb 2021
- Concentration: All groups: 2 x approximately 500 mg
- Homogeneity: All groups: 2 x approximately 500 mg

All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.

Duration of treatment / exposure:

Males were treated for a minimum of 28 days, up to and including the day before scheduled necropsy. This included a minimum of two weeks prior to mating and during the mating period.
Females were treated for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females were not dosed during littering.

The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 will assume the day of the animal being replaced).

Pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

Animals were dosed at 500 mg/kg/day for Days 1-5. On Day 6 animals were not dosed, and from Day 7 onwards animals were treated at a dose level of 400 mg/kg/day.

No. of animals per sex per dose:

10 per sex per dose

Control animals:

no

Details on study design:

- Dose selection rationale: The dose levels were selected based on information provided by the Sponsor (28-day repeated dose toxicity study with oral administration of NBDI in rats, reference ECHA dossier), and in an attempt to produce graded responses to the test item. In the 28-day study
decreased body weight gain up to 13% was recorded in males at 500 mg/kg/day. In females at 500 mg/kg/day increased absolute and relative liver weight (12 and 17%) and decreased absolute and relative ovary weight (18 and 14%) were recorded. In a OECD422 study with XDI (read-across) body weight was significantly lower in males at 200 mg/kg/day throughout the administration period and in females at the same dose level on Day 15 of administration, throughout pregnancy, and on Days 0 and 6 of lactation.
- Animal assignment: Random

Terminal procedures F0:
- Males (which sire or fail to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Females which deliver: PND 14-16.
- Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27. Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
- Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

Examinations

Parental animals: Observations and examinations:

MORTALITY CHECKS: Yes
- Time schedule: At least twice daily throughout the study.
- Procedure: Animals will be observed for general health/mortality and moribundity. Animals will not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals will be observed at least once daily, up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females will be weighed on the first day of treatment (prior to dosing) and weekly thereafter. Mated females will be weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. In order to monitor the health status animals may be weighed more often.

FOOD CONSUMPTION
- Food consumption: Yes
- Time schedule: Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study.

Oestrous cyclicity (parental animals):

Daily vaginal lavage will be performed beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal lavage will continue for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage will also be taken to determine the stage of estrus. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously.

Estrous cycles will be evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures.

Sperm parameters (parental animals):

Not reported.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after a minimum of 28 days of administration.
- Maternal animals: All surviving animals, PND 14-16.
- All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted overnight.
- Method of euthanasia: deep anesthesia using isoflurane and followed by exsanguination.

GROSS NECROPSY
- All animals were subjected to a full postmortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites was recorded for all paired females.
In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

ORGAN WEIGHTS
The Epididymis and Testes were weighed.

Tissue collection and preservation:
- The following organs were collected and preserved: Cervix, epididymis, coagulation gland, mammary gland, parathyroid gland, pituitary gland, prostate, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.

Postmortem examinations (offspring):

SACRIFICE
- Method of euthanasia: Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-16) were euthanized by an intraperitoneal injection of sodium pentobarbital.
- Unscheduled deaths: Recognizable fetuses of females that died spontaneously or were euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation.
- Scheduled euthanasia: On PND 4, the surplus pups were euthanized by decapitation. All remaining pups were euthanized on PND 14-16.

GROSS NECROPSY
- 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.

Statistics:

No statistical analysis was performed.

Reproductive indices:

Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Group 1 (200 mg/kg/day): Incidental rales and red staining of the snout have been recorded in males. Hypersensitivity to touch was recorded in two males on Days 18-19 and/or Days 41-42 of treatment. In all males hunched posture and piloerection were recorded starting in Week 4. Piloerection, hunched posture and rales were also recorded before dosing. Incidental rales and hunched posture were recorded in females. On Day 19 of treatment two females showed hypersensitivity to touch. Piloerection was recorded in all females starting during the mating period and was also recorded before dosing. At 200 mg/kg/day one male and two females were sacrificed in extremis on respectively Day 6 of treatment, Day 14 post-coitum and Day 8 of lactation. The male animal showed rales, gasping and lethargy and non-collapsed lungs were recorded at necropsy. This mortality was possibly related to the oral gavage procedure, although a relationship to the test item (e.g. irritation) could not be excluded.
The two females showed rales, gasping, piloerection, red discharge from the snout, pale appearance and/or lethargy. In one female severe body weight loss was noted. An accentuated lobular pattern of the liver was recorded in one female; no macroscopic findings were noted in the second female. For these two mortalities, a relation to treatment with the test item could not be excluded.

Group 2 (500/ 400 mg/kg/day): Lethargy, laboured respiration and red discharge from the nose have been incidentally recorded in males. On Day 35 of treatment one male showed hypersensitivity to touch. Diarrhoea was recorded in males from Days 9-14. Hunched posture and/or piloerection was recorded in all males starting on Day 3. Rales were recorded in 8/10 males on one or multiple treatment days, starting in Week 2 of treatment. Hunched posture, rales, piloerection and hypersensitivity to touch were also recorded before dosing. In females, slow breathing or gasping and red discharge from the snout were incidentally recorded. Rales were recorded in 7/10 females on one or multiple treatment days, starting in Week 1. On Days 35 and 37-40 of treatment one female showed hypersensitivity to touch. Diarrhoea was recorded in females on Days 9, 10 and 13. Piloerection and/or hunched posture were recorded in all females starting on Day 3.
In Group 2 two males and one female were sacrificed in extremis during the first 5 days of treatment (at 500 mg/kg/day) showing rales, gasping, laboured respiration, piloerection, diarrhoea, red discharge from the snout and/or ptosis. Severe body weight loss was recorded in the two males. Necropsy findings included irregular surface of the stomach in all three animals. The male mortalities were considered related to treatment with the test item. The female had non-collapsed lungs, which may suggest a gavage-related death, although a relationship to the test item (e.g. irritation) could not be excluded. These three animals were replaced by reserve animals and the dose level of Group 2 was lowered to 400 mg/kg/day from Day 7 onwards.
In Group 2 at 500/400 mg/kg/day one male and four females were sacrificed in extremis or with a total litter loss.
Two females were sacrificed in extremis on respectively Day 14 of treatment and Day 6 of lactation showing piloerection, breathing difficulties, pale appearance, red discharge from the snout and/or alopecia. Necropsy findings included non-collapsed lungs, emaciated appearance, pelvic dilation, accentuated lobular liver pattern, irregular surface of the stomach and/or gas in the gastrointestinal tract. These mortalities were possibly related to the oral gavage procedure, although a relationship to the test item (e.g. irritation) could not be excluded.
Two females and one male were sacrificed in extremis on Day 10 of treatment (both females)
and Day 35 of treatment (male) with breathing difficulties, pale appearance, hunched posture,
piloerection, diarrhoea, red staining from the snout, face grimas and/or hypersensitivity to
touch. In the male body weight loss was recorded. At necropsy irregular surface of the
glandular mucosa was recorded in both females. These premature deaths were considered
related to treatment with the test item.

Salivation was seen after dosing in all animals of all dose groups during most of the treatment period. This was considered to be a physiological response rather than a sign of systemic toxicity.
Any 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality occurred during the study period at both dose levels.
Group 1 (200 mg/kg/day): One male was sacrificed in extremis on Day 6 of treatment with rales, gasping and lethargic behaviour. This animal had non-collapsed lungs at necropsy but no other macroscopic findings were recorded. This mortality was possibly gavage-related, although a relationship to the test item (e.g. irritation) could not be excluded.
One female was sacrificed in extremis on Day 39 of treatment (Day 14 post-coitum) showing gasping, red discharge from the snout and a pale appearance. At necropsy an accentuated lobular pattern of the liver was recorded, as well as early resorptions in the uterus. One female was sacrificed in extremis on Day 55 of treatment (Lactation Day 8) with 16% body weight loss, rales, piloerection and lethargic behaviour. No findings were recorded at necropsy. For these two mortalities, a relation to treatment with the test item could not be excluded.
Group 2 (500/400 mg/kg/day): During the first 5 days of treatment (at 500 mg/kg/day) three Group 2 animals were sacrificed in extremis. One male was sacrificed on Day 3 with rales, piloerection, diarrhoea and red discharge from the snout, and a body weight loss of 14% (recorded in study raw data). Irregular surface of the forestomach and reddish discolouration of the caecum were recorded at necropsy. One male and one female were sacrificed on Day 5 with similar signs of toxicity. A body weight loss of 14% was recorded in the male. Necropsy findings included irregular surface of the stomach. These premature deaths were considered related to treatment with the test item. One female non-collapsed lungs, which may suggest a gavage-related death. A relationship to the test
item (e.g. irritation) could however not be excluded. The three animals were replaced by reserve animals and the dose level of Group 2 was lowered to 400 mg/kg/day from Day 7 onwards.
On Day 14 of treatment, one female was sacrificed in extremis. This animal was showing hunched posture, piloerection, gasping, had a pale appearance and red staining of the skin. At necropsy non collapsed lungs, emaciated appearance and pelvic dilation in the kidney were recorded. One female was sacrificed in extremis on Lactation Day 6. Piloerection, pale appearance, gasping and alopecia were recorded and at necropsy non-collapsed lungs, irregular surface of the stomach, gas in the gastrointestinal tract and an accentuated lobular liver pattern were observed. Non-collapsed lungs may suggest a gavage-related death in these animals, although a relationship to the test item (e.g. irritation) could not be excluded.
On Day 10 of treatment two females were sacrificed in extremis with severe breathing difficulties, pale appearance, hunched posture, piloerection, diarrhoea, red staining from the snout and/or face grimas. Irregular surface of the glandular mucosa was recorded at necropsy.
On Day 35 of dosing, one male was sacrificed in extremis. This animal was showing hunched posture, rales, piloerection, red staining of the nose and hypersensitivity to touch and it had lost >8% body weight in the last week. No findings were recorded at necropsy. These premature deaths were considered related to treatment with the test item.
One female was sacrificed with a total litter loss on Lactation Day 1. This female had a yellow staining of the urine, rales and piloerection the days preceding death. An irregular surface of the forestomach was recorded at necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group 1 (200 mg/kg/day): One male lost 2% body weight on Day 8 and one male lost 1% body weight on Day 15. The other males gained weight, but body weight gain was low compared to values expected for rats of this age and strain throughout the treatment period. Absolute body weights were within expected ranges.
Two females lost 1-3% body weight on Days 5, 8 and 15. One female has lost 1% body weight on Day 15. Mean body weight gain in females was within ranges expected for rats of this age and strain.
Body weight gain during the post-coitum period was considered normal, and body weight gain during lactation was within normal ranges as well.
Group 2 (500/400 mg/kg/day): All animals lost weight or did not gain body weight on Day 5. On Day 8 7/8 males gained weighed again, and on Day 15 5/8 remaining males were back to start weight or above start weight. 2/8 males were back on their start weight at start of the mating period. Mean body weight gain was low compared to values expected for rats of this age and strain throughout the treatment period. Absolute body weights were on the lower end of normal biological variation.
On Day 8 all females gained weight or remained on the same weight. 6/9 remaining females were on or above start weight on Day 8. On Day 15 all remaining females (6) were above start weight, and body weight gain was within ranges expected for rats of this age and strain.
Body weight gain during the post-coitum period was slightly low. This was mainly caused by one female, which was not pregnant, but the individual body weight gain values in the other females were slightly low as well. Body weight gain during lactation was considered normal.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption at 200 and 500/400 mg/kg bw/day was slightly low in males and females during the premating and mating period. This was considered test item-related. Food consumption during post-coitum and lactation was within ranges expected for rats of this age and strain.

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 200 mg/kg bw/day.
At 500/400 mg/kg bw/day estrous cycle length and regularity were affected by treatment. Out of seven females included in the study throughout the complete premating period, four females were noted with an acyclic and one female was noted with an irregular estrous cycle. This was considered to be related to treatment with the test item.
As three females were sacrificed during the premating period estrous cycle could not be evaluated.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility indices were considered unaffected by treatment with the test item up to 500/400 mg/kg bw/day.
The fertility indices were 100% and 86% for the 200 and 500/400 mg/kg bw/day groups, respectively. A total of 1/7 females at 500/400 mg/kg bw/day was not pregnant.

Details on results (P0)

Mating index: Mating index was considered not to be affected by treatment with the test item. All paired females showed evidence of mating.
Precoital time: Precoital time was considered not to be affected by treatment with the test item. Most females showed evidence of mating within 4 days.
Number of implantation sites: Mean number of implantation sites was considered unaffected by treatment with the test item at 200 mg/kg bw/day.
At 500/400 mg/kg bw/day mean number of implantation sites was lower compared to the 200 mg/kg/day group (mean 10.0 (n=6) versus 12.9 (n=10), respectively) and slightly low compared to values expected for rats of this age and strain. A relation to treatment with the test item could not be excluded.

Note: As three females were sacrificed in the premating period, and one other female was not pregnant, reproduction data is available of only 6 females in the 500/400 mg/kg/day dose group. For estrous cycle, mating index and precoital time data from 7 females was available. For all parameters with data from 6 females only, toxicological evaluation in the high dose group was therefore not conclusive.

Gestation index/duration: Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
Except for one female at 500/400 mg/kg/day with a total litter loss, all pregnant females had live offspring. The gestation indices were 90% and 83% for the 200 and 500/400 mg/kg/day groups, respectively.

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.

Effect levels (P0)

Key result

Dose descriptor:

other: Upper dose level EOGRTs

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Besides the three mortalities, of which one was considered gavage-related and not test item-related, no signs of severe toxicity were recorded at 200 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered low compared to values expected for rats of this age and strain starting at 200 mg/kg bw/day.
Post-implantation survival index was 82% and 85% for the 200 and 500/400 mg/kg bw/day groups, respectively. A relation to treatment with the test item could not be excluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of pups at 200 mg/kg bw/day were within ranges expected for rats of this age and strain.
Body weights of male and female pups at 500/400 mg/kg bw/day were below normal ranges from Day 7 onwards. This was considered related to treatment with the test item.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item up to 500/400 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, one male pup was missing its tail apex (first observed on Day 6). The days before the pup was missing its tail apex it showed a black tail apex. Furthermore, in the 400 mg/kg bw/day group, five out of eight pups in one litter showed beginning autolysis. The other three pups from tis litter were cannibalised except for the head. No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Litter size: Litter size was considered not affected by treatment with the test item at 200 mg/kg bw/day. At 500/400 mg/kg bw/day litter size was considered affected by treatment with the test item. Live litter sizes were 11.7 and 7.2 living pups/litter for the 200 and 500/400 mg/kg bw/day groups, respectively.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item at 200 mg/kg bw/day. At 500/400 mg/kg bw/day live birth index was low, caused by one female with a total litter loss on Lactation Day 1. The live birth indices were 100% and 84% for the 200 and 500/400 mg/kg bw/day groups, respectively.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of live offspring on Day 1 was considered not affected by treatment with the test item up to 500/400 mg/kg bw/day. Viability indices were 99 and 100% for the 200 and 500/400 mg/kg bw/day groups, respectively. One pup at 200 mg/kg/day was missing on PND 3. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups 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 considered not to be affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13. The lactation indices were 89% and 86% for the 200 and 500/400 mg/kg bw/day groups, respectively. Respectively 8 and 5 pups were sacrificed in the 200 and 500/400 mg/kg bw/day groups because the dam was sacrificed in extremis. This was unrelated to the condition of the pups. When excluding these pups, lactation indices were 100% for both groups.
- Sex ratio: Sex ratio was considered not to be affected by treatment with the test item up to 500/400 mg/kg/day.

Any other information on results incl. tables

DOSE FORMULATION ANALYSES

Accuracy
The concentrations analyzed in the formulations of Groups 1 and 2 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeneity
The formulations of Group 1 and Group 2 were homogeneous (i.e. coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this Dose Range Finder study, a simplified reproduction/developmental toxicity screening test, a dose level of 500/400 mg/kg bw/day was not tolerated by Wistar Han Rats based on the available data. Signs of parental and reproduction toxicity recorded at 500/400 mg/kg bw/day were considered too severe to allow 400 mg/kg/day as a high dose in the subsequent extended one generation reproductive toxicity study (Test Facility Study No. 20263211).
For the extended one generation reproductive toxicity study the high dose level should produce some signs of toxicity, but no obvious suffering. Besides the three mortalities, of which one was considered gavage-related and not test item-related, no signs of severe toxicity were recorded at 200 mg/kg bw/day. Therefore, the dose levels for the extended one generation reproductive toxicity were selected to be 40, 80 and 200 mg/kg bw/day.
Furthermore, it is proposed to initiate the extended one generation reproductive toxicity study with 28 animals/sex instead of 25 animals/sex in the high dose group to ensure that sufficient animals are available at start of the mating period for generation of litters.

Executive summary:

The objective of this study was to select dose levels for the extended one generation reproductive toxicity study in Wistar Han rats (Test Facility Study No. 20263211).
The potential toxic effects of NBDI when given orally by gavage for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated. The dose levels in this study were selected to be 200 and 500 mg/kg bw/day, based on information by the Sponsor (28-day repeated dose toxicity study with oral administration of NBDI in rats, reference ECHA dossier). From Day 7 onwards the high dose level was lowered to 400 mg/kg bw/day.

The study design was as follows:

Group No.	Test item id.	Dose Level (mg/kg bw/day)	Dose volume (mL/kg)	Dose concentration (mg/mL)	Males	Females
1	NBDI	200	4	500	10	10
2	NBDI	500/400*	4	125/100*	10	10

*Animals were dosed at 500 mg/kg bw/day for Days 1-5. On Day 6 animals were not dosed, and from Day 7 onwards animals were treated at a dose level of 400 mg/kg bw/day.

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: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle, gross necropsy findings and organ weights. 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).
Formulation analyses confirmed that formulations of test item in Corn oil were prepared accurately and homogenously.

Parental toxicity
Parental toxicity was observed at 200 and 500/400 mg/kg bw/day. At 200 mg/kg bw/day one male and two females were sacrificed in extremis on respectively Day 6 of treatment, Day 14 post-coitum and Day 8 of lactation. The male animal showed rales, gasping and lethargy and non-collapsed lungs were recorded at necropsy. This mortality was possibly related to the oral gavage procedure, although a relationship to the test item (e.g. irritation) could not be excluded. The two females showed rales, gasping, piloerection, red discharge from the snout, pale appearance and/or lethargy. In one female severe body weight loss was  noted. An accentuated lobular pattern of the liver was recorded in one female; no macroscopic findings were noted in the second female. For these two mortalities, a relation to treatment with the test item could not be excluded.

At 500/400 mg/kg bw/day two males and one female were sacrificed in extremis during the first 5 days of treatment showing rales, gasping, laboured respiration, piloerection, diarrhoea, red discharge from the snout and/or ptosis. Severe body weight loss was recorded in the two males. Necropsy findings included irregular surface of the stomach in all three animals. The male mortalities were considered related to treatment with the test item. The female had non-collapsed lungs, which may suggest a gavage-related death, although a relationship to the test item (e.g. irritation) could not be excluded. These three animals were replaced by reserve animals and the dose level was lowered to 400 mg/kg bw/day from Day 7 onwards. At 500/400 mg/kg bw/day one male and four females were sacrificed in extremis or with a total litter loss. Two females were sacrificed in extremis on respectively Day 14 of treatment and Day 6 of lactation showing piloerection, breathing difficulties, pale appearance, red discharge from the snout and/or alopecia. Necropsy findings included non-collapsed lungs, emaciated appearance, pelvic dilation, accentuated lobular liver pattern, irregular surface of the stomach and/or gas in the gastrointestinal tract. These mortalities were possibly related to the oral gavage procedure, although a relationship to the test item (e.g. irritation) could not be excluded. Two females and one male were sacrificed in extremis on Day 10 of treatment (both females) and Day 35 of treatment (male) with breathing difficulties, pale appearance, hunched posture, piloerection, diarrhoea, red staining from the snout, face grimas and/or hypersensitivity to touch. In the male body weight loss was recorded. At necropsy irregular surface of the glandular mucosa was recorded in both females. These premature deaths were considered related to treatment with the test item.
Test item-related clinical signs were observed at 200 and 500/400 mg/kg bw/day. At 200 mg/kg bw/day, hunched posture and piloerection were recorded in all males starting at Week 4. Incidental rales, red staining from the snout and hypersensitivity to touch were recorded in males and/or females. Females showed incidental hunched posture and piloerection was recorded in all females starting during the mating period.
At 500/400 mg/kg bw/day, diarrhoea was recorded in males and females on one or several days during Week 2 of treatment. Lethargy, laboured respiration, slow breathing, gasping, hypersensitivity to touch and/or red discharge from the nose were incidentally recorded in males and females. Hunched posture and/or piloerection was recorded in all males and females starting on Day 3. Rales were recorded in 8/10 males and 7/10 females on one or multiple treatment days, starting in Week 2 and Week 1 of treatment, respectively.
Test item-related changes in body weight and body weight gain were recorded at 200 and 500/400 mg/kg bw/day.
At 200 mg/kg bw/day mean body weight gain was slightly low compared with values expected for rats of this age and strain, but absolute body weights were within expected ranges.
At 500/400 mg/kg bw/day all animals lost weight or did not gain body weight on Day 5 of treatment, which mostly recovered between Days 8 and 15 of treatment. Mean body weight gain was low compared to values expected for rats of this age and strain in males throughout the treatment period and in females during the post-coitum period. Absolute body weights in males were on the lower end of normal biological variation. Food consumption at 200 and 500/400 mg/kg bw/day was slightly low in males and females during the premating and mating period. This was considered test item-related. Test item-related gross findings were noted in animals surviving up to scheduled necropsy: one male at 200 mg/kg bw/day had a diaphragmatic hernia of the liver and cysts in the kidney in one male. One male each at 500/400 mg/kg bw/day showed greenish discolouration of the kidney or irregular surface of the forestomach and dark red discolouration of the mesenteric lymph nodes. Gas in the gastrointestinal tract and foci on mesenteric lymph nodes was observed in one female at 500/400 mg/kg bw/day. No toxicologically significant changes were noted in testes and epididymides weights.

Reproductive toxicity
No reproduction toxicity was observed up to 200 mg/kg bw/day. Note that reproduction data is available of only 6 females in the 500/400 mg/kg bw/day dose group. For estrous cycle, mating index and precoital time data from 7 females was available. For all parameters with data from 6 females only, toxicological evaluation in the high dose group was therefore not conclusive. Based on the available data, estrous cycle length and regularity and number of implantation sites were affected by treatment at 500/400 mg/kg bw/day, as only 2/7 females had a normal estrous cycle and mean number of implantation sites was slightly low. Based on the available data, no treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices and precoital time).

Developmental toxicity
Note that developmental data is available of only 5 females in the 500/400 mg/kg bw/day dose group. For gestation index and duration and post-implantation survival index data from 6 females was available. Toxicological evaluation in the high dose group was therefore not conclusive.
One female at 500/400 mg/kg bw/day was sacrificed with a total litter loss on Lactation Day 1. This female had a yellow staining of the urine, rales and piloerection the days preceding death. An irregular surface of the forestomach was recorded at necropsy. Based on the available data, developmental toxicity was observed starting at 200 mg/kg bw/day.
Post-implantation survival index was considered low compared to values expected for rats of this age and strain at 200 and 500/400 mg/kg bw/day. At 500/400 mg/kg bw/day, litter size, live birth index and body weight of pups were considered affected by treatment with the test item.
Based on the available data, no toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, gestation index and duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention and macroscopic examination).

In conclusion, based on the results of this Dose Range Finder study, a simplified reproduction/developmental toxicity screening test, a dose level of 500/400 mg/kg bw/day was not tolerated by Wistar Han Rats based on the available data. Signs of parental and reproduction toxicity recorded at 500/400 mg/kg bw/day were considered too severe to allow 400 mg/kg bw/day as a high dose in the subsequent extended one generation reproductive toxicity study (Test Facility Study No. 20263211).
For the extended one generation reproductive toxicity study the high dose level should produce some signs of toxicity, but no obvious suffering. Besides the three mortalities, of which one was considered gavage-related and not test item-related, no signs of severe toxicity were recorded at 200 mg/kg bw/day. Therefore, the dose levels for the extended one generation reproductive toxicity were selected to be 40, 80 and 200 mg/kg bw/day.
Furthermore, it is proposed to initiate the extended one generation reproductive toxicity study with 28 animals/sex instead of 25 animals/sex in the high dose group to ensure that sufficient animals are available at start of the mating period for generation of litters.