N-bromosuccinimide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2017 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650 Combined repeated dose toxicity study with the reproduction/developmental toxicity screening

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-week old males and 13-week old females
- Weight at study initiation: 267-304 g for males and 201-228 g for females
- Fasting period before study: no
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm) during premating period; cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm) during mating; during the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); during the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet (ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 41-65
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Nov 2017 To:19 Jan 2018

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared daily as a solution in water for dose concentrations up to 13.3 mg/ml and as a suspension in water for the higher dose concentrations of 20 mg/ml (in the DRF). Animals were dosed within 6 hours after adding the vehicle to the test item. To achieve maximal solubility in the vehicle, formulations were heated to a maximum temperature of 50±5°C for maximally 15 minutes to obtain visual homogeneity. Formulations were released for dosing when they have obtained a temperature of 40°C or lower. 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 density of the test item and no adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.


- VEHICLE
- Justification for use and choice of vehicle: Analytical work has been previously undertaken at the sponsor using NMR, which shows that N-bromosuccinimide is chemically stable in water
- Concentration in vehicle: maximum 13.3 mg/ml
- Amount of vehicle (if gavage): 15 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in week 1 and analysed for concentration in all groups and homogeneity (top, middle and bottom) at 10 and 200 mg/kg bw/d.

Duration of treatment / exposure:

Min of 28 days: Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy. Females that delivered were treated for 50 - 55 days (most females) or 64 days (one control female), 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 42 - 55 days.

Frequency of treatment:

7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finding study with oral exposure of N-BROMOSUCCINIMIDE in the rat (3 females/dose) at 202 and 400 mg/kg bw/d. Main clinical signs observed were lethargy, hunched posture, piloerection of the fur, hypothermia, ptosis and breathing difficulties. Severe body weight loss and severely reduced food intake were noticed during day 1-5 of treatment. Animals treated with 400 mg/kg bw/d were euthanized for humane reasons at day 5 of treatment. At dose level 200 mg/kg bw/d some clinical signs were noted and normal body weights and food consumption levels were observed for the duration of the treatment period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once daily

BODY WEIGHT:
- Time schedule for examinations: first day of treatment (prior to dosing), and weekly thereafter. Mated females were 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, 8 males at 200 mg/kg bw/d were also weighed on Day 12 of treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was 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: subjective appraisal was maintained during the study

HAEMATOLOGY and CLINICAL CHEMISTRY:
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight for animal welfare reasons, so that the separation of the lactating females from their pups could be avoided.
- How many animals: 5/sex/dose
- Parameters according to OECD 422 were examined, including total T4 was conducted for F0-males, F0-females, PND 14-16 pups, and PND 4 pups, and TSH was conducted for F0-males, F0-females and PND 14-16 pups.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: 5 males during Week 5 (see deviation in Appendix 8) of treatment and 5 females during the last week of lactation (i.e. PND 11, 12 and 13). These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind limb grip strength, locomotor activity

OTHER: For all non-selected males, one blood sample was collected on Day 29 of treatment, after 1 hour of exposure to determine bromide in plasma.

Sacrifice and pathology:

Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25, 26 or 27.
Without evidence of mating: 25 days after the last day of the mating period.
Female with total litter loss: Animal was found dead on PND 2.
All males surviving to scheduled necropsy were fasted (overnight with a maximum of approximately 24 hours) before their scheduled necropsy. Water was available. F0-females were not fasted.

GROSS PATHOLOGY: All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: organs of 5 scheduled euthanasia animals/sex/dose according to OECD 422
HISTOPATHOLOGY: according to OECD 422 for 5 animals/sex/dose and unscheduled deaths; reproductive organs for males that failed to sire and females that failed to deliver pups and female with total litter loss (also mammary gland). For remaining animals gross lesions were examined.
For the testes of all selected males of control and at 200 mg/kg bw/d, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

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% and 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 comparison 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
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of deceased animals are noted in the Mortality section below.
Rales were noted in four surviving females at 200 mg/kg, mostly in the post-coitum period. Three of them showed rales on only one occasion, the fourth female showed rales on six days.
Salivation was noted among animals of the 200 mg/kg group from treatment Day 5 onwards. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (mostly slight) and the time of occurrence (i.e. after dosing), and may be related to the irritancy of the test item.
No additional clinical signs were noted during the weekly arena observations. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 200 mg/kg there were five premature decedents (three males and two females) which were considered to be related to treatment with the test item.
Two males had lost nearly 20% of their initial weight during the first treatment week and were euthanized for humane reasons on study Day 8. They showed no clinical signs of toxicity. One male showed rales and/or piloerection on treatment Days 19-21 (but no weight loss) and was humanely sacrificed on treatment Day 21. One female was euthanized due to delivery difficulties on study Day 37 (post-coitum Day 20) when she showed hunched posture, piloerection and a slightly pale appearance. A few days earlier she showed rales. Between post-coitum Days 17-20 she had lost 6% of her weight and her food consumption was severely reduced. At necropsy, her uterus contained 15 living fetuses (all without abnormalities) and one early resorption. The moribundity of these four animals was due to erosion/ulceration (marked to massive) and associated inflammation (moderate to marked) in the esophagus.
Another female was found dead on study Day 41. At dosing on the previous day, it was noted that she was no longer pregnant (total litter loss). No signs of toxicity or difficult parturition were noted and her body weight gain and food consumption were normal. Necropsy findings consisted of advanced autolysis and a reduced size of the spleen. At microscopic examination, moderate inflammation, including foci with mineralization, was noted at the hilus of the lung (i.e. at the visceral pleura side, outside the lung parenchyma). This was regarded as the cause of her death. Despite the fact that no lesions were noted in the esophagus of this animal (most likely dependent on the plane of section), the inflammation at the lung hilus most likely reflects an inflammatory process extending from the esophagus (like noted in the other early deaths).
Additionally, decedents showed an increase in granulocytes in the bone marrow of sternum and femur which was moderate or marked in the males and minimal in one female. Bone marrow of the female found death showed no abnormalities (sternum) or was autolytic (femur).
Most of the remaining morphologic alterations in decedents were similar as in survivors and are, therefore, presented together with those of survivors.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 200 mg/kg, body weight loss occurred in the two males which were euthanized at Day 8 (nearly 20% of their initial weight) and, to a lesser extent, in a surviving male (8% loss between Day 1-8). Two other survivors showed reduced weight gain between treatment Days 1-8. After Day 8, weight gain of 200 mg/kg males was normal. Mean body weights of males at 200 mg/kg did not differ significantly from those of controls.
In females at 200 mg/kg, statistically significantly lower mean body weights (nearly 10% difference from control values) and body weight gain were noted at Days 4 and 7 of the lactation period. The lower mean values resulted from slight weight loss/no weight gain in 3/5 females between lactation Days 1-4 or Days 1-7. Body weights and weight gain of 200 mg/kg females during the pre-mating and gestation periods were considered not to be affected by treatment. The slightly lower mean weight gain (not statistically significant) during the last week of gestation could be explained by the weight loss of one decedent and the lower weight gain of another that had only one implantation site.
No treatment-related changes in body weight (gain) were observed in males and females treated up to 45 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at 200 mg/kg, mean food consumption between treatment Days 1-8 was reduced for the cage housing the two males that were euthanized on Day 8 (differences from controls: about 30 and 20% before and after correction for body weight, respectively). Food consumption of the surviving 200 mg/kg males was considered not to be affected by treatment.
In females at 200 mg/kg, lower mean food consumption values (before and after correction for body weight) were noted throughout the lactation period. Compared to controls, mean absolute food consumption was about 40, 25 and 15% lower during lactation Days 1-4, 4-7 and 7-13, respectively (statistically significant, except for the last interval). The lower mean values resulted from the reduced food consumption of the three 200 mg/kg females which showed slight weight loss/no weight gain in the first week of the lactation period. Food consumption of the other two lactating females of this dose group was normal. Food consumption of 200 mg/kg females during the pre-mating and gestation periods was considered not to be affected by treatment. The slightly lower food consumption (not statistically significant) between Days 17-20 of gestation could be explained by the severely reduced food consumption of one decedent and the lower value for another female which had only one implantation site (her food intake towards the end of gestation was similar to that of the non-pregnant control females).
No treatment-related changes in food consumption before or after correction for body weight were noted in males and females treated up to 45 mg/kg.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In females at 200 mg/kg, the mean number of total white blood cells was statistically significantly higher compared to controls (41% difference). The highest value (exceeding the normal range) was noted in female no. 80 which also had an increased number of neutrophils, which was likely related to the inflammation seen in the esophagus and glandular stomach of this female (at marked and moderate degree, respectively).
No treatment-related changes in hematology parameters were noted in male rats.
Coagulation parameters were considered not to be affected by treatment up to 200 mg/kg.
An isolated, statistically significant difference noted in females (higher activated partial thromboplastin time (APTT) at 45 mg/kg) was considered to be unrelated to treatment due to the lack of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly increased plasma concentrations of chloride were noted in males at 45 and 200 mg/kg (relative differences from controls: 5 and 15%, respectively, exceeding the upper limit of the historical control range ).
A statistically significantly higher mean plasma chloride level was also observed in females at 45 mg/kg (5% difference from controls). It was noted that chloride concentrations in two control females were below the normal range, whereas the values in two 45 mg/kg females (and one 200 mg/kg female) exceeded this range . The lack of a dose-related trend in combination with a slightly low concurrent control value in females makes it less likely that the slightly higher mean chloride value in 45 mg/kg females reflected a test item-related change. Clinical chemistry results showed elevated plasma concentrations of chloride in males at 45 and 200 mg/kg (difference from controls: 5 and 15%, respectively). Plasma chloride was measured by means of an Ion Selective Electrode (ISE) which is not perfectly specific for the chloride ion. Bromide, a constituent of the test item, is known to interfere with the chloride determination, causing falsely high concentrations of chloride (Dimeski et al., 2010). The presence of bromide in plasma of rats treated with the test item was confirmed by bioanalysis (non-GLP). Therefore, the elevated chloride levels noted in treated rats were considered to be the result of interference of the analysis by bromide rather than an adverse effect of the test item on plasma chloride.
The other statistically significant variations noted in clinical chemistry parameters were regarded as not toxicologically relevant due to direction of the change (lower bilirubin at 200 mg/kg in females), or unrelated to treatment due to the lack of a dose-related trend (lower inorganic phosphate at 45 mg/kg in males, higher potassium at 10 and/or 45 mg/kg in both sexes).
Mean serum T4 levels of F0-males were statistically significantly decreased at 45 and 200 mg/kg (relative differences from controls: 27 and 38%, respectively). The mean values at these dose levels were below the historical control range. Mean serum T4 levels of F0-females were statistically significantly decreased at 10, 45 and 200 mg/kg (relative differences from controls: 28%, 26% and 38%, respectively). The mean T4 values at dose 200 mg/kg were in the lower limit of the historical control range.
For mean serum TSH levels for F0-males a dose related trend is observed with a statistically significant increase at 200 mg/kg (relative difference from control: x2.7 fold). The mean values at these dose levels were within the historical control range . For F0-females, no statistically significant changes were observed for mean serum TSH levels. All values were within the historical control range.
Historical control data:
chloride (mmol/L) in fasted male Wistar Han rats (period 2015 - December 2017): mean = 102, P5 - P95 = 100 - 105 (n=304)
chloride (mmol/L) in non-fasted female Wistar Han rats (period 2017 - wk 02 2018): mean = 102, P5 - P95 = 99 - 104 (n=55)
T4 (µg/dL) in Wistar Han rats (period 2015 - December 2017): male rats: mean = 4.65, P5 - P95 = 3.01 - 6.52 (n=724); female rats: mean = 3.45, P5 - P95 = 1.70 - 5.06 (n=55).
TSH (µIU/mL) in Wistar Han rats (period 2015 - December 2017): male rats: mean = 0.170, P5 - P95 = 0.044 - 0.462 (n=75). female rats: mean = 0.256, P5 - P95 = 0.045 - 0.724 (n=45).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were noted for functional observations up to 200 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was considered not to be affected by treatment. It was noted that mean hind limb grip strength in 200 mg/kg males was 24% lower than that of concurrent controls. This intergroup difference was not attributed to treatment as it was not statistically significant and all values at 200 mg/kg remained in the historical control range.
The variation in motor activity did not indicate a relation with treatment. All examined groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related lower thymus weights (absolute and relative to body weight; not statistically significant) were observed at 200 mg/kg in females (abs +4, -6 and -23% relative to control; rel +4, -9 and -21% relative to control).
The statistically significant differences in thyroid weights noted in females (lower absolute weights at 45 and 200 mg/kg, lower relative weight at 45 mg/kg) were considered to be unrelated to treatment due to the lack of a dose-related trend. Mean relative thyroid weights were similar in all groups of treated females despite a 20-fold difference between the lowest and the highest dose level. Moreover, mean relative thyroid weights in treated females were similar to the historical control mean.
Historical control data for thyroid to body weight ratio (%) in non-fasted female Wistar Han rats (period 2017-wk 2 2018): mean = 0.005; P5 - P95 = 0.004 - 0.007 (n=118).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed at 200 mg/kg in the forestomach and esophagus of rats of both sexes and in the thymus of females. These findings consisted of:
• Forestomach: Irregular surface in 2/10 males (1 unscheduled, 1 scheduled) and in 1/10 female (unscheduled). Microscopic correlate: squamous cell hyperplasia with hyperkeratosis
• Forestomach: Several dark red foci in 1/10 males (unscheduled). Microscopic correlate: squamous cell hyperplasia with hyperkeratosis and erosion.
• Esophagus: Thickened in 1/10 males (unscheduled) and in 2/10 females (1 unscheduled, 1 scheduled). Microscopic correlate: erosion/ulceration and inflammation.
• Esophagus: Adhesions in 2/10 males (unscheduled). Microscopic correlate: erosion/ ulceration and inflammation.
• Thymus: Reduced size in 2/10 females (1 unscheduled and 1 scheduled). Microscopic correlate: lymphoid atrophy.
• Thymus: Gelatinous in 1/10 females (unscheduled). Microscopic correlate: lymphoid atrophy.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were observed in the thyroid gland of males and in the thymus, stomach, esophagus, trachea and/or lung of both sexes.
Thyroid gland:
Follicular cell hypertrophy was noted at increased incidence and severity (up to slight) at 200 mg/kg in males.
Thymus:
Lymphoid atrophy was noted at increased severity at 200 mg/kg in both sexes. Severities were up to slight in scheduled sacrificed rats and up to marked in the decedents.
Forestomach:
Lesions were noted up to slight degrees at 200 mg/kg in males and, less frequently, females, and consisted of erosion/ulceration, squamous cell hyperplasia, inflammatory cell infiltrate and (sub) mucosal edema. The lesions occurred in decedents and survivors.
Glandular stomach:
Erosion/ulceration, inflammatory cell infiltrate and edema, all up to moderate severities, were noted at 200 mg/kg in one female (survivor) and a few males (one decedent and two survivors). Additional findings consisted of minimal mucosal hypertrophy in both males that were euthanized on Day 8, and slight hemorrhages in a few rats (two decedents, one survivor).
Esophagus:
Erosion/ulceration (marked or massive) and inflammation (moderate or marked) were noted at 200 mg/kg in a few males (decedents) and females (one survivor, one decedent). Myofiber degeneration (minimal) was noted in one surviving 200 mg/kg male.
Trachea:
Erosion/ulceration (moderate) and/or inflammatory cell infiltrate (up to slight) were noted in a few 200 mg/kg females (one survivor, both decedents). Epithelial alteration (loss of microvilli, up to slight) was noted at 200 mg/kg in one male (decedent) and three females (two survivors, one decedent).
Lung:
Two males (decedents) and a few females (one survivor, two decedents) at 200 mg/kg showed erosion/ulceration (slight) and hyperplasia/hypertrophy of the bronchus epithelium (minimal), acute bronchioalveolar inflammation (slight), inflammatory cell infiltrate of the lung hilus (up to moderate) and/or presence of granuloma (slight).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) at 200 mg/kg. These changes caused moribundity/mortality of five animals

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

lungs

oesophagus

stomach

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Dose formulation analysis:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation≤ 10%).

Bromide in plasma:

A dose dependent increase in bromide concentration was observed. Bromide levels were below the detection limit of 50 µg/mL in male control animals, one hour after dosing on Day 29 of treatment. In 4 out of 5 tested male animals treated with 10 mg/kg, bromide concentrations were slightly above the detection limit of 50 µg/mL with bromide concentrations between 51.1 and 54.9 µg/mL measured one hour after exposure on Day 29 of treatment. Bromide levels increased to on average 236 µg/mL (about 4.5x higher compared to dose 10 mg/kg) when animals were treated with 45 mg/kg and to 855 µg/mL (about 16.4x higher compared to dose 10 mg/kg) when treated with 200 mg/kg.

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following parental NOAEL for N-bromosuccinimide was established: 45 mg/kg bw/d, based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) at 200 mg/kg bw/d. These changes caused moribundity/mortality of five animals.

Executive summary:

A 28-day repeated dose toxicity study combined with screening for reproduction/developmental toxicity was performed in Wistar Han rats according to OECD 422 and GLP. Rats were exposed to 0, 10, 45 or 200 mg/kg bw/d N-bromosuccinimide in water. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy. Females that delivered were treated for 50 - 55 days (most females) or 64 days (one control female), 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 42 - 55 days. The following parental parameters were evaluated in this study: mortality/moribundity, clinical signs, functional tests, body weight, food consumption, estrous cycle length and regularity, clinical pathology, plasma concentration of bromide (non-GLP), serum level of thyroid hormones T4 and TSH (F0-rats of both sexes), gross necropsy findings, organ weights and histopathologic examination. 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 weight, anogenital distance, areola/nipple retention, serum levels of thyroid hormones (T4 in PND 4 and PND 14-16 pups, TSH in PND 14-16 pups) and macroscopy). Formulation analysis showed that formulations were prepared accurately and homogeneously. At 200mg/kg bw/d, there were five test item-related unscheduled deaths (two males on Day 8, one male on Day 21, one female on Day 37 and one female on Day 41 of treatment). Four of these were due to erosion/ulceration (marked to massive) and associated inflammation (moderate to marked) of the esophagus and one was related to inflammation of the lung hilus (outside of the lung; this likely reflected an inflammatory process extending from the esophagus). Most of the unscheduled deaths showed weight loss and reduced food consumption and two of them showed clinical signs of toxicity (rales, piloerection, hunched posture and/or a pale appearance).   

Slight weight loss/reduced weight gain and reduced food consumption also occurred in a few surviving 200 mg/kg males (in the first treatment week) and females (in the first week of the lactation period), one of which had severe lesions in the stomach and esophagus. Slight salivation after dosing,regarded as a physiological response related to the irritant properties of the test item rather than a sign of systemic toxicity, was noted in all rats at 200 mg/kg bw/d. Occasionally, rales were noted in 200 mg/kg bw/d females. Major microscopic findings consisted of lesions indicative of irritating properties of the test item. Lesions were noted in the esophagus, stomach, trachea and lung of 200 mg/kg bw/d treated rats and consisted of:

·    Erosion/ulceration of protective epithelial layers of esophagus, stomach, trachea and bronchus. The most severe lesions occurred in the esophagus (up to massive). Related gross lesions in the esophagus consisted of adhesions and thickened wall.

·    Concomitant inflammation/inflammatory cell infiltrate and/or edema of these organs and their surrounding areas (like lung hilus).

·    Regeneration-like processes, such as hyperplasia of the forestomach epithelium, hypertrophy of the glandular mucosa, epithelial alteration of the trachea epithelium (loss of villi) and hyperplasia/hypertrophy of the bronchus epithelium. Related gross lesions were noted in the forestomach (irregular surface, dark red foci).

In general, these findings were most obvious in the premature decedents. One surviving female also showed severe lesions. Most likely the test item caused irritation by direct exposure in the stomach or indirectly by reflux/regurgitation. This reflux might be due to a combination of a slightly high dose volume (15 ml/kg body weight) and (irritant) test item properties. Given the nature and high severities of the erosion/ulceration and inflammation and the relation with premature deaths, including possible relationship with total litter loss and delivery problems, these findings were regarded as adverse. Increased severity of lymphoid atrophy in the thymus(related with reduced size/gelatinous and lower thymus weights) and an increase in granulocytes in bone marrow were noted in a few males and females at 200 mg/kg bw/d. Since these findings occurred only in rats with severe esophagus lesions (premature decedents and a surviving female) they might be interpreted as secondary effects due to stress.  A subtle increase in the incidence and severity of follicular cell hypertrophy was noted at 200 mg/kg bw/d in males. This finding was regarded as non-adverse based on the low severity (up to slight) and absence of any degenerative change in the thyroid. Serum levels of T4 were decreased in males at 45 and 200 mg/kg bw/d (by about 30 and 40%, respectively).  Serum T4 levels of F0-females were decreased at 10, 45 and 200 mg/kg bw/d (by about 28%, 26% and 38%, respectively). TSH levels were increased of F0-males at dose 200 mg/kg bw/d (by about 2.7 fold) and no changes in TSH levels were noted for F0-females. Possible adversity of the effects on thyroid hormone levels at 200 mg/kg could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL. Hematology results showed an increase in total white blood cells in females at 200 mg/kg bw/d. The increase was most marked in the female that had inflammation in the esophagus (marked) and stomach (moderate). She also had an increased number of neutrophils. Clinical chemistry results showed elevated plasma concentrations of chloride in males at 45 and 200 mg/kg bw/d (difference from controls: 5 and 15%, respectively). This was considered to be the result of interference of the analysis by bromide (since this is a compound of the test item) rather than an adverse effect of the test item on plasma chloride.

The following parental NOAEL for N-bromosuccinimide was established: 45 mg/kg bw/d, based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) at 200 mg/kg bw/d.  These changes caused moribundity/mortality of five animals.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650 Combined repeated dose toxicity study with the reproduction/developmental toxicity screening

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-bromosuccinimide

EC Number:

204-877-2

EC Name:

N-bromosuccinimide

Cas Number:

128-08-5

Molecular formula:

C4H4BrNO2

IUPAC Name:

1-bromopyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

Appearance: slightly yellow powder
Storage conditions: in a refrigerator (2-8°C) protected from light and the container flushed with nitrogen

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

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.

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: 10-week old males and 13-week old females
- Weight at study initiation: 267-304 g for males and 201-228 g for females
- Fasting period before study: no
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm) during premating period; cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm) during mating; during the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); during the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 41-65
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 16 Nov 2017 To: 19 Jan 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared daily as a solution in water for dose concentrations up to 13.3 mg/ml and as a suspension in water for the higher dose concentrations of 20 mg/ml (in the DRF). Animals were dosed within 6 hours after adding the vehicle to the test item. To achieve maximal solubility in the vehicle, formulations were heated to a maximum temperature of 50±5°C for maximally 15 minutes to obtain visual homogeneity. Formulations were released for dosing when they have obtained a temperature of 40°C or lower. 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 density of the test item and no adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.


- VEHICLE
- Justification for use and choice of vehicle: Analytical work has been previously undertaken at the sponsor using NMR, which shows that N-bromosuccinimide is chemically stable in water
- Concentration in vehicle: maximum 13.3 mg/ml
- Amount of vehicle (if gavage): 15 ml/kg

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. Two males that had proven to sire, were cohabited with an additional female as 2 males were sacrificed moribund prior to the start of the mating period.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected in week 1 and analysed for concentration in all groups and homogeneity (top, middle and bottom) at 10 and 200 mg/kg bw/d.

Duration of treatment / exposure:

Min of 28 days: Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy. Females that delivered were treated for 50 - 55 days (most females) or 64 days (one control female), 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 42 - 55 days.

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finding study with oral exposure of N-BROMOSUCCINIMIDE in the rat (3 females/dose) at 202 and 400 mg/kg bw/d. Main clinical signs observed were lethargy, hunched posture, piloerection of the fur, hypothermia, ptosis and breathing difficulties. Severe body weight loss and severely reduced food intake were noticed during day 1-5 of treatment. Animals treated with 400 mg/kg bw/d were euthanized for humane reasons at day 5 of treatment. At dose level 200 mg/kg bw/d some clinical signs were noted and normal body weights and food consumption levels were observed for the duration of the treatment period.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule:
twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
once daily

BODY WEIGHT:
- Time schedule for examinations:
first day of treatment (prior to dosing), and weekly thereafter. Mated females were 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, 8 males at 200 mg/kg bw/d were also weighed on Day 12 of treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was 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: subjective appraisal was maintained during the study

HAEMATOLOGY and CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
before necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0-females were not fasted overnight for animal welfare reasons, so that the separation of the lactating females from their pups could be avoided.
- How many animals:
5/sex/dose
- Parameters according to OECD 422 were examined, including total T4 was conducted for F0-males, F0-females, PND 14-16 pups, and PND 4 pups, and TSH was conducted for F0-males, F0-females and PND 14-16 pups.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations:
5 males during Week 5 (see deviation in Appendix 8) of treatment and 5 females during the last week of lactation (i.e. PND 11, 12 and 13). These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).
- Dose groups that were examined:
all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind limb grip strength, locomotor activity

OTHER: For all non-selected males, one blood sample was collected on Day 29 of treatment, after 1 hour of exposure to determine bromide in plasma.

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 (pre-test 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.

Sperm parameters (parental animals):

For the testes of all selected males of control and at 200 mg/kg bw/d, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, to reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]

Postmortem examinations (parental animals):

Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25, 26 or 27.
Without evidence of mating: 25 days after the last day of the mating period.
Female with total litter loss: Animal was found dead on PND 2.
All males surviving to scheduled necropsy were fasted (overnight with a maximum of approximately 24 hours) before their scheduled necropsy. Water was available. F0-females were not fasted.

GROSS PATHOLOGY: All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: organs of 5 scheduled euthanasia animals/sex/dose according to OECD 422
HISTOPATHOLOGY: according to OECD 422 for 5 animals/sex/dose and unscheduled deaths; reproductive organs for males that failed to sire and females that failed to deliver pups and female with total litter loss (also mammary gland). For remaining animals gross lesions were examined.

Postmortem examinations (offspring):

Recognizable fetuses of female that was euthanized for humane reasons (post-coitum Day 20) were examined externally, sexed (both externally and internally), and euthanized by decapitation.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
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.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin.

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% and 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 comparison 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
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). 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:

Mating (%): [Number of females mated/number of females paired] x 100
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 live offspring on Day 1 after littering] x 100
Lactation index (%): [Number of live offspring on Day 13 after littering/Number of 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):

Clinical signs of deceased animals are noted in the Mortality section below.
Rales were noted in four surviving females at 200 mg/kg, mostly in the post-coitum period. Three of them showed rales on only one occasion, the fourth female showed rales on six days.
Salivation was noted among animals of the 200 mg/kg group from treatment Day 5 onwards. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (mostly slight) and the time of occurrence (i.e. after dosing), and may be related to the irritancy of the test item.
No additional clinical signs were noted during the weekly arena observations. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 200 mg/kg there were five premature decedents (three males and two females) which were considered to be related to treatment with the test item.
Two males had lost nearly 20% of their initial weight during the first treatment week and were euthanized for humane reasons on study Day 8. They showed no clinical signs of toxicity. One male showed rales and/or piloerection on treatment Days 19-21 (but no weight loss) and was humanely sacrificed on treatment Day 21. One female was euthanized due to delivery difficulties on study Day 37 (post-coitum Day 20) when she showed hunched posture, piloerection and a slightly pale appearance. A few days earlier she showed rales. Between post-coitum Days 17-20 she had lost 6% of her weight and her food consumption was severely reduced. At necropsy, her uterus contained 15 living fetuses (all without abnormalities) and one early resorption. The moribundity of these four animals was due to erosion/ulceration (marked to massive) and associated inflammation (moderate to marked) in the esophagus.
Another female was found dead on study Day 41. At dosing on the previous day, it was noted that she was no longer pregnant (total litter loss). No signs of toxicity or difficult parturition were noted and her body weight gain and food consumption were normal. Necropsy findings consisted of advanced autolysis and a reduced size of the spleen. At microscopic examination, moderate inflammation, including foci with mineralization, was noted at the hilus of the lung (i.e. at the visceral pleura side, outside the lung parenchyma). This was regarded as the cause of her death. Despite the fact that no lesions were noted in the esophagus of this animal (most likely dependent on the plane of section), the inflammation at the lung hilus most likely reflects an inflammatory process extending from the esophagus (like noted in the other early deaths).
Additionally, decedents showed an increase in granulocytes in the bone marrow of sternum and femur which was moderate or marked in the males and minimal in one female. Bone marrow of the female found death showed no abnormalities (sternum) or was autolytic (femur).
Most of the remaining morphologic alterations in decedents were similar as in survivors and are, therefore, presented together with those of survivors.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 200 mg/kg, body weight loss occurred in the two males which were euthanized at Day 8 (nearly 20% of their initial weight) and, to a lesser extent, in a surviving male (8% loss between Day 1-8). Two other survivors showed reduced weight gain between treatment Days 1-8. After Day 8, weight gain of 200 mg/kg males was normal. Mean body weights of males at 200 mg/kg did not differ significantly from those of controls.
In females at 200 mg/kg, statistically significantly lower mean body weights (nearly 10% difference from control values) and body weight gain were noted at Days 4 and 7 of the lactation period. The lower mean values resulted from slight weight loss/no weight gain in 3/5 females between lactation Days 1-4 or Days 1-7. Body weights and weight gain of 200 mg/kg females during the pre-mating and gestation periods were considered not to be affected by treatment. The slightly lower mean weight gain (not statistically significant) during the last week of gestation could be explained by the weight loss of one decedent and the lower weight gain of another that had only one implantation site.
No treatment-related changes in body weight (gain) were observed in males and females treated up to 45 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at 200 mg/kg, mean food consumption between treatment Days 1-8 was reduced for the cage housing the two males that were euthanized on Day 8 (differences from controls: about 30 and 20% before and after correction for body weight, respectively). Food consumption of the surviving 200 mg/kg males was considered not to be affected by treatment.
In females at 200 mg/kg, lower mean food consumption values (before and after correction for body weight) were noted throughout the lactation period. Compared to controls, mean absolute food consumption was about 40, 25 and 15% lower during lactation Days 1-4, 4-7 and 7-13, respectively (statistically significant, except for the last interval). The lower mean values resulted from the reduced food consumption of the three 200 mg/kg females which showed slight weight loss/no weight gain in the first week of the lactation period. Food consumption of the other two lactating females of this dose group was normal. Food consumption of 200 mg/kg females during the pre-mating and gestation periods was considered not to be affected by treatment. The slightly lower food consumption (not statistically significant) between Days 17-20 of gestation could be explained by the severely reduced food consumption of one decedent and the lower value for another female which had only one implantation site (her food intake towards the end of gestation was similar to that of the non-pregnant control females).
No treatment-related changes in food consumption before or after correction for body weight were noted in males and females treated up to 45 mg/kg.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In females at 200 mg/kg, the mean number of total white blood cells was statistically significantly higher compared to controls (41% difference). The highest value (exceeding the normal range) was noted in female no. 80 which also had an increased number of neutrophils, which was likely related to the inflammation seen in the esophagus and glandular stomach of this female (at marked and moderate degree, respectively).
No treatment-related changes in hematology parameters were noted in male rats.
Coagulation parameters were considered not to be affected by treatment up to 200 mg/kg.
An isolated, statistically significant difference noted in females (higher activated partial thromboplastin time (APTT) at 45 mg/kg) was considered to be unrelated to treatment due to the lack of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly increased plasma concentrations of chloride were noted in males at 45 and 200 mg/kg (relative differences from controls: 5 and 15%, respectively, exceeding the upper limit of the historical control range ).
A statistically significantly higher mean plasma chloride level was also observed in females at 45 mg/kg (5% difference from controls). It was noted that chloride concentrations in two control females were below the normal range, whereas the values in two 45 mg/kg females (and one 200 mg/kg female) exceeded this range . The lack of a dose-related trend in combination with a slightly low concurrent control value in females makes it less likely that the slightly higher mean chloride value in 45 mg/kg females reflected a test item-related change. Clinical chemistry results showed elevated plasma concentrations of chloride in males at 45 and 200 mg/kg (difference from controls: 5 and 15%, respectively). Plasma chloride was measured by means of an Ion Selective Electrode (ISE) which is not perfectly specific for the chloride ion. Bromide, a constituent of the test item, is known to interfere with the chloride determination, causing falsely high concentrations of chloride (Dimeski et al., 2010). The presence of bromide in plasma of rats treated with the test item was confirmed by bioanalysis (non-GLP). Therefore, the elevated chloride levels noted in treated rats were considered to be the result of interference of the analysis by bromide rather than an adverse effect of the test item on plasma chloride.
The other statistically significant variations noted in clinical chemistry parameters were regarded as not toxicologically relevant due to direction of the change (lower bilirubin at 200 mg/kg in females), or unrelated to treatment due to the lack of a dose-related trend (lower inorganic phosphate at 45 mg/kg in males, higher potassium at 10 and/or 45 mg/kg in both sexes).
Mean serum T4 levels of F0-males were statistically significantly decreased at 45 and 200 mg/kg (relative differences from controls: 27 and 38%, respectively). The mean values at these dose levels were below the historical control range. Mean serum T4 levels of F0-females were statistically significantly decreased at 10, 45 and 200 mg/kg (relative differences from controls: 28%, 26% and 38%, respectively). The mean T4 values at dose 200 mg/kg were in the lower limit of the historical control range.
For mean serum TSH levels for F0-males a dose related trend is observed with a statistically significant increase at 200 mg/kg (relative difference from control: x2.7 fold). The mean values at these dose levels were within the historical control range . For F0-females, no statistically significant changes were observed for mean serum TSH levels. All values were within the historical control range.
Historical control data:
chloride (mmol/L) in fasted male Wistar Han rats (period 2015 - December 2017): mean = 102, P5 - P95 = 100 - 105 (n=304)
chloride (mmol/L) in non-fasted female Wistar Han rats (period 2017 - wk 02 2018): mean = 102, P5 - P95 = 99 - 104 (n=55)
T4 (µg/dL) in Wistar Han rats (period 2015 - December 2017): male rats: mean = 4.65, P5 - P95 = 3.01 - 6.52 (n=724); female rats: mean = 3.45, P5 - P95 = 1.70 - 5.06 (n=55).
TSH (µIU/mL) in Wistar Han rats (period 2015 - December 2017): male rats: mean = 0.170, P5 - P95 = 0.044 - 0.462 (n=75). female rats: mean = 0.256, P5 - P95 = 0.045 - 0.724 (n=45).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were noted for functional observations up to 200 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was considered not to be affected by treatment. It was noted that mean hind limb grip strength in 200 mg/kg males was 24% lower than that of concurrent controls. This intergroup difference was not attributed to treatment as it was not statistically significant and all values at 200 mg/kg remained in the historical control range.
The variation in motor activity did not indicate a relation with treatment. All examined groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

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):

Test item-related microscopic findings were observed in the thyroid gland of males and in the thymus, stomach, esophagus, trachea and/or lung of both sexes.
Thyroid gland:
Follicular cell hypertrophy was noted at increased incidence and severity (up to slight) at 200 mg/kg in males.
Thymus:
Lymphoid atrophy was noted at increased severity at 200 mg/kg in both sexes. Severities were up to slight in scheduled sacrificed rats and up to marked in the decedents.
Forestomach:
Lesions were noted up to slight degrees at 200 mg/kg in males and, less frequently, females, and consisted of erosion/ulceration, squamous cell hyperplasia, inflammatory cell infiltrate and (sub) mucosal edema. The lesions occurred in decedents and survivors.
Glandular stomach:
Erosion/ulceration, inflammatory cell infiltrate and edema, all up to moderate severities, were noted at 200 mg/kg in one female (survivor) and a few males (one decedent and two survivors). Additional findings consisted of minimal mucosal hypertrophy in both males that were euthanized on Day 8, and slight hemorrhages in a few rats (two decedents, one survivor).
Esophagus:
Erosion/ulceration (marked or massive) and inflammation (moderate or marked) were noted at 200 mg/kg in a few males (decedents) and females (one survivor, one decedent). Myofiber degeneration (minimal) was noted in one surviving 200 mg/kg male.
Trachea:
Erosion/ulceration (moderate) and/or inflammatory cell infiltrate (up to slight) were noted in a few 200 mg/kg females (one survivor, both decedents). Epithelial alteration (loss of microvilli, up to slight) was noted at 200 mg/kg in one male (decedent) and three females (two survivors, one decedent).
Lung:
Two males (decedents) and a few females (one survivor, two decedents) at 200 mg/kg showed erosion/ulceration (slight) and hyperplasia/hypertrophy of the bronchus epithelium (minimal), acute bronchioalveolar inflammation (slight), inflammatory cell infiltrate of the lung hilus (up to moderate) and/or presence of granuloma (slight).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment.
Except for one 200 mg/kg female which was acyclic, all females had regular cycles, mostly of four days. The one female had a normal precoital interval (evidence of mating after one day of cohabitation) and a normal litter. Moreover, there were no treatment-related changes in the morphology of the female reproductive tract. Therefore, the acyclicity of this female was considered not to reflect an effect of the test item on estrous cyclicity. Extended di-estrus during pairing occurred in two control females (one which was not pregnant and one with a normal litter), one female at 10 mg/kg (normal litter) and one female at 200 mg/kg (not mated). For the female at 10 mg/kg, the extended di-estrus could be explained by the continuation of vaginal lavages after mating had occurred (mating was overlooked). The extended di-estrus at 200 mg/kg was not attributed to treatment due to its incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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

Reproductive performance:

no effects observed

Description (incidence and severity):

One couple at 200 mg/kg showed no evidence of mating. The females of four couples were not pregnant despite evidence of mating: two of the control group, one at 45 mg/kg and one at 200 mg/kg. No abnormalities were seen in the reproductive organs which could account for their unsuccessful mating/non-pregnancy.
One female at 200 mg/kg had one implantation site but no offspring. The hemorrhagic debris in the uterus lumen and mucification of the vagina epithelium might be related to, but cannot explain, the lack of offspring. It was noted that the male of this couple was also paired with another female which showed no evidence of mating.
One female at 200 mg/kg showed delivery difficulties and was therefore euthanized. Her difficult parturition was considered to be related with a poor health condition caused by a severe esophagus lesion.
Another female at 200 mg/kg had total litter loss at PND 1 and was found dead the next day. There was no evident cause for the total litter loss. It might be explained by the noted inflammatory cell infiltrate with mineralization of the lung hilus, which could represent an extending inflammation from the esophagus.

Details on results (P0)

Mating index was considered not to be affected by treatment. Except for one female of the 200 mg/kg group, all females showed evidence of mating. The mating indices were 90% for the 200 mg/kg group and 100% for the other groups. This incidental case of unsuccessful mating, without supportive morphological changes in reproductive organs, was considered to be unrelated to treatment.
Precoital time was not affected by treatment. All mated females treated with the test item showed evidence of mating within four days. This was also true for most females of the control group. Two control females showed evidence of mating at the end of the 14-day mating period.
Number of implantation sites was considered not to be affected by treatment. One pregnant female at 200 mg/kg had only one implantation site (no offspring). Such a low number of implantation sites occasionally occurs in untreated controls. The other pregnant females at 200 mg/kg had normal numbers of implantation sites. Therefore, the low number of implantation sites in the one female was regarded as unrelated to treatment.
Fertility index was not affected by treatment. Except for two control females, one female at 45 mg/kg and one female at 200 mg/kg, all mated females were pregnant. The fertility indices were 80, 100, 90 and 89% for the control, 10, 45 and 200 mg/kg groups, respectively. These incidental cases of non-pregnancy, all without related histopathology changes in reproductive organs, were considered to be unrelated to treatment as their incidence showed no dose-related trend.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) atnges caused moribundity/mortality of five animals

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

lungs

oesophagus

stomach

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
One pup at 200 mg/kg that was euthanized for humane reasons at PND 13 showed abnormal gait and hypotonia of the left hind leg on the day of euthanasia. As these abnormalities were not observed in any other pups of females administered the test item, they were considered not to be test item-related.
The nature and incidence of the other clinical signs observed incidentally (alopecia) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Gestation index and duration of gestation was not affected by treatment at dose 10 and 45 mg/kg. Gestation index was 100% in the control, 10 and 45 m/kg groups. All pregnant females of these groups had living pups.
A low gestation index (63%) was noted at 200 mg/kg. At this dose level, 3/8 pregnant females had no live offspring: one had implantation sites only; a second one had total litter loss at PND 1 (she was found dead the next day); a third female was euthanized at post-coitum Day 20 due to delivery difficulties (at necropsy, her uterus contained 15 living foetuses, all without abnormalities). Two had adverse changes in the lungs or esophagus causing their death/moribundity.
A low (60%) post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was noted at 200 mg/kg. This was due to the high numbers of unaccounted for sites (number of implantation sites minus total number of pups born) of two females:one (13 implantation sites, no pups found) and another (euthanized due to delivery difficulties; 16 implantation sites, no pups delivered, 15 living pups in uterus at necropsy). These females had adverse changes in the lungs or esophagus causing their death/moribundity. The other 200 mg/kg females had no or very low numbers of unaccounted for sites. Post-implantation survival was normal at the dose 10 and 45 mg/kg. The indices were 93, 91 and 93% at 0, 10 and 45 mg/kg, respectively.
For one female at 45 mg/kg the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99% for the 45 mg/kg group and 100% for dose 10 mg/kg.
At first litter check, one pup of the 45 mg/kg group was found dead. This incidental pup mortality was unrelated to treatment. One female at 200 mg/kg had total litter loss at PND 1 (as no pups of this female were found, she was not included in the calculation of the live birth index).
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. No pups died on PND 2-4, resulting in a viability index of 100% for all groups.
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. The lactation index was 100% for dose 10 and 45 mg/kg.
One pup of at 200 mg/kg was euthanized for humane reasons at PND 13. This incidental breeding loss was within the range considered normal for pups of this age and judged to be unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of male and female pups was reduced at 200 mg/kg throughout the lactation period, resulting in 19 % (males) or 24% (females) lower mean body weights at PND 13 (statistically significant). At birth (PND 1), mean body weights of 200 mg/kg pups, particularly of females, were slightly lower than those of control pups (relative differences: 5% (males) and 9% (females); not statistically significant).
Body weights of pups at 10 and 45 mg/kg were considered not to be affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female PND 14-16 pups at 45 mg/kg showed statistically significantly lower serum T4 levels than control pups (relative differences: 22% (males) and 25% (females). The mean T4 values at 45 mg/kg and 200 mg/kg were slightly below the historical control range.
A dose related trend for increased mean serum TSH levels was noted for male PND 14-16 pups, but no statistical significance was achieved (relative differences: 13%, 29% and 36% for dose 10, 45 and 200 mg/kg, respectively). All mean serum TSH levels were within the historical control range . Mean serum TSH levels for female PND 14-16 pups up to dose 45 mg/kg remained within the historical control range and were out of this range at dose 200 mg/kg.

Historical control data for serum total T4 (µg/dL) in PND 14-16 pups (period 2015- January 2018):
male PND 14-16 pups: mean = 6.26, P5 - P95 = 4.59 - 8.48 (n=83).
female PND 14-16 pups: mean = 5.81, P5 - P95 = 4.67 - 8.06 (n=83).

Historical control data for serum TSH (µIU/mL) in PND 13-15 pups (period 2015- January 2018):
male PND 13-15 pups: mean = 0.148, P5 - P95 = 0.047 - 0.327 (n=28).
female PND 13-15 pups: mean = 0.136, P5 - P95 = 0.064 - 0.210 (n=26).

No changes were noted in serum T4 levels for PND 4 pups.

Details on results (F1)

Other endpoints:
Sex ratio:
Sex ratio was considered not to be affected by treatment.
The statistically significant difference noted at 45 mg/kg (%males/females 44/56 versus 62/38 in the control group) was due to the unusually high proportion of male pups in the control group, i.e. at the upper end of the historical control range (Beekhuijzen et al., 2014). Sex ratio in all treated groups remained well within this range.

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

Areola/nipple retention:
Treatment up to 45 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Any other information on results incl. tables

Dose formulation analysis:

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation. It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks.The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation≤ 10%).

Bromide in plasma:

A dose dependent increase in bromide concentration was observed. Bromide levels were below the detection limit of 50 µg/mL in male control animals, one hour after dosing on Day 29 of treatment. In 4 out of 5 tested male animals treated with 10 mg/kg, bromide concentrations were slightly above the detection limit of 50 µg/mL with bromide concentrations between 51.1 and 54.9 µg/mL measured one hour after exposure on Day 29 of treatment. Bromide levels increased to on average 236 µg/mL (about 4.5x higher compared to dose 10 mg/kg) when animals were treated with 45 mg/kg and to 855 µg/mL (about 16.4x higher compared to dose 10 mg/kg) when treated with 200 mg/kg.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of N-BROMOSUCCINIMIDE were established:
Parental NOAEL: 45 mg/kg bw/d, based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) at 200 mg/kg bw/d. These changes caused moribundity/mortality of five animals.
Reproduction NOAEL: at least 200 mg/kg bw/d.

Executive summary:

A 28 -day repeated dose toxicity study combined with screening for reproduction/developmental toxicity was performed in Wistar Han rats according to OECD 422 and GLP. Rats were exposed to 0, 10, 45 or 200 mg/kg bw/d N-bromosuccinimide in water.

Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy. Females that delivered were treated for 50 - 55 days (most females) or 64 days (one control female), 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 42 - 55 days.

The following parental parameters were evaluated in this study: mortality/moribundity, clinical signs,functional tests,body weight, food consumption, estrous cycle length and regularity,clinical pathology, plasma concentration of bromide (non-GLP),serum level of thyroid hormones T4 and TSH (F₀-rats of both sexes), gross necropsy findings, organ weights and histopathologic examination.

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 weight, anogenital distance, areola/nipple retention, serum levels of thyroid hormones (T4 in PND 4 and PND 14-16 pups, TSH in PND 14-16 pups) and macroscopy).

Formulation analysis showed that formulations were prepared accurately and homogeneously.

At 200 mg/kg bw/d , there were five test item-related unscheduled deaths (two males on Day 8, one male on Day 21, one female on Day 37 and one female on Day 41 of treatment). Four of these were due to erosion/ulceration (marked to massive) and associated inflammation (moderate to marked) of the esophagus and one was related to inflammation of the lung hilus (outside of the lung; this likely reflected an inflammatory process extending from the esophagus). Most of the unscheduled deaths showed weight loss and reduced food consumption and two of them showed clinical signs of toxicity (rales, piloerection, hunched posture and/or a pale appearance).   

Slight weight loss/reduced weight gain and reduced food consumption also occurred in a few surviving 200 mg/kg bw/d males (in the first treatment week) and females (in the first week of the lactation period), one of which had severe lesions in the stomach and esophagus.

Slight salivation after dosing,regarded as a physiological response related to the irritant properties of the test item rather than a sign of systemic toxicity, was noted in all rats at 200 mg/kg bw/d. Occasionally, rales were noted in 200 mg/kg bw/d females.

Major microscopic findings consisted of lesions indicative of irritating properties of the test item. Lesions were noted in the esophagus, stomach, trachea and lung of 200 mg/kg bw/d treated rats and consisted of:

·    Erosion/ulceration of protective epithelial layers of esophagus, stomach, trachea and bronchus. The most severe lesions occurred in the esophagus (up to massive). Related gross lesions in the esophagus consisted of adhesions and thickened wall.

·    Concomitant inflammation/inflammatory cell infiltrate and/or edema of these organs and their surrounding areas (like lung hilus).

·    Regeneration-like processes, such as hyperplasia of the forestomach epithelium, hypertrophy of the glandular mucosa, epithelial alteration of the trachea epithelium (loss of villi) and hyperplasia/hypertrophy of the bronchus epithelium. Related gross lesions were noted in the forestomach (irregular surface, dark red foci).

In general, these findings were most obvious in the premature decedents. One surviving female also showed severe lesions.

Most likely the test item caused irritation by direct exposure in the stomach or indirectly by reflux/regurgitation. This reflux might be due to a combination of a slightly high dose volume (15 ml/kg body weight) and (irritant) test item properties. Given the nature and high severities of the erosion/ulceration and inflammation and the relation with premature deaths, including possible relationship with total litter loss and delivery problems, these findings were regarded as adverse.

Increased severity of lymphoid atrophy in the thymus(related with reduced size/gelatinous and lower thymus weights) and an increase in granulocytes in bone marrow were noted in a few males and females at 200 mg/kg bw/d. Since these findings occurred only in rats with severe esophagus lesions (premature decedents and a surviving female) they might be interpreted as secondary effects due to stress. 

A subtle increase in the incidence and severity of follicular cell hypertrophy was noted at 200 mg/kg bw/d in males. This finding was regarded as non-adverse based on the low severity (up to slight) and absence of any degenerative change in the thyroid. 

Serum levels of T4 were decreased in males at 45 and 200 mg/kg bw/d (by about 30 and 40%, respectively).  Serum T4 levels of F₀-females were decreased at 10, 45 and 200 mg/kg bw/d (by about 28%, 26% and 38%, respectively). TSH levels were increased of F₀-males at dose 200 mg/kg bw/d (by about 2.7 fold) and no changes in TSH levels were noted for F₀-females. Possible adversity of the effects on thyroid hormone levels at 200 mg/kg bw/d could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL. 

Hematology results showed an increase in total white blood cells in females at 200 mg/kg bw/d. The increase was most marked in the female that had inflammation in the esophagus (marked) and stomach (moderate). She also had an increased number of neutrophils. 

Clinical chemistry results showed elevated plasma concentrations of chloride in males at 45 and 200 mg/kg bw/d (difference from controls: 5 and 15%, respectively). This was considered to be the result of interference of the analysis by bromide (since this is a compound of the test item) rather than an adverse effect of the test item on plasma chloride.

The following parental NOAEL for N-bromosuccinimide was established: 45 mg/kg bw/d, based on adverse changes in the esophagus, stomach, trachea and lung (ulceration/erosion of protective epithelial layers and associated inflammation) at 200 mg/kg bw/d.  These changes caused moribundity/mortality of five animals. Based on no effects on reproduction observed, the NOAEL for reproduction is 200 mg/kg bw/d.