N'-(1,3-dimethylbutylidene)-3-hydroxy-2-naphthohydrazide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Treatment with BMH by oral gavage in male and female Wistar Han rats according to OECD guideline 421 at dose levels of 50, 150 and 1000 mg/kg revealed parental toxicity at 150 and 1000 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2012-May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) approximately 11 weeks.
- Fasting period before study: no
- Housing: pre-mating: housed in groups of 5 animals/sex/cage in Macrolon plastic cages; mating: one male and one female in Macrolon plastic cages; post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages; Lactation: Pups were kept with the dam until termination in Macrolon plastic cages.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap-water.
- Acclimation period: at least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 January 2013 To:18 February 2013

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Dose volume: 5 mL/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe and on a
28-day toxicity study in rats (NOTOX Project 300623).

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occured
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on two occasions (18 January and 07 February 2013) during the treatment phase on samples according to a validated method (NOTOX Project 300689). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

once daily for 7 days per week

Remarks:

Doses / Concentrations:
50, 150, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 28-day toxicity study in rats (NOTOX Project 300623). In that study, dose levels of 50, 150 and 1000 mg/kg were tested. The low dose of 50 mg/kg was defined as the NOAEL. At 150 mg/kg, effects included decreased alanine aminotransferase activity and increased cholesterol and bilirubin values. At 1000 mg/kg, additional findings included decreased prothrombin time and glucose values, increased aspartate aminotransferase enzyme activities, increased (relative) kidney and liver weights and microscopic evidence of midzonal/centrilobular hypertrophy in the liver. Cortical hyaline droplets in kidneys were seen at all dose levels but were regarded as non-adverse as they were not accompanied by microscopic evidence of kidney damage. In addition, based on the findings in the 28-day toxicity study blood sampling for clinical biochemistry and weight determination of the liver and kidneys were added to the repro screening study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: YES
- Time schedule: 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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: Blood samples were collected on the day of necropsy from 5 selected animals/sex/group under anaesthesia using isoflurane for determination of the following clinical biochemical parameters: ALAT, ASAT, ALP, total protein, Alb, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorg P.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

Yes, PND 1-4 (mortality/viability, clinical signs, body weights, sex)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: all surviving animals on lactation days 5-7

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Epididymides, kidneys, liver and testes were weighed from all F0 animals on the scheduled day of necropsy.
Histopathology was performed on all organs/tissues according to OECD guidelines, including liver and kidneys.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (number of females mated/ number of females paired) x 100
Fertility index (%) =(number of pregnant females/number of females paired) x 100
Conception index (%) = (number of pregnant females/number of females mated) x 100
Gestation index (%) = (number of females bearing live pups/number of pregnant females) x 100
Duration of gestation = number of days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

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
Percentage of postnatal loss = (number of dead pups before planned necropsy/number of live pups at first litter check) x 100
Viability index = (number of dead pups before planned necropsy/number of pups born alive) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Salivation seen after dosing for all animals of the treated groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Body weights, body weight gain and food consumption of treated animals remained in the same range as controls over the treatment period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation
sites were unaffected by treatment. One female at 1000 mg/kg was not pregnant; this is within normal limits.

ORGAN WEIGHTS (PARENTAL ANIMALS): At 1000 mg/kg, liver and kidneys weights (absolute and relative to body weight) were statistically significantly increased for both sexes. Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals. Also terminal body weights of females were unaffected up to 1000 mg/kg.

GROSS PATHOLOGY (PARENTAL ANIMALS): A soft yellowish nodule at the epididymides was observed in one control male, one male from the 150 mg/kg bw/day group and 3 males from the 1000 mg/kg bw/day group. In the control male and 2 high dose males histopathological examination showed these nodules to be granulomas. These findings are within normal limits for this type of study, and not considered treatment related.

HISTOPATHOLOGY (PARENTAL ANIMALS): Males: Kidney, tubular basophilia was present at increased incidence and severity in 5/10 (3 minimal, 1 slight, 1 moderate) males at 150 mg/kg and in 8/10 (1 minimal, 5 slight, 2 moderate) males at 1000 mg/kg, compared to minimal degrees in 4/10 control and 1/10 50 mg/kg treated males. Kidney, hyaline droplets were present at increased incidence and/or severity in 10/10 (2 minimal, 4 slight, 4 moderate) males at 150 mg/kg and in 10/10 (7 moderate, 3 marked) at 1000 mg/kg, compared to 4/10 (3 minimal, 1 slight) control and 10/10 (6 minimal, 4 slight) 50 mg/kg treated males. Kidney, inflammation lymphocytic was present in 4/10 (4 minimal) males treated at 1000 mg/kg.
Females: Kidney, hyaline casts were present at increased incidence and severity in 3/10 (3 slight) females treated at 1000 mg/kg, compared to 1/10 females (1 minimal) treated at 150 mg/kg.
Males and females: Liver, centrilobular hypertrophy was present in 3/10 males (3 minimal) at 150 mg/kg and in 9/10 males (7 minimal, 2 slight) and 8/10 females (8 minimal) treated at 1000 mg/kg.
Other findings of note: Kidney, adenoma, bilateral, was present in 1/10 1000 mg/kg treated male. This adenoma resembled the amphophilic-vacuolar variant, which is a spontaneously occurring renal tumor type and not chemically induced.

OTHER FINDINGS (PARENTAL ANIMALS): clinical biochemistry: At 1000 mg/kg, clinical biochemistry parameters were affected by treatment. Statistically significant
changes at the high dose included increased levels of total bilirubin, cholesterol and inorganic phosphate for males, and increased concentration of total bilirubin for females. The statistically significant changes noted for aspartate aminotransferase (high dose males) and urea (mid dose females) were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. In addition, the increased levels for alkaline phosphatase (not statistically significant) for females of all treated groups when compared to the control group were caused by a relatively low mean value of the concurrent control group, and as all values fell within normal limits, were not considered toxicologically relevant.

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg)

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
Mortality: One pup at 50 mg/kg, five pups at 150 mg/kg and six pups at 1000 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a clear dose-related trend and remained within the range considered normal for pups of this age.
Observations: Incidental clinical symptoms of pups consisted of cold or pale appearance, black discolouration of the abdomen, and scabbing of the head (confirmed at macroscopic examination). Autolysis was noted for two dead pups. The nature and incidence of these findings remained within the range considered
normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Accuracy of preparation

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with the target concentration (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulations.

Homogeneity

The formulations of Group 2 and Group 4 were determined to be homogeneous (i.e. coefficient of variation ≤10%).

Stability

Analysis (after storage) of the Group 2 and Group 4 formulations yielded a relative difference of ≤10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

Treatment with BMH by oral gavage in male and female Wistar Han rats according to OECD guideline 421 at dose levels of 50, 150 and 1000 mg/kg revealed parental toxicity at 150 and 1000 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day.

Executive summary:

In a study according to OECD guideline 421, BMH was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-46 days).

Parental toxicity was observed at 150 and 1000 mg/kg bw/day. At 150 mg/kg bw/day treatment related changes consisted of histopathological changes in the male kidneys (increased incidence and severity of tubular basophilia and hyaline droplets) and male livers (centrilobular hypertrophy). At 1000 mg/kg bw/day treatment related changes comprised increased levels of total bilirubin, cholesterol and inorganic phosphate for males, and increased concentration of total bilirubin for females. Liver and kidneys weights were increased for both sexes. In addition, microscopic examination revealed morphologic alterations in the kidneys for males (increased incidence and severity of tubular basophilia and hyaline droplets, and lymphocytic inflammation) and females (increased incidence and severity of hyaline casts), and in the livers of both sexes (centrilobular hypertrophy).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg bw/day was derived. The reproduction and developmental NOAEL was considered to be at least 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a study according to OECD guideline 421, BMH was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-46 days).

Parental toxicity was observed at 150 and 1000 mg/kg bw/day. At 150 mg/kg bw/day treatment related changes consisted of histopathological changes in the male kidneys (increased incidence and severity of tubular basophilia and hyaline droplets) and male livers (centrilobular hypertrophy). At 1000 mg/kg bw/day treatment related changes comprised increased levels of total bilirubin, cholesterol and inorganic phosphate for males, and increased concentration of total bilirubin for females. Liver and kidneys weights were increased for both sexes. In addition, microscopic examination revealed morphologic alterations in the kidneys for males (increased incidence and severity of tubular basophilia and hyaline droplets, and lymphocytic inflammation) and females (increased incidence and severity of hyaline casts), and in the livers of both sexes (centrilobular hypertrophy).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg bw/day was derived. The reproduction and developmental NOAEL was considered to be at least 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The study is performed according to OECD and OPPTS guidelines and in accordance with GLP principles.

Effects on developmental toxicity

Description of key information

In conclusion, treatment with BMH by oral gavage in male and female Wistar Han rats according to OECD guideline 421 at dose levels of 50, 150 and 1000 mg/kg revealed parental toxicity at 150 and 1000 mg/kg bw/day, with maternal toxicity at 1000 mg/kg bw/day only. No  developmental toxicity was observed for treatment up to and including 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2012-May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

other: Wistar Han

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) approximately 11 weeks.
- Fasting period before study: no
- Housing: pre-mating: housed in groups of 5 animals/sex/cage in Macrolon plastic cages; mating: one male and one female in Macrolon plastic cages; post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages. Lactation Pups were kept with the dam until termination in Macrolon plastic cages.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap-water.
- Acclimation period: at least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 January 2013 To:18 February 2013

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on two occasions (18 January and 07 February 2013) during the treatment phase on samples according to a validated method (NOTOX Project 300689). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occured
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum

Duration of treatment / exposure:

Males were exposed for 29 days. Females were exposed for 41-46 days.

Frequency of treatment:

once daily for 7 days per week

Duration of test:

Until day 4 of lactation.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on a 28-day toxicity study in rats (NOTOX Project 300623). In that study, dose levels of 50, 150 and 1000 mg/kg were tested. The low dose of 50 mg/kg was defined as the NOAEL. At 150 mg/kg, effects included decreased alanine aminotransferase activity and increased cholesterol and bilirubin values. At 1000 mg/kg, additional findings included decreased prothrombin time and glucose values, increased aspartate aminotransferase enzyme activities, increased (relative) kidney and liver weights and microscopic evidence of midzonal/centrilobular hypertrophy in the liver. Cortical hyaline droplets in kidneys were seen at all dose levels but were regarded as non-adverse as they were not accompanied by microscopic evidence of kidney damage. In addition, based on the findings in the 28-day toxicity study blood sampling for clinical biochemistry and weight determination of the liver and kidneys were added to the repro screening study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: YES
- Time schedule: 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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: Blood samples were collected on the day of necropsy from 5 selected animals/sex/group under anaesthesia using isoflurane for determination of the following clinical biochemical parameters: ALAT, ASAT, ALP, total protein, Alb, total bilirubin, urea, creatinine, glucose, choleaterol, sodium, potassium, chloride, calcium, inorg P.
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Epididymides, kidneys, liver and testes were weighed from all F0 animals on the scheduled day of necropsy.
Histopatholy was performed on all organs/tissues according to OECD guidelines.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were
evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Indices:

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
Percentage of postnatal loss = (number of dead pups before planned necropsy/number of live pups at first litter check) x 100
Viability index = (number of dead pups before planned necropsy/number of pups born alive) x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 1000 mg/kg treatment related changes comprised increased concentration of total bilirubin for females. Liver and kidneys weights were increased for both sexes. In addition, microscopic examination revealed morphologic alterations in the kidneys for females (increased incidence and severity of hyaline casts), and in the livers of both sexes (centrilobular hypertrophy).

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg)

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Accuracy of preparation

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with the target concentration (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulations.

Homogeneity

The formulations of Group 2 and Group 4 were determined to be homogeneous (i.e. coefficient of variation ≤10%).

Stability

Analysis (after storage) of the Group 2 and Group 4 formulations yielded a relative difference of ≤10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

In conclusion, treatment with BMH by oral gavage in male and female Wistar Han rats according to OECD guideline 421 at dose levels of 50, 150 and 1000 mg/kg revealed parental toxicity at 150 and 1000 mg/kg bw/day, with maternal toxicity at 1000 mg/kg bw/day only. No developmental toxicity was observed for treatment up to and including 1000 mg/kg bw/day.

Executive summary:

In a study according to OECD guideline 421, BMH was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-46 days).

Parental toxicity was observed at 150 and 1000 mg/kg bw/day. At 150 mg/kg bw/day treatment related changes consisted of histopathological changes in the male kidneys (increased incidence and severity of tubular basophilia and hyaline droplets) and male livers (centrilobular hypertrophy). At 1000 mg/kg bw/day treatment related changes comprised increased levels of total bilirubin, cholesterol and inorganic phosphate for males, and increased concentration of total bilirubin for females. Liver and kidneys weights were increased for both sexes. In addition, microscopic examination revealed morphologic alterations in the kidneys for males (increased incidence and severity of tubular basophilia and hyaline droplets, and lymphocytic inflammation) and females (increased incidence and severity of hyaline casts), and in the livers of both sexes (centrilobular hypertrophy).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg bw/day was derived, with a maternal NOAEL of 150 mg/kg bw/day. The reproduction and developmental NOAEL was considered to be at least 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a study according to OECD guideline 421, BMH was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-46 days).

Parental toxicity was observed at 150 and 1000 mg/kg bw/day. At 150 mg/kg bw/day treatment related changes consisted of histopathological changes in the male kidneys (increased incidence and severity of tubular basophilia and hyaline droplets) and male livers (centrilobular hypertrophy). At 1000 mg/kg bw/day treatment related changes comprised increased levels of total bilirubin, cholesterol and inorganic phosphate for males, and increased concentration of total bilirubin for females. Liver and kidneys weights were increased for both sexes. In addition, microscopic examination revealed morphologic alterations in the kidneys for males (increased incidence and severity of tubular basophilia and hyaline droplets, and lymphocytic inflammation) and females (increased incidence and severity of hyaline casts), and in the livers of both sexes (centrilobular hypertrophy).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg bw/day was derived, with a maternal NOAEL of 150 mg/kg bw/day. The reproduction and developmental NOAEL was considered to be at least 1000 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
The study is performed according to OECD and OPPTS guidelines and in accordance with GLP principles.

Justification for classification or non-classification

Based on the available information on BMH in animal studies, no relevant reproductive or developmental toxicity has been observed. Based on these observations, BMH needs not to be classified for reproductive and developmental toxicity according to

-Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

-Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.