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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): BMH
- Physical state: light brown crystal powder

Test animals

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

Administration / exposure

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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

1

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

2

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

Results (fetuses)

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).

Effect levels (fetuses)

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)

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.