Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
(screening, post-natal development)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-Jan-2019 to 29-Nov-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Toxic effect type:
dose-dependent

The screening study for reproductive / developmental toxicity performed in Wistar rats according to the OECD TG 422 did not show toxicologically significant changes in the reproductive parameters investigated (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The NOAEL for reproductive toxicity was considered to be at least 1000 mg/kg/day.

Endpoint conclusion:
no adverse effect observed
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Effects on the post-natal development were investigated in a screening study for reproductive / developmental toxicity performed in Wistar rats according to the OECD TG 422.

Body weights of female and male pups were decreased from PND 4 at the dose 1000 mg/kg/day, compared to the control group. As no recovery in body weights was or could be observed beyond this time point of development, these decreases were considered adverse.

The NOAEL for developmental toxicity was established at 300 mg/kg/day.

Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, a NOAEL of 300 mg/kg/day was determined considering lower body weights observed in pups at PND14 -16 at the dose 1000 mg/kg/day. There were no other effects considered toxicologically significant.

No hazard classification is considered necessary according to the CLP criteria in EU Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

Reason / purpose:
reference to same study
Reference

In an OECD 422 test guideline study, Wistar Han rats were treated with 1,3-Bis (4-hydroxy benzoyl) benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, 1% aqueous carboxymethyl cellulose, alone. Males were treated for a total of 29 days. Females that delivered offspring were treated for a total of 50-63 days. Females that failed to deliver pups were treated for 43-54 days. The main finding in parental animals was a retinal atrophy of the eyes in 2/5 females at the highest dose level tested (1000 mg/kg/day), at minimal degree. Retinal atrophy is a finding which is occasionally observed in control rats, however, as the lesion was not present in the control group of the current study, a relation with treatment cannot be excluded. Since the eye can be seen as part of the central nervous system with very limited to no regenerative capacity, this finding was considered adverse.

There were no toxicologically significant changes in any reproductive parameters examined.

In the offspring, developmental toxicity was observed at the highest dose level tested (1000 mg/kg/day). Body weights of female and male pups were decreased from PND 4. As no recovery in body weights was or could be observed beyong this time point, these decreases were considered adverse.

A shift towards more males than females was observed at 1000 mg/kg/day and was considered non adverse in absence of effects on anogenital distance, thyroid hormone levels and nipple retention. As the shift towards more males than females was present in most litters (7 out of 8 litters), a test item related effect could not be excluded.

Toxic effect type:
dose-dependent
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable study
System:
eye
Organ:
retina
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Retinal atrophy of the eyes at minimal degree was observed in two females of the high dose group (1000 mg/kg/day).

Retinal atrophy is a finding which is occasionally observed in control rats, however, as the lesion was not present in the control group of the current study, a relation with treatment cannot be excluded. Since the eye can be seen as part of the central nervous system with very limited to no regenerative capacity, this finding was considered adverse. The relevance is unknown.

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, a NOAEL of 300 mg/kg/day was determined considering parental toxicity (retinal atrophy in females) at the dose 1000 mg/kg/day.

No hazard classification is considered necessary according to the CLP criteria in EU Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD422
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Identification: 1,3-Bis (4-hydroxy benzoyl) benzene
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 01 April 2021 (expiry date)
- Batch # MMHBB-002/18

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males were 12-14 weeks old; females were 13-14 weeks old.
- Weight at study initiation: males: 330-371g / females: 199-235g
- Fasting period before study: no
- Housing: up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon plastic cages, MIV type, height 18 cm)
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: males: 8 days before the start of the dosing; females: 8 days prior to start of the pre-test period.

DETAILS OF FOOD AND WATER QUALITY:
Food: Pelleted rodent diet was provided ad libitum; The feed was analyzed by the supplier for nutritional components and environmental contaminants
Water: Municipal tap water was freely available to each animal via water bottles; Periodic analysis of the water was performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 18-24°C; actual daily mean temperatures during the study period was 20-22°C
- Humidity (%): target: 40-70%; actual daily mean relative humidity was 35-54%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

IN-LIFE DATES: From: 13-FEB-2019 (females receipt) To: 09-MAY-2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
dosing formulations were prepared weekly as a suspension, filled out in daily proportions and stored in the refrigerator protected from light. Prior to dosing formulation were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. The dosing formulations were stirred continuously during dose administration.
Adjustment was made for specific gravity of the test item (1.29).
Dose volume was based on most recent body weight measurement.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqueous carboxymethyl cellulose was selected following trials to find the suitable vehicle and formulation procedure.
- Concentration in vehicle: 1% v/v
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no.: 18C01-U02-044711
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days or until detection of mating
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 post-coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, In case less than 9 females per group have shown evidence of mating, each non-mated female may be re-mated once with a male for a maximum of 7 days (if possible). A male of the same group having previously shown evidence of mating (non-selected male if possible, see section 14) will be used for re-mating.
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: none relevant to the mating procedure.
Duration of treatment / exposure:
Males: 29 days
Females that delivered were treated for 50-63 days
Females which failed to deliver were treated for 43-54 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Frequency of treatment:
Daily administration by gavage
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle: 1% aqueous CMC
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health conditions/mortality, in the morning and at the end of the workday

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dosing, then once weekly throuhout treatment. The observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly, except for males and females housed together for mating, and females without evidence of mating

WATER CONSUMPTION AND COMPOUND INTAKE:
- no quantitative investigation done for this study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice of females which delivered: on PND 14-16
- Sacrifice of females which failed to deliver: With evidence of mating: Post-coitum Day 25-27
- Organs examined: see section 7.5.1 and 7.8.1
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Not relevant
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
Not relevant
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric analysis:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-parametric analysis: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test)

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
- Reproductive indices
Mating index (%) (# females mated/ # females paired) *100
Fertility index (%) (# pregnant females / # females mated) *100
Precoital time : Number of days between initiation of cohabitation and confirmation of mating
Gestation index (%) (# females with living pups on Day 1 / # pregnant females) *100
Duration of gestation : Number of days between confirmation of mating and the beginning of parturition

- Offspring viability indices
Post-implantation survival index (%): (total # of offspring born / Total number of uterine implantation sites) * 100
Live birth index (%): (# of live offspring on Day 1 after littering / Total # of offspring born) * 100
Numbers and Percentage live males and females at First Litter Check (%)
Viability index (%): (# of live offspring on Day 4 before culling / # live offspring on Day 1 after littering) * 100
Lactation index (%): (# of live offspring on Day 13 after littering / # live offspring on Day 4 after culling)*100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was unaffected by treatment with the test item up to 300 mg/kg/day.
In females at 1000 mg/kg/day, absolute and relative food consumption levels were decreased (0.82-0.91x compared with controls) during lactation Days 1-7. As differences were only minimal and no changes were observed in body weights or body weight gain, the decreased food consumption was considered not toxicologically relevant.
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Description (incidence and severity)
No toxicologically relevant changes were noted in clinical biochemistry parameters after treatment with the test item up to 1000 mg/kg/day.
In females at 1000 mg/kg/day, urea levels were decreased (0.82x compared with controls) and glucose levels were increased (1.38x). As the opposite direction of change (urea) would be expected
in case of toxicity, the difference was caused by a slightly low control value (glucose) and as mean values remained within normal range, these changes were considered not toxicologically relevant.
-------------------------------------------------
Historical control data for female Wistar Han rats (period 2017-2018):
Urea (mmol/L): mean = 10.1; P5 - P95 = 8.40 - 12.00 (n=210).
Glucose (mmol/L): mean = 7.55; P5 - P95 = 5.84 - 10.16 (n=210)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Retinal atrophy, possibly test item-related, were noted in the eyes of the 1000 mg/kg/day group females (see details in section 7.5.1).
This finding can occasionally be seen in controls as well at very low incidences, but was absent in the concurrent controls. Therefore a possible relation with treatment cannot be excluded. Since the eye, can be seen as part of the central nervous system with very limited to no regenerative capacity, this finding was considered adverse.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
There was 1/10 couples (Male No. 18 and Female No. 58) of the 100 mg/kg/day group and two couples of the 1000 mg/kg/day group (Male No. 36 and Female No. 76, and Male No. 38 and Female No.78) with no offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Details on maternal toxic effects:
Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females.
No deficiencies in maternal care were observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
ophthalmological examination

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Summary in Table 1. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Viability indices were 99, 100, 100 and 99% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
One pup of the control group and one pup at 1000 mg/kg/day were found dead on PND 2. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
- Sex ratio was considered affected by treatment with the test item at 1000 mg/kg/day. (see Table 1)
At 1000 mg/kg/day, a male/female ratio of 62/38 was observed. With the exception of one litter (7 out of 8 litters), all litters of this group had more males than females.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
post-natal pup body weight was lower in the high dose group (Summary in Table 2).
Body weights of pups were considered unaffected by treatment up to 300 mg/kg/day.
Body weights of pups were decreased in both sexes from PND 4 and were statistically significant decreased for female pups to 0.92-0.93x from PND 7 onwards and for male pups to 0.93x compared with controls at PND 13.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
additional data obn post-natal development in section 7.8.1

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
no macroscopic findings

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

Table 1 - Litter data

 

 group 1

controls

 group 2

100 mg/kg/day

 group 3

300 mg/kg/day

 group 4

1000 mg/kg/day

Total number of litters   10  9  10  8
 Dead pups at first litter check  0  0  0  0

 Live pups at first litter check

Total offspring born

118  96  118  90
 Mean litter size+SD  11.8+1.3  10.7+2.1  11.8+1.9  11.3+3.3
 M/F (%)  51/49  45/55  49/51  62/38
 Postnatal loss (Day 0 - 4)  1M  0  0  1M
 Living pups Day 4 (after culling)  80  72  80  60
 Mean litter size+SD  8.0+0.0  8.0+0.0  8.0+0.0  7.5+1.4
 Breeding loss (Day 5 - 13)  0  0  0  0
 Living pups PND 13  80  72  80  60
 M/F (%)  49/51  47/53  53/48  52/48
 Mean litter size+SD   8.0+0.0  8.0+0.0    8.0+0.0  7.5+1.4
 Post-implantation survival index (%)  91  83  89  88
 Live birth index (%)  100  100  100  100
 viability index (%)  99  100  100  99
 Lactation index (%)  100  100  100  100

Table 2 - pups body weights (grams)

 Day  Sex

 

 Group 1

Control

 Group 2

100 mg/kg/day

 Group 3

300 mg/kg/day

 Group 4

1000 mg/kg/day

   Number of litters  10  9 (°)  10  8
 1  M  Mean bw+SD  6.5 + 0.5  6.8 + 0.6  6.8 + 0.5  6.2 + 0.4
   F  Mean bw+SD  6.2 + 0.5  6.3 + 0.4  6.4 + 0.4  6.2 + 0.4
   M+F Mean bw+SD  6.3 + 0.5  6.5 + 0.5  6.6 + 0.5  6.2 + 0.4
 4  M Mean bw+SD  9.9 +1.0  10.3 + 1.1  10.2 + 1.0  9.2 + 0.8
   Mean bw+SD  9.7 + 0.9  9.7 + 0.7  9.8 + 0.9  9.1 + 0.5
   M+F Mean bw+SD   9.8 + 0.9  9.9 + 0.8  10.0 + 1.0  9.2 + 0.6
 7  M Mean bw+SD   16.7 + 1.4  16.5 + 1.2  16.6 + 1.4  15.2 + 1.1
   F  Mean bw+SD  16.3 + 1.2  15.6 + 1.0  16.0 + 1.2  15.0 + 1.1*
   M+F  Mean bw+SD  16.5 + 1.2  16.0 + 0.9  16.3 + 1.2  15.1 + 1.0*
 13  M  Mean bw+SD  32.3 + 2.2  31.4 + 0.9  31.4 + 2.3  29.7 + 1.8*
   F  Mean bw+SD  31.5 + 1.5  30.2 + 1.7  30.7 + 1.5  29.2 + 1.9*
   M+F  Mean bw+SD  31.9 + 1.8  30.8 + 1.1  31.1 + 1.8  29.5 + 1.8*

(°) except for day 1, due to the missing first litter check for one female (mean bw was based on only 8 litters)

* Dunnett-test based on pooled variance significant at 5% level

Applicant's summary and conclusion

Conclusions:
Post-natal development was assessed in this screenig sudy up to PND14. Developmental toxicity was observed at the highest dose level tested (1000 mg/kg/day): Body weights of female and male pups were decreased from PND 4. As no recovery in body weights was or could be observed, these decreases were considered adverse.
A shift towards more males than females was observed at 1000 mg/kg/day and was considered non adverse in absence of effects on anogenital distance, thyroid hormone levels and nipple retention. However, as the shift towards more males than females was present in most litters (7 out of 8 litters), a test item related effect could not be excluded.
Executive summary:

The objectives of this study were to determine the potential toxic effects of 1,3-Bis (4-hydroxy benzoyl)benzene when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the Dose Range Finder.

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

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

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

Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose were prepared accurately and homogenously.

Parental toxicity was observed at the highest dose level tested (1000 mg/kg/day): retinal atrophy of the eyes at minimal degree was observed in two females. Retinal atrophy is a finding which is occasionally observed in control rats, however, as the lesion was not present in the control group of the current study, a relation with treatment cannot be excluded. Since the eye can be seen as part of the central nervous system with very limited to no regenerative capacity, this finding was considered adverse.

At 1000 mg/kg/day, a test item-related decrease in grip strength of the hind leg, total movements and ambulations was observed in females. As all values remained within normal range and in absence of correlating microscopic findings, these findings were considered non adverse.

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

Developmental toxicity was observed at the highest dose level tested (1000 mg/kg/day):Body weights of female and male pups were decreased from PND 4. As no recovery in body weights was or could be observed, these decreases were considered adverse.

A shift towards more males than females was observed at 1000 mg/kg/day and was considered non adverse in absence of effects on anogenital distance, thyroid hormone levels and nipple retention. As the shift towards more males than females was present in most litters (7 out of 8 litters), a test item related effect could not be excluded.

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 (NOAEL) for 1,3-Bis (4-hydroxy benzoyl) benzene were established:

Parental:                                300 mg/kg/day (based on retinal atrophy of the eyes)

Reproduction:                        at least 1000 mg/kg/day

Developmental:                    300 mg/kg/day (based on decreased body weights of pups)