1,3-phenylenebis((4-hydroxyphenyl)methanone)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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-reference

Reason / purpose for cross-reference:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Remarks:

No deviations to the test guideline. Study plan deviations occured during the conduct of the in-life procedure but were not considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-phenylenebis((4-hydroxyphenyl)methanone)

EC Number:

816-325-3

Cas Number:

5436-05-5

Molecular formula:

C20H14O4

IUPAC Name:

1,3-phenylenebis((4-hydroxyphenyl)methanone)

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MMHBB-002/18
- Expiration date of the lot/batch: 01-APR-2021
- Purity test date: 09-APR-2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: a stability testing was performed prior to the study. The formulation in 1% aqueous carboxymethylcellulose was stable for at least 24 hours at room temperature under normal light conditions, 8 days in the refrigerator and 3
weeks in the freezer (≤ -15°C), over the concentration range 1 to 200 mg/mL (suspensions)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

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: 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 oral 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 analytical verification of doses or concentrations:

Duplicate samples were analysed for concentration, and homogeneity.
Concentration analysis: acceptable if mean sample concentration results were within or equal to +/- 15% for suspensions of target concentration (mean accuracies between 85-115%).
Homogeneity analysis: acceptable if the coefficient of variation (CV) of concentrations was <= 10%
Stability was investigated prior to the in-life study. Stability in the vehicle was demonstrated when prepared and stored under the conditions used.

Duration of treatment / exposure:

Males: 29 days (including a minimum of 14 days prior to mating and during the mating period).
Females that delivered were treated for 50-63 days including 14 days prior to mating (to cover at least 2 complete estrous cycles), time to conception, duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver were treated 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, by gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle: 1% aqueous CMC

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the doses selected for the main study were based on a 10-day dose-range finding study at doses of 500 and 1000 mg/kg bw/day.
- Rationale for animal assignment : random
- Fasting period before blood sampling for clinical biochemistry: parental males were fasted overnight prior to blood sampling and necropsy. Parental females were not fasted overnight.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: F0 observation for the general conditions (health, mortality and moribundity) twice daily, in the morning and the end of the working day. Clinical observations were performed once daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration of the test item and at weekly intervals during the treatment period, after dosing (clinical observations in a standard arena)

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

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly except during mating.
Food consumption of mated females will be measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION : Yes, visual inspection of the water bottles on a regular basis during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males: Yes, fasted overnight with a maximum of 24 hours; Females: No
- How many animals: 5/sex/group
- Parameters listed in Table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy, between 7.00 and 10.30 a.m
- Animals fasted: Yes (males only)
- How many animals: 5/sex/group
- Parameters listed in Table No.2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 Males, during the week 4 of treatment, and 5 females during the last week of lactation (PND 10-13). Tests performed after dosing, after clinical observations (including arena observations, if applicable)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex) / fore- and hind-limb grip strength / motor activity (ambulations)

IMMUNOLOGY: No

OTHER:
- SPERMATOGENESIS: Stage dependent qualitative evaluation in the testis
- ESTROUS CYCLE in females evaluated by examining the vaginal cytology of samples obtained daily by vaginal lavage, starting14 days prior to treatment (pre-test period), during 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 estrous.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (tissue list in attachment 1)
HISTOPATHOLOGY: Yes (tissue list in in attachment 1)

Other examinations:

Thyroid Hormone: T4 analysis in serum from all 10 parental males, and from 2 pups/litter on PND4 and PND14-16

Developmental parameters in F1 pups (see results in IUCLID 7.8):
- anogenital distance on PND1 for all live pups;
- areola/nipple retention on PND13 on all males of each litter

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No effect 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, without effect on body weight or body weight gain. The decreased food consumption was considered not toxicologically relevant.
Limited data was available for the evaluation of food consumption between lactation Days 7-13. Based on body weights and body weight gain, food consumption levels were likely not affected between lactation Days 7-13

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Retinal atrophy (minimal degree) was observed in 2 females of the high dose group (1000 mg/kg/day) (Table 2). 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.
There was no such findings in animals of the 2 other dose groups.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Any changes noted in hematology parameters in males achieving a level of statistical significance when compared to controls, were considered unrelated to administration of the test item due to the minimal magnitude of the change and absence of a dose response.
Coagulation parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Clinical biochemistry findings:

no effects observed

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.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observations are summarised in Table 1.
Functional observation parameters were unaffected by treatment with the test item up to 300 mg/kg/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg/day
Females, 1000 mg/kg/day: Grip strength of the hind leg, total movements and ambulations were decreased (0.78x, 0.61x and 0.51x, respectively), with a statistical significance. All values remained within normal range.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Any differences, including those that reached statistical significance (absolute weight of thyroid gland of 1000 mg/kg/day group males, without microscopic correlate) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic findings.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Possible test item-related microscopic findings were noted in the eyes of the 1000 mg/kg/day group females: minimal retinal atrophy was noted in 2 females out of 5 examined. (Table 2)
The retinal atrophy of eyes observed at minimal degree in 1000 mg/kg/day females, is a finding that occasionally can be seen in control rats at very low incidence. In the current study, this lesion was absent in the control group and from the other dose groups, and present at a somewhat higher incidence (2 out of 5) than in the internal historical control range data (i.e.: in females an incidence of 0.6%, at a minimal degree). Therefore a possible relation with treatment with the test item 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 was 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Spermatogenesis: The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present (with the exception of one male of the 1000 mg/kg/day group which had a small epididymis without sperm).

- Estrous cycle: Length and regularity of the estrous cycle were considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Most females had regular cycles of 4 to 5 days. Female Nos. 59 (100 mg/kg/day), 64 and 65 (300 mg/kg/day) had an irregular cycle (all with normal litter). One female (No. 69, 1000 mg/kg/day), had an extended estrus (with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

- Thyroid hormone (Table 3): Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day

Reproduction and developmental data are described in the section 7.8 of IUCLID-Toxicity to Reproduction

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

ophthalmological examination

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

eye

Organ:

retina

Treatment related:

yes

Dose response relationship:

no

Any other information on results incl. tables

Table 1 - Functional observations - grip strength (end of treatment)

		Group 1 Control	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Males
Grip strength forelimb	Mean + SD	1133  + 275	1401  + 168	1281  + 82	1397  + 232
Grip strength hindlimb	Mean + SD	528  + 55	546  + 84	545  + 68	568  + 80
Total movements	Mean + SD	2901 + 788	3587 + 1540	3622 + 958	2638 + 633
Ambulations	Mean + SD	539 + 185	652 + 309	679 + 222	408 + 83
Females
Grip strength forelimb	Mean + SD	1038 + 61	982  + 159	886  + 84	923 + 119
Grip strength hindlimb	Mean + SD	537 + 45	466  + 67	511  + 48	417 + 90 *
Total movements	Mean + SD	3920 + 749	3502 + 1650	3006 + 733	2399 + 336*
Ambulations	Mean + SD	1063 + 169	857 + 391	734 + 177	539 + 88*

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

* indicates a p-value <0.05

Historical control data for female Wistar Han rats (period 2015-2018):

Grip strength hind leg: mean = 560; P5 - P95 = 369 - 789 (n=400).

Total movements: mean = 3372; P5 - P95 = 1529 - 5618 (n=400).

Ambulations: mean = 817; P5 - P95 = 317 - 1440 (n=400).

Table 2: Incidence of retinal atrophy in parental females

		Females
Dose levels	mg/kg/day	0	100	300	1000
number of animals	N	5	5	5	5
Eyes	mean SD	4.02 0.72	4.25 0.59	4.63 1.10	4.33 0.88
Retinal atrophy	grade 1: minimal	-	-	-	2
	Total affected	-	-	-	2
	mean grade	-	-	-	1.0

Table 3: summary of T4 analyses in parental males

		Males
Dose levels	mg/kg/day	0	100	300	1000
number of animals	N	10	10	10	10
Total  T4 (µg/dL)	mean SD	4.02 0.72	4.25 0.59	4.63 1.10	4.33 0.88

Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)

Historical control data for male Wistar Han rats (period 2017-2018):

Total T4 (µg/dL): mean = 4.51; P5-P95 = 2.85 - 6.37 (males, n= 557).

Applicant's summary and conclusion

Conclusions:

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 main finding in parental animals was a retinal atrophy of the eyes in 2 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.

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)