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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Cross-reference 1

Reason / purpose for cross-reference:

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD422

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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

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

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

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

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

ophthalmological examination

Key result

Abnormalities:

no effects observed

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

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

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

no macroscopic findings

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

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
	F	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

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)

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)

Deviations:

no

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: 1-APR-2021
- Purity test date: 09-APR-2018

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han rat is an accepted rodent species for regulatory toxicity testing. The laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 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

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.

Analytical verification of doses or concentrations:

yes

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 / 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 dose selection was defined following a 10-day dose range-finding study at doses of 500 and 1000 mg/kg/day
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: parental males were fasted overnight before sample collection and necropsy. Parental females were not fasted overnight.

Examinations

Parental animals: Observations and examinations:

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

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 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, but only 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 Repeated dose toxicity summary (IUCLID section 7.5.1) 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 Repeated dose toxicity summary (IUCLID section 7.5.1) 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

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Estrous cycle determination was performed during pre-test period in all females. A total of 40 females with regular estrous cycles were selected for the study.
The vaginal cytology of samples obtained daily by vaginal lavage was examined, 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.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testis weight, epididymis weight
A qualitative evaluation of the testis was conducted on H&E stained sections from all control and high dose animals. Testes were evaluated to assess the progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis). Any cell- or stage-specificity of testicular findings were noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter were kept (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other.
Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities; possible cause of death was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 29 days of treatment
- Maternal animals: All surviving animals on PND13

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in attachment were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 14-16 days of age.
- These animals were subjected to postmortem macroscopic examinations

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGHTS: No

Blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin (not examined). The pups selected for blood sampling were the same pups as selected for thyroid preservation.

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.

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: P0 (first parental generation)

General toxicity (P0)

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.

Food efficiency:

not examined

Haematological findings:

no effects observed

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.

-------------------------------------------------
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

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.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. 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 1 male from the high dose group, Male No. 32).
Male No. 32 had one small epididymis without sperm (reduced sperm, grade 5) related with agenesis of one testis. The contralateral organs were normal. This was considered as an incidental finding, unrelated to treatment with the test item.

Reproductive performance:

no effects observed

Description (incidence and severity):

No effects on mating index (100%), precoital time (all females showed evidence of mating within 13 days), number of implantation sites, fertility index, gestation index, duration of gestation. (Table 1)

Details on results (P0)

Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Litter data: Table 2

Effect levels (P0)

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 (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

eye

Organ:

retina

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For one pup at 1000 mg/kg/day, the left hind leg was paralyzed. The nature and incidence of this finding was considered within background range for pups of this age, and was therefore considered not to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Summary in Table 3.
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.

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No effect observed on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

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 2)
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.

- Thyroid hormone levels (serum T4) in male and female pups PND 14-16 (Table 4): no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1 - Reproduction parameters

	group 1 controls	group 2 100 mg/kg/day	group 3 300 mg/kg/day	group 4 1000 mg/kg/day
Females paired	10	10	10	10
Females mated	10	10	10	10
Pregnant females	10	9	10	8
Females with living pups on day 1	10	9	10	8
Mating index (%)	100	100	100	100
Fertility index (%)	100	90	100	80
Gestation index (%)	100	100	100	100
Median precoital time (days)	2	3	3	4
Mean precoital time (days)	2.0	3.6	2.3	4.8
Duration of gestation (days)	21.4	21.6	21.8	21.3
Number of implantation sites	130	115	132	102
Mean number of implantation sites	13.0	12.8	13.2	12.8

       Historical control - Fertility index (%): mean = 95 ; P5 - P95 = 80 - 100 (n=120)

Table 2 - 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 3 - 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
	F	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

Table 4: summary of T4 analyses in PND 14-16 pups

		Males				Females
Dose levels	mg/kg/day	0	100	300	1000	0	100	300	1000
number of animals	N	10	9	10	8	10	8	10	8
Total  T4 µg/dL	mean SD	6.18 1.19	6.09 0.73	5.96 1.00	5.89 1.13	5.62 1.13	6.20 1.08	6.03 0.73	5.51 0.42

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

Historical control data for Wistar Han rats PND14 -16 pups (period 2017-2018):

Total T4 (µg/dL), males: mean = 5.93; P5-P95 = 4.28 - 8.03 (males, n = 499).

Total T4 (µg/dL), females: mean = 5.57; P5-P95 = 4.04 - 7.50 (females, n = 499).

Applicant's summary and conclusion

Conclusions:

The study did not show toxicologically significant changes in the reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
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)