4,4'-bis(diethylamino)benzophenone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2017 - 10 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The following functional groups are common across the target and source substance: aryl groups and carbonyl groups. It is the scientific hypothesis of this read-across justification that the presence of these functional groups dictates the toxicological potential of the target substance. The target and sources substances both pass Lipinski’s rule of five and are therefore considered to have the potential to be absorbed into the body via the oral route. The breakdown products within the body are likely to be similar, or the same, as shown in the table below.
Breakdown products:
Source substance - Benzophenone, Toluene, Hippuric acid or ortho-Cresol
Target substance - Benzophenone, Diethylamine (x2)

The target and source substance are expected to degrade in a similar way within the body and are expected to generate similar if not the same major metabolites. The molecular weight of the target and source substance are similar and are both below 500 daltons. The water solubility and partition coefficients of the target and source substances are comparable. Both the target and source substances pass Lipinski’s rule of five indicating that they may be orally absorbed and therefore are available within the body and may act in a similar toxic way.

2. SOURCE AND TARGET CHEMICAL
The source and target substances are composed of the same functional groups bound together in similar ways. The molecular weight of the target and source substances are similar and they are expected to be absorbed in the body in similar ways. The breakdown products of the substance are expected to be the same or very similar and are expected to have the same potential for acute toxicity.
The target and source substance contains very low levels of unidentified impurities. These impurities are considered not to affect the classification and labelling of the substance due to the very low levels at which they occur. No information on impurities present in the source test materials was available in the literature sources.
The impact of “impurities” is therefore considered not to affect the reliability of the read-across prediction.


3. ANALOGUE APPROACH JUSTIFICATION
Due to the similarities of the source and target substance with regards to physico-chemical properties, Lipinski’s rule of 5, and the fact that the target substance is expected to breakdown into structurally similar molecules in the body, the target substance is expected to behave in a substantially similar manner to the source substance. The target substance is therefore predicted NOAEL 300 mg/kg bw in the OECD 422 study when conducted in the rats. By extension, the target substance is considered not to fulfil the criteria for reproductive toxicity under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

4. DATA MATRIX
See appended full justification document.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: a decrease in the number of implantation sites at 1000 mg/kg.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other: reduced post-implantation survival and decreased live litter size

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-phenylbenzophenone

EC Number:

218-345-2

EC Name:

4-phenylbenzophenone

Cas Number:

2128-93-0

Molecular formula:

C19H14O

IUPAC Name:

{[1,1'-biphenyl]-4-yl}(phenyl)methanone

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Batch No.of test material:
20161118
- Expiration date of the lot/batch:
22 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature protected from light

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
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
not specified - checking with lab.
- Age and weight at study initiation:
males were 10 weeks old and weighed between 273 and 316 g and females were 13 weeks old and weighed between 202 and 248 g.
- Housing:
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam.
- Diet (e.g. ad libitum):
ad libitum
- Water (e.g. ad libitum):
ad libitum
- Acclimation period:
8 days prior to start of the pre-test period (females) or 10 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
18 to 24°C
- Humidity (%):
40 to 70%
- Air changes (per hr):
Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light):
12 hour light/12 hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis for concentration, homogeneity and stability were undertaken on duplicate samples.

Duration of treatment / exposure:

Once daily oral gavage 7 days a week for a minimum of 29 days.
Males were treated for 29 days (2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy).
Females that delivered were treated for 50 - 56 days (most females) or 64 days (one female of Group 3), (14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy).
Females which failed to deliver or had a total litter loss were treated for 39-43 days (Group 4) or 41-54 days (Groups 2 and 3).

Frequency of treatment:

Once daily oral gavage 7 days a week for a minimum of 29 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

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/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes


HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), 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 estrus. This was done for all females, except for one female with total litter loss.

Sperm parameters (parental animals):

Parameters examined in P male parental generation: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter of equal sex; excess pups were killed and discarded. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, anogenital distance (AGD), presence of nipples/areolae in male pups and clinical biochemistry.

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Statistics:

All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted up to treatment with 1000 mg/kg.

Mortality:

no mortality observed

Description (incidence):

One female (no. 73) at 1000 mg/kg was sacrificed at PND 1 due to total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight gain of females at 1000 mg/kg were statistically significantly reduced at Days 17 and 20 of the post-coitum period. At the end of this period, mean body weight of 1000 mg/kg females was 19% lower than that of controls. This could be explained by the different physiological status (former pregnancy but no live offspring).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Females at 1000 mg/kg consumed less food than controls between Days 17-20 of the post-coitum period (statistically significant). This correlated with their lower body weight and was related to the different physiological status (former pregnancy but no live offspring).
No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 1000 mg/kg and females treated at 100 mg/kg.

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

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The lower number of neutrophils and mean corpuscular volume (MCV) noted in 1000 mg/kg females were related to the physiological status of these females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 1000 mg/kg bw, statistically significant differences were noted for alkaline phosphatase (ALP) activity (lower) and chloride (higher). These findings were not accompanied by adverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg bw males remained within the historical control ranges . As such, these clinical pathology changes were regarded as non-adverse.

In lactating females, isolated statistically significant differences were noted for glucose (lower at 100 and 300 mg/kg bw) and sodium (higher at 300 mg/kg bw). These differences were considered to be unrelated to treatment as they showed no dose-related trend (glucose) and/or remained within normal limits.

Clinical chemistry values in females treated at 1000 mg/kg bw showed the following statistically significant differences from concurrent controls, all considered to be related to the difference in physiological status (percentage differences were not calculated due to the difference in physiological status). It cannot be determined if values in 1000 mg/kg bw females remained within the normal range, since no historical control data is available for females with implantation sites only. Query with lab.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Query with lab.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Substance-related microscopic findings were noted in the thyroid glands of both sexes and adrenal glands of females.
In the thyroid gland, follicular cell hypertrophy was observed at increased incidence and severity (up to slight) in 300 mg/kg males and in 1000 mg/kg rats of both sexes.
In the adrenal gland, inflammatory cell infiltrate (minimal) was observed in 1000 mg/kg females. This infiltrate mainly consisted of lymphocytes and was located at the zona fasciculata (and sometimes zona reticularis) of the adrenal cortex.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment.
Any effects seen were incidental and in the absence of a dose-related incidence, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were no morphological findings in the reproductive organs of males which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Couples without offspring at 100 and 300 mg/kg bw:
One couple (out of 10) at 100 mg/kg bw and 1/10 couples at 300 mg/kg bw had no offspring. No abnormalities were seen in the reproductive organs which could account for their lack of offspring.

Couples without offspring at 1000 mg/kg bw:
Females of the 1000 mg/kg group had no offspring (except for one female which had two dead pups at first litter check). There were 2/9 females without evidence of former pregnancy and 7/9 females with evidence of former pregnancy. The latter seven females showed small/old implantation sites, none to minimal lobuloalveolar development of the mammary gland and histologic proof of a normal estrus cycle. The lack of healthy offspring in the 1000 mg/kg bw group was considered to be test item-related.

Mating index and precoital time was not affected by treatment.
The mean number of implantation sites was statistically significantly decreased at 1000 mg/kg bw (9.5 versus 13.0 in controls).
Treatment at 100 or 300 mg/kg bw did not affect the number of implantation sites.
The fertility indices were 100, 90, 90 and 80% for the control, 100, 300 and 1000 mg/kg bw groups, respectively.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: a decrease in the number of implantation sites at 1000 mg/kg.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The single 1000 mg/kg female with offspring had only dead pups (two) at first litter check (total litter loss).
At 300 mg/kg, there were four pups that died postnatally (versus 0 in the control group; this difference was statistically significant).
Two pups went missing (presumably cannibalized) at PND 2 (litter nos. 62 and 69), one was found dead at PND 2 (litter no. 62) and one was euthanized for humane reasons at PND 1 (litter no. 68). At 100 mg/kg, one pup was euthanized for humane reasons (PND 3, litter no. 53). This postnatal loss remained within the range considered normal for pups of this age, however, was in the upper limit of considered normal range and may be test item related together with the other findings in developmental data. Therefore a treatment related effect cannot be excluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg, mean body weights of male and female pups were lower compared to controls from PND 4 (9% difference) until the end of the lactation period (about 17% difference at PND 13). Statistical significance was achieved from PND 7 onwards. Mean body weight of male 300 mg/kg pups was also lower at birth (PND 1) whereas birth weight of female 300 mg/kg pups was close to the control value (8 and 5% difference, respectively).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were judged to be unaffected by treatment up to 300 mg/kg (T4 measurements were not conducted at higher doses).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other: reduced post-implantation survival and decreased live litter size

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Organ:

not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of the substance were established:
Parental NOAEL: at least 1000 mg/kg.
Reproduction NOAEL: 300 mg/kg, based on a decrease in the number of implantation sites at 1000 mg/kg.
Developmental NOAEL: Below 100 mg/kg, based on reduced post-implantation survival and decreased live litter size starting at 100 mg/kg.

Executive summary:

The potential toxic effects of the substance when given orally by gavage for a minimum of 28 days to Wistar Han rats and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development was determined in a 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422. The dose levels were 0, 100, 300 and 1000 mg/kg/day, based on the results of the dose range finder. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). Reproductive toxicity was observed at 1000 mg/kg as indicated by a reduced number of implantation sites. There were no morphological findings in the reproductive organs of either sex which could explain these effects. Developmental toxicity was observed at 100, 300 and 1000 mg/kg.  At 1000 mg/kg, none of the eight pregnant females had healthy offspring. One of them had two dead pups at first litter check and seven had no offspring but showed proof of former pregnancy. At 100 and 300 mg/kg, post-implantation survival index and live litter size were reduced.  In conclusion, based on the results of this study, the parental NOAEL was determined to be at least 1000 mg/kg. Reproduction NOAEL was 300 mg/kg, based on a decrease in the number of implantation sites at 1000 mg/kg. Developmental NOAEL was considered to be below 100 mg/kg, based on reduced post-implantation survival and decreased live litter size starting at 100 mg/kg. At 1000 mg/kg no pups were available for evaluation of postnatal development.